ALBERT

Description: Background: Mutations in the PTRF gene, coding for cavin-1, cause congenital generalized lipodystrophy type 4 (CGL4) associated with myopathy. In CGL4, symptoms are variable comprising, in addition to myopathy, smooth and skeletal muscle hypertrophy, cardiac arrhythmias, and skeletal abnormalities. Secondary features are atlantoaxial instability, acanthosis nigricans, hepatomegaly, umbilical prominence and metabolic abnormalities related to insulin resistance, such as diabetes mellitus, hyperlipidemia and hepatic steatosis.Case presentationWe describe a 3 year-old child of Moroccan origin with mild muscle phenotype, mainly characterized by mounding, muscle pain, hyperCKemia and mild caveolin 3 reduction on muscle biopsy. No CAV3 gene mutation was detected; instead we found a novel mutation, a homozygous single base pair deletion, in the PTRF gene. Only after detection of this mutation a mild generalized loss of subcutaneous fat, at first underestimated, was noticed and the diagnosis of lipodystrophy inferred. Conclusions: The PTRF gene should be investigated in patients with hyperCKemia, mild myopathy associated with spontaneous or percussion-induced muscle contractions like rippling or mounding, and no CAV3 mutation. The analysis should be performed even if cardiac or metabolic alterations are absent, particularly in young patients in whom lipodystrophy may be difficult to ascertain.

Description: Background: Type 2 diabetes mellitus is increasing dramatically in sub-Saharan Africa, and genetic predisposition is likely involved in that. Yet, genetic variants known to confer increased susceptibility among Caucasians are far from being established in African populations. In Ghanaian adults, we examined associations of several of these polymorphisms with type 2 diabetes. Methods: A hospital-based case--control study on type 2 diabetes (and hypertension) was conducted in Kumasi, Ghana. TCF7L2 rs7903146, KCNJ11 rs5219, PPARgamma rs1801282 and CAPN10 rs3842570, rs3792267, and rs5030952 were typed and associations with type 2 diabetes and phenotypic traits examined. Results: 675 patients with type 2 diabetes and 377 controls were compared. The minor allele frequency of the TCF7L2 (T) allele was 0.33. In the multivariate model, this allele increased the risk of type 2 diabetes by 39% (95% confidence interval (CI), 1.07-1.81; p = 0.014). The minor alleles KCNJ11 (G) and PPARgamma (G) were practically absent (each, 0.001). Minor allele frequencies of CAPN10 were for -43 (A) 0.11 and for -63 (C) 0.46. These variants showed no significant associations with type 2 diabetes. Two CAPN10 haplotypes tended to protect against type 2 diabetes: 211 (aOR, 0.32; 95%CI, 0.03-1.92; p = 0.31) and 221 (aOR, 0.73; 95%CI, 0.48-1.10; p = 0.13). Conclusions: In urban Ghana, the frequency of the TCF7L2 rs7903146 (T) allele is comparable to the one in Caucasians; the association with type 2 diabetes is slightly weaker. The risk allele KCNJ11 (G) and the protective allele PPARgamma (G) are virtually absent. The potential influence of comparatively rare CAPN10 haplotypes on type 2 diabetes risk in this population requires further evaluation. Large-scale genetic studies among native Africans aiming at fine-mapping the candidate genes are needed to identify the actual factors involved in their increased susceptibility to type 2 diabetes.

Description: Background: Asthma genome-wide association studies (GWAS) have identified several asthma susceptibility genes with confidence; however the relative contribution of these genetic variants or single nucleotide polymorphisms (SNPs) to clinical endpoints (as opposed to disease diagnosis) remains largely unknown. Thus the aim of this study was to firstly bridge this gap in knowledge and secondly investigate whether these SNPs or those that are in linkage disequilibrium are likely to be functional candidates with respect to regulation of gene expression, using reported data from the ENCODE project. Methods: Eleven of the key SNPs identified in eight loci from recent asthma GWAS were evaluated for association with asthma and clinical outcomes including percent predicted FEV1, bronchial hyperresponsiveness (BHR) to methacholine, severity defined by British Thoracic Society steps and positive response to skin prick test using the family based association test additive model in a well characterised UK cohort consisting of 370 families with at least two asthmatic children. Results: GSDMB SNP rs2305480 (Ser311Pro) was associated with asthma diagnosis (p = 8.9x10-4), BHR (p = 8.2x10-4) and severity (p = 1.5x10-4) with supporting evidence from a second GSDMB SNP rs11078927 (intronic). SNPs evaluated in IL33, IL18R1, IL1RL1, SMAD3, IL2RB, PDE4D, CRB1 and RAD50 did not show association with any phenotype tested when corrected for multiple testing. Analysis using ENCODE data provide further insight into the functional relevance of these SNPs. Conclusions: Our results provide further support for the role of GSDMB SNPs in determining multiple asthma related phenotypes in childhood asthma including associations with lung function and disease severity.

Description: Background: Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as large genomic rearrangements. However, a large number of mutations, including missense, silent, and intronic variants, are classified as variants of unknown clinical significance. Methods: Intronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing. Results: We describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c.588 + 5G 〉 A, MLH1 c.677 + 3A 〉 T, MLH1 c.1732-2A 〉 T, MSH2 c.1276 + 1G 〉 T, and MSH2 c.1662-2A 〉 C), while four mutations had no effect on splicing compared to wild type (MLH1 c.117-34A 〉 T, MLH1 c.1039-8 T 〉 A, MSH2 c.2459-18delT, and MSH6 c.3439-16C 〉 T). Conclusions: In conclusion, we classify five MLH1/MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and their family members.

Description: Background: APOAI, a member of the APOAI/CIII/IV/V gene cluster on chromosome 11q23-24, encodes a major protein component of HDL that has been associated with serum lipid levels. The aim of this study was to determine the genetic association of polymorphisms in the APOAI promoter region with plasma lipid levels in a cohort of healthy Kuwaiti volunteers. Methods: A 435 bp region of the APOAI promoter was analyzed by re-sequencing in 549 Kuwaiti samples. DNA was extracted from blood taken from 549 healthy Kuwaiti volunteers who had fasted for the previous 12 h. Univariate and multivariate analysis was used to determine allele association with serum lipid levels. Results: The target sequence included a partial segment of the promoter region, 5'UTR and exon 1 located between nucleotides -141 to +294 upstream of the APOAI gene on chromosome 11. No novel single nucleotide polymorphisms (SNPs) were observed. The sequences obtained were deposited with the NCBI GenBank with accession number [GenBank: JX438706]. The allelic frequencies for the three SNPs were as follows: APOAI rs670G = 0.807; rs5069C = 0.964; rs1799837G = 0.997 and found to be in HWE. A significant association (p 〈 0.05) was observed for the APOAI rs670 polymorphism with increased serum LDL-C. Multivariate analysis showed that APOAI rs670 was an independent predictive factor when controlling for age, sex and BMI for both LDL-C (OR: 1.66, p = 0.014) and TC (OR: 1.77, p = 0.006) levels. Conclusion: This study is the first to report sequence analysis of the APOAI promoter in an Arab population. The unexpected positive association found between the APOAI rs670 polymorphism and increased levels of LDL-C and TC may be due to linkage disequilibrium with other polymorphisms in candidate and neighboring genes known to be associated with lipid metabolism and transport.

Description: Background: We investigated a potential link between genetic polymorphisms in genes XRCC1 (Arg399Gln), OGG1 (Ser326Cys), XRCC3 (Thr241Met), and XRCC4 (Ile401Thr) with the level of DNA damage and repair, accessed by comet and micronucleus test, in 51 COPD patients and 51 controls. Methods: Peripheral blood was used to perform the alkaline and neutral comet assay; and genetic polymorphisms by PCR/RFLP. To assess the susceptibility to exogenous DNA damage, the cells were treated with methyl methanesulphonate for 1-h or 3-h. After 3-h treatment the % residual damage was calculated assuming the value of 1-h treatment as 100%. The cytogenetic damage was evaluated by buccal micronucleus cytome assay (BMCyt). Results: COPD patients with the risk allele XRCC1 (Arg399Gln) and XRCC3 (Thr241Met) showed higher DNA damage by comet assay. The residual damage was higher for COPD with risk allele in the four genes. In COPD patients was showed negative correlation between BMCyt (binucleated, nuclear bud, condensed chromatin and karyorrhexic cells) with pulmonary function and some variant genotypes. Conclusion: Our results suggest a possible association between variant genotypes in XRCC1 (Arg399Gln), OGG1 (Ser326Cys), XRCC3 (Thr241Met), and XRCC4 (Ile401Thr), DNA damage and progression of COPD.

Description: Background: Current genetic test algorithms for Charcot Marie Tooth (CMT) disease are based on family details and comprehensive clinical and neurophysiological data gathered under ideal conditions for clinical assessment. However, in a diagnostic laboratory setting relying on external test requisitions and patient samples, such conditions are not always met. Our objective was therefore to perform a retrospective evaluation of the data given in laboratory request forms and to assess their quality and applicability with regard to the recommended algorithms for CMT diagnostics. As we are the main test centre for CMT in Norway our results also provide an overview of the spectrum of gene defects in the Norwegian CMT population. Methods: Genetic testing was performed according to polyneuropathy type; demyelinating/mixed: PMP22 duplication, MPZ, EGR2, LITAF, NEFL, PMP22, GJB1, axonal: MFN2, MPZ, NEFL, and GJB1. Results: Diagnostic testing of index patients was requested in 435 of the 549 cases. Seventy-two (16.6%) positive molecular genetic findings were made. The majority (94.6%) of mutation positive cases showed disease onset before 50 years of age. PMP22 (duplication), MPZ, GJB1 and MFN2 mutations constituted 95.8% of the positive findings. Within the nerve conduction study groups, mutation detection rates were; demyelinating 33.8%; mixed 29.0%; axonal 8.8%; unspecified 16.5%. Conclusion: We suggest a simplified algorithm intended for referral centres, dealing with DNA/blood samples, which involves the assessment of age at onset and neurophysiological data followed by testing of four genes; PMP22 (duplication), MPZ, GJB1 and MFN2. Patients negative for mutations in those four genes should be subjected to evaluation at an interdisciplinary inherited neuropathy clinic with the capacity for extended molecular genetic analysis by next generation sequencing.

Description: Background: Tumor-specific, coordinate expression of cancer-testis (CT) genes, mapping to the X chromosome, is observed in more than 60% of non-small cell lung cancer (NSCLC) patients. Although CT gene expression has been unequivocally related to DNA demethylation of promoter regions, the underlying mechanism leading to loss of promoter methylation remains elusive. Polymorphisms of enzymes within the 1-carbon pathway have been shown to affect S-adenosyl methionine (SAM) production, which is the sole methyl donor in the cell. Allelic variants of several enzymes within this pathway have been associated with altered SAM levels either directly, or indirectly as reflected by altered levels of SAH and Homocysteine levels, and altered levels of DNA methylation. We, therefore, asked whether the five most commonly occurring polymorphisms in four of the enzymes in the 1-carbon pathway associated with CT gene expression status in patients with NSCLC. Methods: Fifty patients among a cohort of 763 with NSCLC were selected based on CT gene expression status and typed for five polymorphisms in four genes known to affect SAM generation by allele specific q-PCR and RFLP. Results: We identified a significant association between CT gene expression and the MTHFR 677 CC genotype, as well as the C allele of the SNP, in this cohort of patients. Multivariate analysis revealed that the genotype and allele strongly associate with CT gene expression, independent of potential confounders. Conclusions: Although CT gene expression is associated with DNA demethylation, in NSCLC, our data suggests this is unlikely to be the result of decreased MTHFR function.

Description: Background: Vitamin D deficiency rickets is common in China. Genetic factors may play an important role in the susceptibility to rickets. Our study aimed to identify the relationship between three vitamin D-related genes (group specific component [GC], cytochrome P450, family 2, subfamily R, polypeptide 1 (CYP2R1), and 7-dehydrocholesterol reductase/nicotinamide-adenine dinucleotide synthetase 1 (DHCR7/NADSYN1) and rickets in Han Chinese children from northeastern China. Methods: A total of 506 Han children from northeastern China were enrolled in the current study. Twelve SNPs in three candidate genes were genotyped using the SNaPshot assay. Linear regression was used to examine the effect of 12 single-nucleotide polymorphisms (SNPs) on the risk of rickets. Results: In our case--control cohort, six alleles of the 12 SNPs conferred a significantly increased risk of rickets in GC (rs4588 C, P = 0.003, OR: 0.583, 95% CI: 0.412-0.836; rs222020 C, P = 0.009, OR: 1.526, 95% CI:1.117-2.0985; rs2282679 A, P = 0.010, OR: 0.636, 95% CI :0.449-0.900; and rs2298849 C, P = 0.001, OR: 1.709, 95% CI:1.250-2.338) and in CYP2R1 (rs10741657 G, P = 0.019, OR: 1.467, 95% CI:1.070-2.011; and rs2060793 G, P = 0.023, OR: 0.689, 95% CI:0.502-0.944). The results remained significant after adjustment for sex and body mass index. We further analyzed the effect of genotypes under three different genetic models. After using Bonferroni's method for multiple corrections, rs4588, rs2282679, and rs2298849 of the GC gene were significantly associated with rickets under the dominant (P =0.003 for rs4588, P =0.024 for rs2282679, and P =0.005 for rs2298849) and additive models (P = 0.006 for rs4588, P = 0.024 for rs2282679, and P = 0.005 for rs2298849). Haplotype analysis showed that the CAT haplotype of the GC gene (P = 0.005) and the GAA haplotype of the CYP2R1 gene (P = 0.026) were associated with susceptibility to rickets. Conclusions: This case--control study confirmed the strong effect of GC and CYP2R1 loci on rickets in Han children from northeastern China.

Description: Background: Chromosomal instability is a hallmark of human cancer caused by errors in mitotic control and chromosome segregation. STAG2 encodes a subunit of the cohesion complex that participates in mitotic chromatid separation and was recently found to show low expression and inactivating mutations in Ewing's sarcoma, melanoma and glioblastoma.In the childhood tumor neuroblastoma (NB) segmental chromosomal alterations are associated with poor prognosis whereas tumors displaying whole chromosome gains and losses have a much better prognosis.MethodAs the genetic contribution to aneuploidy is unknown in NB, we investigated the presence of STAG2 mutations through sequence analysis of all 33 coding exons in 37 primary NB tumors.Results and conclusionAs no STAG2 mutation was detected in this study, we conclude that inactivating mutation of STAG2 is not likely causative to neuroblastoma aneuploidy.

Description: Background: Mutations within the C-terminal region of the COL6A1 gene are only detected in Ullrich/Bethlem patients on extremely rare occasions.Case presentationHerein we report two Brazilian brothers with a classic Ullrich phenotype and compound heterozygous for two truncating mutations in COL6A1 gene, expected to result in the loss of the alpha1(VI) chain C2 subdomain. Despite the reduction in COL6A1 RNA level due to nonsense RNA decay, three truncated alpha1 (VI) chains were produced as protein variants encoded by different out-of-frame transcripts. Collagen VI matrix was severely decreased and intracellular protein retention evident. Conclusion: The altered deposition of the fibronectin network highlighted abnormal interactions of the mutated collagen VI, lacking the alpha1(VI) C2 domain, within the extracellular matrix, focusing further studies on the possible role played by collagen VI in fibronectin deposition and organization.

Description: Background: Cystic fibrosis (CF) is a monogenic disease caused by CFTR gene mutations, with clinical expression similar to complex disease, influenced by genetic and environmental factors. Among the possible modifier genes, those associated to metabolic pathways of glutathione (GSH) have been considered as potential modulators of CF clinical severity. In this way it is of pivotal importance investigate gene polymorphisms at Glutamate-Cysteine Ligase, Catalytic Subunit (GCLC), Glutathione S-transferase Mu 1 (GSTM1), Glutathione S-transferase Theta 1 (GSTT1), and Glutathione S-transferase P1 (GSTP1), which have been associated to the GSH metabolic pathway and CF clinical severity.MethodA total of 180 CF's patients were included in this study, which investigated polymorphisms in GCLC and GST genes (GCLC -129C〉T and -3506A〉G; GSTM1 and GSTT1 genes deletion, and GSTP1*+313A〉G) by PCR and PCR-RFLP associating to clinical variables of CF severity, including variables of sex, clinical scores [Shwachman-Kulczycki, Kanga e Bhalla (BS)], body mass index, patient age, age for diagnosis, first clinical symptoms, first colonization by Pseudomonas aeruginosa, sputum's microorganisms, hemoglobin oxygen saturation in the blood, spirometry and comorbidities. The CFTR genotype was investigated in all patients, and the genetic interaction was performed using MDR2.0 and MDRPT0.4.7 software. Results: The analysis of multiple genes in metabolic pathways in diseases with variable clinical expression, as CF disease, enables understanding of phenotypic diversity. Our data show evidence of interaction between the GSTM1 and GSTT1 genes deletion, and GSTP1*+313A〉G polymorphism with CFTR gene mutation classes, and BS (Balance testing accuracy= 0.6824, p= 0.008), which measures the commitment of bronchopulmonary segments by tomography. Conclusion: Polymorphisms in genes associated with metabolism of GSH act on the CF's severity.

Description: Background: Vascular endothelial growth factor A (VEGFA) is a major regulator of both physiological and pathological angiogenesis. Associations between polymorphisms in VEGFA and complex disease have been inconsistent. The parent from whom the allele was inherited may account for these inconsistencies. This study examined the parent of origin effect on the expression of Vegfa. Methods: Two homozygous, inbred mouse strains A/J (AJ) and 129x1/SvJ (129) were crossed to produce reciprocal AJ129 and 129AJ offspring, respectively. RNA was extracted from cardiac tissue of 6 week old male (n = 8) and female (n = 8) parental, and male and female F1 offspring mice (AJ129 n = 8 and 129AJ n = 8). Vegfa and Hif1a expression levels were measured by qPCR and compared between the F1 offspring from the reciprocal crosses. Results: We found significant differences in the expression of Vegfa in F1 offspring (AJ129 and 129AJ mice) of the reciprocal crosses between AJ and 129 mice. Offspring of male AJ mice had significantly higher expression of Vegfa than offspring of male 129 mice (p = 0.006). This difference in expression was not the result of preferential allele expression (allelic imbalance). Expression of Hif1a, a transcriptional regulator of Vegfa expression, was also higher in F1 offspring of an AJ father (p = 0.004). Conclusion: Differences in Vegfa and Hif1a gene expression are likely the result of an upstream angiogenic regulator gene that is influenced by the parent of origin. These results highlight the importance of including inheritance information, such as parent of origin, when undertaking allelic association studies.

Description: Background: Variant Creutzfeldt-Jakob disease is an infectious, neurodegenerative, protein-misfolding disease, of the prion disease family, originally acquired through ingestion of meat products contaminated with bovine spongiform encephalopathy (BSE). Public health concern was increased by the discovery of human-to-human transmission via blood transfusion. This study has verified a novel genetic marker linked to disease risk. Methods: SNP imputation and association testing indicated those genes that had significant linkage to disease risk and one gene was investigated further with Sanger resequencing. Results from variant Creutzfeldt-Jakob disease were compared with those from sporadic (idiopathic) Creutzfeldt-Jakob disease and published controls. Results: The most significant disease risk, in addition to the prion protein gene, was for the phosphatidylinositol-specific phospholipase C, X domain containing 3 (PLCXD3) gene. Sanger resequencing of CJD patients across a region of PLCXD3 with known variants confirmed three SNPs associated with variant and sporadic CJD. Conclusions: These data provide the first highly significant confirmation of SNP allele frequencies for a novel CJD candidate gene providing new avenues for investigating these neurodegenerative prion diseases. The phospholipase PLCXD3 is primarily expressed in the brain and is associated with lipid catabolism and signal transduction.

Description: Background: We report on a patient with genetically confirmed overlapping diagnoses of CMT1A and FSHD. This case adds to the increasing number of unique patients presenting with atypical phenotypes, particularly in FSHD. Even if a mutation in one disease gene has been found, further genetic testing might be warranted in cases with unusual clinical presentation.Case presentationThe reported 53 years old male patient suffered from walking difficulties and foot deformities first noticed at age 20. Later on, he developed scapuloperoneal and truncal muscle weakness, along with atrophy of the intrinsic hand and foot muscles, pes cavus, claw toes and a distal symmetric hypoesthesia. Motor nerve conduction velocities were reduced to 20 m/s in the upper extremities, and not educible in the lower extremities, sensory nerve conduction velocities were not attainable. Electromyography showed both, myopathic and neurogenic changes. A muscle biopsy taken from the tibialis anterior muscle showed a mild myopathy with some neurogenic findings and hypertrophic type 1 fibers. Whole body muscle MRI revealed severe changes in the lower leg muscles, tibialis anterior and gastrocnemius muscles were highly replaced by fatty tissue. Additionally, fatty degeneration of shoulder girdle and straight back muscles, and atrophy of dorsal upper leg muscles were seen. Taken together, the presenting features suggested both, a neuropathy and a myopathy. Patient's family history suggested an autosomal dominant inheritance.Molecular testing revealed both, a hereditary motor and sensory neuropathy type 1A (HMSN1A, also called Charcot-Marie-Tooth neuropathy 1A, CMT1A) due to a PMP22 gene duplication and facioscapulohumeral muscular dystrophy (FSHD) due to a partial deletion of the D4Z4 locus (19 kb). Conclusion: Molecular testing in hereditary neuromuscular disorders has led to the identification of an increasing number of atypical phenotypes. Nevertheless, finding the right diagnosis is crucial for the patient in order to obtain adequate medical care and appropriate genetic counseling, especially in the background of arising curative therapies.

Description: Background: Auditory neuropathy spectrum disorder (ANSD) is a unique form of hearing loss that involves absence or severe abnormality of auditory brainstem response (ABR), but also the presence of otoacoustic emissions (OAEs). However, with age, the OAEs disappear, making it difficult to distinguish this condition from other nonsyndromic hearing loss. Therefore, the frequency of ANSD may be underestimated. The aim of this study was to determine what portion of nonsyndromic hearing loss is caused by mutations of OTOF, the major responsible gene for nonsyndromic ANSD. Methods: We screened 160 unrelated Japanese with severe to profound recessive nonsyndromic hearing loss (ARNSHL) without GJB2 or SLC26A4 mutations, and 192 controls with normal hearing. Results: We identified five pathogenic OTOF mutations (p.D398E, p.Y474X, p.N727S, p.R1856Q and p.R1939Q) and six novel, possibly pathogenic variants (p.D450E, p.W717X, p.S1368X, p.R1583H, p.V1778I, and p.E1803A). Conclusions: The present study showed that OTOF mutations accounted for 3.2--7.3% of severe to profound ARNSHL patients in Japan. OTOF mutations are thus a frequent cause in the Japanese deafness population and mutation screening should be considered regardless of the presence/absence of OAEs.

Description: Background: Delayed neuropsychological sequelae (DNS) are the most severe and clinically intractable complications following acute carbon monoxide (CO) poisoning. Symptoms of DNS often resemble those of Parkinson's disease (PD), suggesting shared neurological deficits. Furthermore, Parkinson protein 2 (PARK2) mutations are associated with PD and other neurodegenerative diseases. The association signal was detected between PARK2 and DNS after acute CO poisoning in our DNA pooling base genome-wide association study. Methods: Two PARK2 single nucleotide polymorphisms (SNPs), rs1784594 (C/T allele) and rs1893895 (G/A allele), selected from DNA pooling base genome-wide association study, were genotyped by in 514 CO poisoning patients using polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLPs). The patient group consisted of 231 patients with DNS and 283 patients with no signs of lasting neurological damage (control population). Results: The frequency of the rs1784594 T allele was significantly lower in the DNS population (OR = 1.42, 95%CI: 1.08 - 1.87), as was the TT vs. CC genotype (OR = 1.95, 95%CI: 1.15 - 3.23) and the TT vs. CT + CC frequency (OR = 1.68, 95%CI: 1.32 - 2.49) compared to controls. Association analysis revealed a significant association between DNS and rs1784594 (P 〈 0.01) but not rs1893895 (P 〉 0.05). In female cases, the T allele frequency of rs1784594 was significantly lower in DNS patients compared to female controls (OR = 1.48, 95%CI: 1.01 - 2.17). Conclusion: These data suggest that the allelic variant of rs1784594 is a risk factor for DNS following acute CO poisoning, especially in females. The PARK2 protein may modulate the susceptibility to DNS, underscoring the importance of examining the relationship between other PARK2 polymorphisms and clinical outcome following CO poisoning.

Description: Background: Multiple genome-wide association studies (GWAS) within European populations have implicated common genetic variants associated with insulin and glucose concentrations. In contrast, few studies have been conducted within minority groups, which carry the highest burden of impaired glucose homeostasis and type 2 diabetes in the U.S. Methods: As part of the 'Population Architecture using Genomics and Epidemiology (PAGE) Consortium, we investigated the association of up to 10 GWAS-identified single nucleotide polymorphisms (SNPs) in 8 genetic regions with glucose or insulin concentrations in up to 36,579 non-diabetic subjects including 23,323 European Americans (EA) and 7,526 African Americans (AA), 3,140 Hispanics, 1,779 American Indians (AI), and 811 Asians. We estimated the association between each SNP and fasting glucose or log-transformed fasting insulin, followed by meta-analysis to combine results across PAGE sites. Results: Overall, our results show that 9/9 GWAS SNPs are associated with glucose in EA (p = 0.04 to 9 x 10-15), versus 3/9 in AA (p= 0.03 to 6 x 10-5), 3/4 SNPs in Hispanics, 2/4 SNPs in AI, and 1/2 SNPs in Asians. For insulin we observed a significant association with rs780094/GCKR in EA, Hispanics and AI only. Conclusions: Generalization of results across multiple racial/ethnic groups helps confirm the relevance of some of these loci for glucose and insulin metabolism. Lack of association in non-EA groups may be due to insufficient power, or to unique patterns of linkage disequilibrium.

Description: Background: The transcription factor Nrf2, encoded by the NFE2L2 gene, is an important regulator of the cellular protection against oxidative stress. Parkinson?s disease is a neurodegenerative disease highly associated with oxidative stress. In a previously published study, we reported associations of NFE2L2 haplotypes with risk and age at onset of idiopathic Parkinson?s disease in a Swedish discovery material and a Polish replication material. Here, we have extended the replication study and performed meta-analyses including the Polish material and four new independent European patient-control materials. Furthermore, all SNPs included in the haplotype windows were investigated individually for associations with Parkinson?s disease in meta-analyses including all six materials. Methods: Totally 1038 patients and 1600 control subjects were studied. Based on previous NFE2L2 haplotype associations with Parkinson?s disease, five NFE2L2 tag SNPs were genotyped by allelic discrimination and three functional NFE2L2 promoter SNPs were genotyped by sequencing. The impact of individual SNPs and haplotypes on risk and age at onset of Parkinson?s disease were investigated in each material individually and in meta-analyses of the obtained results. Results: Meta-analyses of NFE2L2 haplotypes showed association of haplotype GAGCAAAA, including the fully functional promoter haplotype AGC, with decreased risk (OR?=?0.8 per allele, p?=?0.012) and delayed onset (+1.1?years per allele, p?=?0.048) of Parkinson?s disease. These results support the previously observed protective effect of this haplotype in the first study. Further, meta-analyses of the SNPs included in the haplotypes revealed four NFE2L2 SNPs associated with age at onset of Parkinson?s disease (rs7557529 G?〉?A, ?1.0?years per allele, p?=?0.042; rs35652124 A?〉?G, ?1.1?years per allele, p?=?0.045; rs2886161 A?〉?G, ?1.2?years per allele, p?=?0.021; rs1806649 G?〉?A, +1.2?years per allele, p?=?0.029). One of these (rs35652124) is a functional SNP located in the NFE2L2 promoter. No individual SNP was associated with risk of Parkinson?s disease. Conclusion: Our results support the hypothesis that variation in the NFE2L2 gene, encoding a central protein in the cellular protection against oxidative stress, may contribute to the pathogenesis of Parkinson?s disease. Functional studies are now needed to explore these results further.

Description: Background: Most PCR-based diagnostics are still considered time- and labor-intensive due to disparate purification, amplification, and detection steps. Advancements in PCR enzymes and buffer chemistry have increased inhibitor tolerance, facilitating PCR directly from crude samples. Obviating the need for DNA purification, while lacking a concentration step, these direct sample methods are particularly apt for human genetic testing. However, direct PCR protocols have traditionally employed thermal cyclers with slow ramp rates and conservative hold times that significantly increase an assay?s time-to-result. For this proof-of-principle study, our objective was to significantly reduce sample preparation and assay time for a PCR-based genetic test, for myotonic dystrophy type 1 (DM1), by pairing an inhibitor-resistant enzyme mix with a rapid thermal cycler to analyze samples directly in whole blood. Methods: DM1 genetic screening was done with an adapted conventional PCR approach that employed the Streck Philisa? Thermal Cycler, the inhibitor-resistant NEBNext? High-Fidelity 2X PCR Master Mix, and agarose gel electrophoresis or an Agilent 2100 Bioanalyzer for detection. The Gene Link? Myotonic Dystrophy Genemer? Kit was used as a reference assay kit to evaluate the rapid assay. Results: In this work, a rapid and direct PCR assay testing 10% whole blood as template has been developed as an exclusionary screening assay for DM1, a triple-repeat genetic disorder. PCR amplification was completed in 15 minutes using 30 cycles, including in situ hot-start/cell lysis. Out of the 40 donors screened, this assay identified 23 (57.5%) as DM1 negative suggesting no need for further testing. These data are 100% concordant with data collected using the commercially available Gene Link Genemer? Kit per the kit-specific PCR protocol. Conclusions: The PCR assay described in this study amplified DM1 short tandem repeats in 15 minutes. By eliminating sample purification and slower conventional PCR protocols, we demonstrated how adaptation of current PCR technology and chemistries can produce a simple-to-use exclusionary screening assay that is independent of up-front sample prep, improving a clinical lab technician?s time-to-result. We envision this direct and rapid methodology could be applied to other conventional PCR-based genetic tests and sample matrices where genomic DNA is targeted for analysis within a given molecular diagnostic platform.

Description: Background: Inherited thrombocytopenias (IT) are a heterogeneous group of rare diseases characterized by a reduced number of blood platelets. The frequency of IT is probably underestimated because of diagnostic difficulties and because not all the existing forms have as yet been identified, with some patients remaining without a definitive diagnosis. Exome Sequencing has made possible the identification of almost all variants in the coding regions of protein-coding genes, thereby providing the opportunity to identify the disease causing gene in a number of patients with indefinite diagnoses, specifically in consanguineous families.Case presentationFamilial thrombocytopenia with small size platelets was present in several members of a highly consanguineous family from Northern Iraq. Genotyping of all affected, their unaffected siblings and parents, followed by exome sequencing revealed a strong candidate loss of function variant in a homozygous state: a frameshift mutation in the FYB gene. The protein encoded by this gene is known to be a cytosolic adaptor molecule expressed by T, natural killer (NK), myeloid cells and platelets, and is involved in platelet activation and controls the expression of interleukin-2. Knock-out mice were reported to show isolated thrombocytopenia. Conclusion: Inherited thrombocytopenias differ in their presentation, associated features, and molecular etiologies. An accurate diagnosis is needed to provide appropriate management as well as counseling for the individuals and their family members. Exome sequencing may become a first diagnostic tool to identify the molecular basis of undiagnosed familial IT. In this report, the clinical evaluation combined with the power and efficiency of genomic analysis defined the FYB gene as the possible underlying cause of autosomal recessive thrombocytopenia with small platelet size. This is the first report linking pathogenic variants in FYB and thrombocytopenia in humans.

Description: Background: The MedSeq Project is a randomized clinical trial developing approaches to assess the impact of integrating genome sequencing into clinical medicine. To facilitate the return of results of potential medical relevance to physicians and patients participating in the MedSeq Project, we sought to develop a reporting approach for the effective communication of such findings. Methods: Genome sequencing was performed on the Illumina HiSeq platform. Variants were filtered, interpreted, and validated according to methods developed by the Laboratory for Molecular Medicine and consistent with current professional guidelines. The GeneInsight software suite, which is integrated with the Partners HealthCare electronic health record, was used for variant curation, report drafting, and delivery. Results: We developed a concise 5?6 page Genome Report (GR) featuring a single-page summary of results of potential medical relevance with additional pages containing structured variant, gene, and disease information along with supporting evidence for reported variants and brief descriptions of associated diseases and clinical implications. The GR is formatted to provide a succinct summary of genomic findings, enabling physicians to take appropriate steps for disease diagnosis, prevention, and management in their patients. Conclusions: Our experience highlights important considerations for the reporting of results of potential medical relevance and provides a framework for interpretation and reporting practices in clinical genome sequencing.

Description: Background: Cerebral palsy (CP) is a heterogeneous neurodevelopmental disorder associated with intellectual disability in one-third of cases. Recent findings support Mendelian inheritance in subgroups of patients with the disease. The purpose of this study was to identify a novel genetic cause of paraplegic CP with intellectual disability in a consanguineous Pakistani family. Methods: We performed whole-exome sequencing (WES) in two brothers with CP and intellectual disability. Analysis of AP4M1 mRNA was performed using quantitative real-time PCR on total RNA from cultured fibroblasts. The brothers were investigated clinically and by MRI. Results: We identified a novel homozygous AP4M1 mutation c.194_195delAT, p.Y65Ffs*50 in the affected brothers. Quantitative RT-PCR analysis showed markedly reduced AP4M1 mRNA levels suggesting partial non-sense mediated mRNA decay. Several clinical and MRI features were consistent with AP-4 complex deficiency. However, in contrast to previously reported cases with AP4M1 mutations our patients show an aggressive behavior and a relatively late onset of disease. Conclusion: This study shows an AP4M1 mutation associated with aggressive behavior in addition to mild dysmorphic features, intellectual disability, spastic paraparesis and reduced head circumference. Our findings expand the clinical spectrum associated with AP-4 complex deficiency and the study illustrates the importance of MRI and WES in the diagnosis of patients with CP and intellectual disability.

Description: Background: Epidemiological studies have suggested that variants on adiponectin (ADIPOQ) and its receptor ADIPOR1 (adiponectin receptor 1) are associated with colorectal cancer (CRC) risk; however, the results were inconclusive. The aim of the study was to evaluate the associations between the variants on ADIPOQ and ADIPOR1 and the CRC risk with a hospital-based case-control study in the Chinese population along with meta-analysis of available epidemiological studies. Methods: With a hospital-based case-control study of 341 cases and 727 controls, the associations between the common variants on ADIPOQ (rs266729, rs822395, rs2241766 and rs1501299) and ADIPOR1 (rs1342387 and rs12733285) and CRC susceptibility were evaluated. Meta-analysis of the published epidemiological studies was performed to investigate the associations between the variants and CRC risk. Results: For the population study, we found that variant rs1342387 of ADIPOR1 was associated with a reduced risk for CRC [adjusted odds ratio (OR)?=?0.74, 95% confidential intervals (95% CI)?=?0.57-0.97; CT/TT vs. CC]. The meta-analysis also suggested a significant association for rs1342387 and CRC risk; the pooled OR was 0.79 (95% CI?=?0.66-0.95) for the CT/TT carriers compared to CC homozygotes under the random-effects model (Q?=?8.06, df?=?4, P?=?0.089; I2?=?50.4%). The case-control study found no significant association for variants rs266729, rs822395, rs2241766, and rs1501299 on ADIPOQ or variant rs12733285 on ADIPOR1 and CRC susceptibility, which were consistent with results from the meta-analysis studies. Conclusions: These data suggested that variant rs1342387 on ADIPOR1 may be a novel CRC susceptibility factor.

Description: Background: Tourette Syndrome (TS) is a neuropsychiatric disorder in children characterized by motor andverbal tics. Although several genes have been suggested in the etiology of TS, the geneticmechanisms remain poorly understood. Methods: Using cytogenetics and FISH analysis, we identified an apparently balancedt(6,22)(q16.2;p13) in a male patient with TS and obsessive-compulsive disorder (OCD). Inorder to map the breakpoints and to identify additional submicroscopic rearrangements, weperformed whole genome mate-pair sequencing and CGH-array analysis on DNA from theproband. Results: Sequence and CGH array analysis revealed a 400 kb deletion located 1.3 Mb telomeric of thechromosome 6q breakpoint, which has not been reported in controls. The deletion affectsthree genes (GPR63, NDUFA4 and KLHL32) and overlaps a region previously found deletedin a girl with autistic features and speech delay. The proband's mother, also a carrier of thetranslocation, was diagnosed with OCD and shares the deletion. We also describe a furtherpotentially related rearrangement which, while unmapped in Homo sapiens, was consistentwith the chimpanzee genome. Conclusions: We conclude that genome-wide sequencing at relatively low resolution can be used for theidentification of submicroscopic rearrangements.We also show that large rearrangements may escape detection using standard analysis ofwhole genome sequencing data. Our findings further provide a candidate region for TS andOCD on chromosome 6q16.

Description: Background: The 22q11.2 deletion syndrome (22q11.2DS) is caused by hemizygous microdeletions on chromosome 22q11.2 with highly variable physical and neuropsychiatric manifestations. We explored the genotype-phenotype relationship in a relatively large 22q11.2DS cohort treated and monitored in our clinic using comprehensive clinical evaluation and detailed molecular characterization of the deletion. Methods: Molecular analyses in 142 subjects with 22q11.2DS features were performed by FISH and MLPA methods. Participants underwent clinical assessment of physical symptoms and structured psychiatric and cognitive evaluation. Results: Deletions were found in 110 individuals including one with an atypical nested distal deletion which was missed by the FISH test. Most subjects (88.2%) carried the 3Mb typically deleted region and 11.8% carried 4 types of deletions differing in size and location. No statistically significant genotype-phenotype correlations were found between deletion type and clinical data although some differences in hypocalcemia and cardiovascular anomalies were noted.Analysis of the patient with the distal nested deletion suggested a redundancy of genes causing the physical and neuropsychiatric phenotype in 22q11.2DS and indicating that the psychiatric and cognitive trajectories may be governed by different genes. Conclusions: MLPA is a useful and affordable molecular method combining accurate diagnosis and detailed deletion characterization. Variations in deletion type and clinical manifestations impede the detection of significant differences in samples of moderate size, but analysis of individuals with unique deletions may provide insight into the underlying biological mechanisms.Future genotype-phenotype studies should involve large multicenter collaborations employing uniform clinical standards and high-resolution molecular methods.

Description: Background: Rubinstein-Taybi syndrome (RTS) is a rare autosomal dominant disorder (prevalence 1:125,000) characterised by broad thumbs and halluces, facial dysmorphism, psychomotor development delay, skeletal defects, abnormalities in the posterior fossa and short stature. The known genetic causes are point mutations or deletions of the cAMP-response element binding protein-BP (CREBBP) (50-60% of the cases) and of the homologous gene E1A-binding protein (EP300) (5%).Case presentationWe describe, for the first time in literature, a RTS Caucasian girl, 14-year-old, with growth hormone (GH) deficiency, pituitary hypoplasia, Arnold Chiari malformation type 1, double syringomyelic cavity and a novel CREBBP mutation (c.3546insCC). Conclusion: We hypothesize that CREBBP mutation we have identified in this patient could be responsible also for atypical features as GH deficiency and pituitary hypoplasia.

Description: Background: Blood coagulation is an essential determinant of coronary artery disease (CAD). Soluble Endothelial Protein C Receptor (sEPCR) may be a biomarker of a hypercoagulable state. We prospectively investigated the relationship between plasma sEPCR levels and the risk of cardiovascular events (CVE). Methods: We measured baseline sEPCR levels in 1673 individuals with CAD (521 with acute coronary syndrome [ACS] and 1152 with stable angina pectoris [SAP]) from the AtheroGene cohort. During a median follow up of 3.7 years, 136 individuals had a CVE. In addition, 891 of these CAD patients were genotyped for the PROCR rs867186 (Ser219Gly) variant. Results: At baseline, sEPCR levels were similar in individuals with ACS and SAP (median: 111 vs. 115 ng/mL respectively; p=0.20). Increased sEPCR levels were found to be associated with several cardiovascular risk factors including gender (p=0.006), soluble Tissue Factor levels (p=0.0001), diabetes (p=0.0005), and factors reflecting impaired renal function such as creatinine and cystatin C (p

Description: Background: Hypertrophic Cardiomyopathy (HCM) is a genetically heterogeneous disease. One specific mutation in the MYBPC3 gene is highly prevalent in center east of France giving an opportunity to define the clinical profile of this specific mutation. Methods: HCM probands were screened for mutation in the MYH7, MYBPC3, TNNT2 and TNNI3 genes. Carriers of the MYBPC3 IVS20-2A〉G mutation were genotyped with 8 microsatellites flanking this gene. The age of this MYBPC3 mutation was inferred with the software ESTIAGE. The age at first symptom, diagnosis, first complication, first severe complication and the rate of sudden death were compared between carriers of the IVS20-2 mutation (group A) and carriers of all other mutations (group B) using time to event curves and log rank test. Results: Out of 107 HCM probands, 45 had a single heterozygous mutation in one of the 4 tested sarcomeric genes including 9 patients with the MYBPC3 IVS20-2A〉G mutation. The IVS20-2 mutation in these 9 patients and their 25 mutation carrier relatives was embedded in a common haplotype defined after genotyping 4 polymorphic markers on each side of the MYBPC3 gene. This result supports the hypothesis of a common ancestor. Furthermore, we evaluated that the mutation occurred about 47 generations ago, approximately at the 10th century.We then compared the clinical profile of the IVS20-2 mutation carriers (group A) and the carriers of all other mutations (group B). Age at onset of symptoms was similar in the 34 group A cases and the 73 group B cases but group A cases were diagnosed on average 15 years later (log rank test p = 0.022). Age of first complication and first severe complication was delayed in group A vs group B cases but the prevalence of sudden death and age at death was similar in both groups. Conclusion: A founder mutation arising at about the 10th century in the MYBPC3 gene accounts for 8.4% of all HCM in center east France and results in a cardiomyopathy starting late and evolving slowly but with an apparent risk of sudden death similar to other sarcomeric mutations.

Description: Background: Emerging evidence has shown that miRNAs are involved in human carcinogenesis as tumor suppressors or oncogenes. Single nucleotide polymorphisms (SNPs) located in pre-miRNAs may affect the processing and therefore, influence the expression of mature miRNAs. Previous studies generated conflicting results when reporting association between the hsa-miR-196a2 rs11614913 common polymorphism and breast cancer. Methods: This study evaluated the hsa-miR-196a2 rs11614913 SNP in 388 breast cancer cases and 388 controls in Brazilian women. Polymorphism was determined by real-time PCR; control and experimental groups were compared through statistical analysis using the X2 or Fisher's exact tests. Results: The analysis of the SNPs frequencies showed a significant difference between the groups (BC and CT) in regards to genotype distribution (chi2: p = 0.024); the homozygous variant (TC) was more frequent in the CT than in the BC group (p = 0.009). The presence of the hsa-miR-196a2 rs11614913 C/T polymorphism was not associated with histological grades (p = 0.522), axillary lymph node positive status (p = 0.805), or clinical stage (p = 0.670) among the breast cancer patients. Conclusions: The results of this study indicated that the CC polymorphic genotype is associated with a decreased risk of BC and the presence of the T allele was significantly associated with an increased risk of BC.

Description: Background: Lung cancer is a complex polygenic disease. Although recent genome-wide association (GWA) studies have identified multiple susceptibility loci for lung cancer, most of these variants have not been validated in a Chinese population. In this study, we investigated whether a genetic risk score combining multiple. Methods: Five single-nucleotide polymorphisms (SNPs) identified in previous GWA or large cohort studies were genotyped in 5068 Chinese case--control subjects. The genetic risk score (GRS) based on these SNPs was estimated by two approaches: a simple risk alleles count (cGRS) and a weighted (wGRS) method. The area under the receiver operating characteristic (ROC) curve (AUC) in combination with the bootstrap resampling method was used to assess the predictive performance of the genetic risk score for lung cancer. Results: Four independent SNPs (rs2736100, rs402710, rs4488809 and rs4083914), were found to be associated with a risk of lung cancer. The wGRS based on these four SNPs was a better predictor than cGRS. Using a liability threshold model, we estimated that these four SNPs accounted for only 4.02% of genetic variance in lung cancer. Smoking history contributed significantly to lung cancer (P 〈 0.001) risk [AUC = 0.619 (0.603-0.634)], and incorporated with wGRS gave an AUC value of 0.639 (0.621-0.652) after adjustment for over-fitting. This model shows promise for assessing lung cancer risk in a Chinese population. Conclusion: Our results indicate that although genetic variants related to lung cancer only added moderate discriminatory accuracy, it still improved the predictive ability of the assessment model in Chinese population.

Description: Background: Many studies have been carried out to test the hypothesis that the NQO1 C609T polymorphism might be associated with the risk of esophageal cancer. However, the results are poorly consistent, partly due to genetic or other sources of heterogeneity. To investigate the association between this polymorphism and the risk of esophageal cancer, a meta-analysis was performed. Methods: We used odds ratios (ORs) with 95% confidence intervals (CIs) to assess the strength of association. The frequency of the putative risk allele in the controls was estimated by the inverse-variance method. Cochran's Q statistic and the inconsistency index (I2) were used to check heterogeneity. Egger's test and an inverted funnel plot were used to assess the publication bias. Results: Our study included eight published case-control studies about the NQO1 C609T polymorphism and esophageal cancer, including a total of 1,217 esophageal cancer patients and 1,560 controls. Overall, a significant association was found between the NQO1 C609T variant and esophageal cancer under a recessive model (OR = 1.647; 95% CI = 1.233-2.200). Regarding histological type, more significant evidence was found for esophageal squamous cell carcinoma (ESCC) (OR = 2.03; 95% CI = 1.29-3.19) than esophageal adenocarcinoma (EAC) (OR = 1.61; 95% CI = 1.01-2.56) under a recessive model. Conclusions: The meta-analysis suggests that the NQO1 C609T polymorphism considerably increases the risk of esophageal cancer.

Description: Background: There is evidence that one of the key type 2 diabetes (T2D) loci identified by GWAS exerts its influence early on in life through its impact on pediatric BMI. This locus on 10q23 harbors three genes, encoding hematopoietically expressed homeobox (HHEX), insulin-degrading enzyme (IDE) and kinesin family member 11 (KIF11), respectively. Methods: We analyzed the impact of adipogeneis on the mRNA and protein expression levels of these genes in the human adipocyte Simpson-Golabi-Behmel syndrome (SGBS) cell line in order to investigate which could be the culprit gene(s) in this region of linkage disequilibrium. Results: Following activation of differentiation with a PPARgamma ligand, we observed ~20% decrease in IDE, ~40% decrease in HHEX and in excess of 80% decrease in KIF11 mRNA levels when comparing the adipocyte and pre-adipocyte states. We also observed decreases in KIF11 and IDE protein levels, but conversely we observed a dramatic increase in HHEX protein levels. Subsequent time course experiments revealed some marked changes in expression as early as three hours after activation of differentiation. Conclusion: Our data suggest that the expression of all three genes at this locus are impacted during SGBS adipogenesis and provides insights in to the possible mechanisms of how the genes at this 10q23 locus could influence both adipocyte differentiation and susceptibility to T2D through insulin resistance.

Description: Background: Insulin resistance (IR) and endothelial dysfunction are frequently associated in cardiac disease. The T-786[rightwards arrow]C variant in the promoter region of the endothelial nitric oxide synthase (eNOS) gene has been associated with IR in both non-diabetic and diabetic subjects. Aim of the study was to assess the reciprocal relationships between T-786[rightwards arrow]C eNOS polymorphism and IR in ischemic and non-ischemic cardiomyopathy.MethodA group of 132 patients (108 males, median age 65 years) with global left ventricular (LV) dysfunction secondary to ischemic or non-ischemic heart disease was enrolled. Genotyping of T-786[rightwards arrow]C eNOS gene promoter, fasting glucose, insulin, and insulin resistance (defined as HOMA-IR index 〉 2.5) were determined in all patients. Results: Genotyping analysis yielded 37 patients homozygous for the T allele (TT), 70 heterozygotes (TC) and 25 homozygous for C (CC). Patients with CC genotype had significantly higher systemic arterial pressure, blood glucose, plasma insulin and HOMA index levels than TT. At multivariate logistic analysis, the history of hypertension and the genotype were the only predictors of IR. In particular, CC genotype increased the risk of IR (CI% 1.4-15.0, p 〈 0.01) 4.5-fold. The only parameter independently associated with the extent of LV dysfunction and the presence of heart failure (HF) was the HOMA index (2.4 CI% 1.1-5.6, p 〈 0.04). Conclusions: T-786[rightwards arrow]C eNOS polymorphism was the major independent determinant of IR in a population of patients with ischemic and non-ischemic cardiomyopathy. The results suggest that a condition of primitive eNOS lower expression can predispose to an impairment of glucose homeostasis, which in turn is able to affect the severity of heart disease.

Description: Background: Deletions and duplications of the PAFAH1B1 and YWHAE genes in 17p13.3 are associatedwith different clinical phenotypes. In particular, deletion of PAFAH1B1 causes isolatedlissencephaly while deletions involving both PAFAH1B1 and YWHAE cause Miller-Diekersyndrome. Isolated duplications of PAFAH1B1 have been associated with milddevelopmental delay and hypotonia, while isolated duplications of YWHAE have beenassociated with autism. In particular, different dysmorphic features associated withPAFAH1B1 or YWHAE duplication have suggested the need to classify the patient clinicalfeatures in two groups according to which gene is involved in the chromosomal duplication. Methods: We analyze the proband and his family by classical cytogenetic and array-CGH analyses. Theputative rearrangement was confirmed by fluorescence in situ hybridization. Results: We have identified a family segregating a 17p13.3 duplication extending 329.5 kilobases byFISH and array-CGH involving the YWHAE gene, but not PAFAH1B1, affected by a milddysmorphic phenotype with associated autism and mental retardation. We propose thatBHLHA9, YWHAE, and CRK genes contribute to the phenotype of our patient. The smallchromosomal duplication was inherited from his mother who was affected by a bipolar andborderline disorder and was alcohol addicted. Conclusions: We report an additional familial case of small 17p13.3 chromosomal duplication includingonly BHLHA9, YWHAE, and CRK genes. Our observation and further cases with similarmicroduplications are expected to be diagnosed, and will help better characterise the clinicalspectrum of phenotypes associated with 17p13.3 microduplications.

Description: Background: Continuing developments in genetic testing technology together with research revealing gene-disease associations have brought closer the potential for genetic screening of populations. A major concern, as with any screening programme, is the response of the patient to the findings of screening, whether the outcome is positive or negative. Such concern is heightened for genetic testing, which it is feared may elicit stronger reactions than non-genetic testing. Methods: This paper draws on thematic analysis of 113 semi-structured interviews with 39 patients being tested for familial hypercholesterolaemia (FH), an inherited predisposition to early-onset heart disease. It examines the impact of disease risk assessments based on both genetic and non-genetic information, or solely non-genetic information. Results: The impact of diagnostic testing did not seem to vary according to whether or not genetic information was used. More generally, being given a positive or negative diagnosis of FH had minimal discernible impact on people's lives as they maintained the continuity of their beliefs and behaviour. Conclusions: The results suggest that concerns about the use of genetic testing in this context are unfounded, a conclusion that echoes findings from studies in this and other health contexts.

Description: Background: A recent, large genome-wide association study (GWAS) of European ancestry individuals has identified multiple genetic variants influencing serum lipids. Studies of the transferability of these associations to African Americans remain few, an important limitation given interethnic differences in serum lipids and the disproportionate burden of lipid-associated metabolic diseases among African Americans. Methods: We attempted to evaluate the transferability of 95 lipid-associated loci recently identified in European ancestry individuals to 887 non-diabetic, unrelated African Americans from a population-based sample in the Washington, DC area. Additionally, we took advantage of the generally reduced linkage disequilibrium among African ancestry populations in comparison to European ancestry populations to fine-map replicated GWAS signals. Results: We successfully replicated reported associations for 10 loci (CILP2/SF4, STARD3, LPL, CYP7A1, DOCK7/ANGPTL3, APOE, SORT1, IRS1, CETP, and UBASH3B). Through trans-ethnic fine-mapping, we were able to reduce associated regions around 75 % of the loci that replicated. Conclusions: Between this study and previous work in African Americans, 40 of the 95 loci reported in a large GWAS of European ancestry individuals also influence lipid levels in African Americans. While there is now evidence that the lipid-influencing role of a number of genetic variants is observed in both European and African ancestry populations, the still considerable lack of concordance highlights the importance of continued ancestry-specific studies to elucidate the genetic underpinnings of these traits.

Description: Background: One of the genes suggested to play an important role in the pathophysiology of bipolar disorder (BPD) is PDLIM5, which encodes LIM domain protein. Our main objective was to examine the effect of olanzapine treatment on PDLIM5 mRNA expression in the peripheral blood leukocytes of BPD patients. Methods: We measured the expression of PDLIM5 mRNA from 16 patients with BPD Type I after 0, 4, and 8 weeks of treatment with olanzapine using quantitative real-time PCR. The Young Mania Rating Scale was used to evaluate the severity of manic symptoms in BPD patients. We also compared PDLIM5 mRNA expression in treatment-naive BPD patients with that in healthy control subjects. Results: No significant difference was found in PDLIM5 mRNA expression between patients before olanzapine treatment and following 4 and 8 weeks of treatment (p〉0.05). Although we observed a significant reduction in the severity of manic symptoms in all BPD patients (p 0.05). Interestingly, PDLIM5 mRNA expression differed significantly between treatment-naive BPD patients and healthy control subjects (p=0.002). Conclusion: PDLIM5 mRNA expression did not appear to be a reflection of the efficacy of olanzapine in reducing the manic symptoms of BPD. The significant difference in expression of PDLIM5 mRNA in the peripheral blood leukocytes of treatment-naive BPD patients versus that of healthy control subjects, however, suggests that it may be a good biological marker for BPD.

Description: Background: The majority of non-syndromic colorectal cancers (CRCs) can be described as a complex disease. A two-stage case--control study on CRC susceptibility was conducted to assess the influence of the ancestral alleles in the polymorphisms previously associated with nutrition-related complex diseases. Methods: In stage I, 28 single nucleotide polymorphisms (SNPs) were genotyped in a hospital-based Czech population (1025 CRC cases, 787 controls) using an allele-specific PCR-based genotyping system (KASPar(R)). In stage II, replication was carried out for the five SNPs with the lowest p values. The replication set consisted of 1798 CRC cases and 1810 controls from a population-based German study (DACHS). Odds ratios (ORs) and 95% confidence intervals (CIs) for associations between genotypes and CRC risk were estimated using logistic regression. To identify signatures of selection, Fay-Wu's H and Integrated Haplotype Score (iHS) were estimated. Results: In the Czech population, carriers of the ancestral alleles of AGT rs699 and CYP3A7 rs10211 showed an increased risk of CRC (OR 1.26 and 1.38, respectively; two-sided p

Description: Background: To explore the association of ALOX5AP single nucleotide polymorphisms (SNPs) andhaplotype with the occurrence of cerebral infarction in the Han population of northern China. Methods: Blood samples were collected from 236 patients of Han ancestry with a history of cerebralinfarction and 219 healthy subjects of Han ancestry with no history of cerebral infarction orcardiovascular disease. Applied Biosystems(R) TaqMan(R) SNP Genotyping Assays for SNPgenotyping were used to determine the genotypes of 7 ALOX5AP SNP alleles (rs4073259,rs4769874, rs9315050, rs9551963, rs10507391, rs9579646, and rs4147064). Results: One SNP allele (A) of rs4073259 was significantly associated with development of cerebralinfarction (P = 0.049). In comparison to control groups, haplotype rs9315050&rs9551963AAAC [OR (95 % CI) =1.53 (1.02-2.29)], and genotypes rs4147064 CT [OR (95 % CI)=1.872 (1.082-3.241)], and rs9551963 AC [OR (95 % CI) = 2.015 (1.165-3.484)] increasedthe risk of cerebral infarction in patients with hypertension. Genotype rs9579646 GG [OR (95% CI) = 2.926 (1.18-7.251)] increased the risk of, while rs4073259 GG [OR (95 %CI) = 0.381 (0.157-0.922)] decreased the risk of cerebral infarction in patients with diabetes. Conclusion: These results suggest the ALOX5AP SNP A allele in rs4073259 and genotype rs9579646 GG,rs9551963 AC, and haplotype rs9315050 & rs9551963 AAAC were associated with anincreased risk of ischemic stroke in the Han population, while rs4073259 GG was associatedwith a decreased risk.

Description: Background: The UMODL1 gene was found to be associated with high myopia in Japanese. This studyaimed to investigate this gene for association with high myopia in Chinese. Methods: Two groups of unrelated Han Chinese from Hong Kong were recruited using the samecriteria: Sample Set 1 comprising 356 controls (spherical equivalent, SE, within +/-1 diopter orD) and 356 cases (SE [less than or equal to] 8D), and Sample Set 2 comprising 394 controls and 526 cases.Fifty-nine tag single nucleotide polymorphisms (SNPs) were selected and genotyped forSample Set 1. Four SNPs were followed up with Sample Set 2. Both single-marker andhaplotype analyses were performed with cases defined by different SE thresholds. Secondaryphenotypes were also analyzed for association with genotypes. Results: Data filtering left 57 SNPs for analysis. Single-marker analysis did not reveal any significantdifferences between cases and controls in the initial study. However, haplotype GCT formarkers rs220168-rs220170-rs11911271 showed marginal significance (empirical P = 0.076;SE [less than or equal to] 12D for cases), but could not be replicated in the follow-up study. In contrast, nonsynonymousSNP rs3819142 was associated with high myopia (SE [less than or equal to] 10D) in the follow-upstudy, but could not be confirmed using Sample Set 1. The SNP rs2839471, positive in theoriginal Japanese study, gave negative results in all our analyses. Exploratory analysis ofsecondary phenotypes indicated that allele C of rs220120 was associated with anteriorchamber depth (adjusted P = 0.0460). Conclusions: Common UMODL1 polymorphisms were unlikely to be important in the geneticsusceptibility to high myopia in Han Chinese.

Description: Background: Neural tube defects (NTDs) are common birth defects (~1 in 1000 pregnancies in the US andEurope) that have complex origins, including environmental and genetic factors. A low levelof maternal folate is one well-established risk factor, with maternal periconceptional folicacid supplementation reducing the occurrence of NTD pregnancies by 50-70%. Gene variantsin the folate metabolic pathway (e.g., MTHFR rs1801133 (677 C 〉 T) and MTHFD1rs2236225 (R653Q)) have been found to increase NTD risk. We hypothesized that variants inadditional folate/B12 pathway genes contribute to NTD risk. Methods: A tagSNP approach was used to screen common variation in 82 candidate genes selectedfrom the folate/B12 pathway and NTD mouse models. We initially genotypedpolymorphisms in 320 Irish triads (NTD cases and their parents), including 301 cases and341 Irish controls to perform case-control and family based association tests. Significantlyassociated polymorphisms were genotyped in a secondary set of 250 families that included229 cases and 658 controls. The combined results for 1441 SNPs were used in a joint analysisto test for case and maternal effects. Results: Nearly 70 SNPs in 30 genes were found to be associated with NTDs at the p 〈 0.01 level. Theten strongest association signals (p-value range: 0.0003-0.0023) were found in nine genes(MFTC, CDKN2A, ADA, PEMT, CUBN, GART, DNMT3A, MTHFD1 and T (Brachyury)) andincluded the known NTD risk factor MTHFD1 R653Q (rs2236225). The single strongestsignal was observed in a new candidate, MFTC rs17803441 (OR = 1.61 [1.23-2.08],p = 0.0003 for the minor allele). Though nominally significant, these associations did notremain significant after correction for multiple hypothesis testing. Conclusions: To our knowledge, with respect to sample size and scope of evaluation of candidatepolymorphisms, this is the largest NTD genetic association study reported to date. The scaleof the study and the stringency of correction are likely to have contributed to real associationsfailing to survive correction. We have produced a ranked list of variants with the strongestassociation signals. Variants in the highest rank of associations are likely to include trueassociations and should be high priority candidates for further study of NTD risk.

Description: Background: The Toll-like receptor proteins are important in host defense and initiation of the innate andadaptive immune responses. A number of studies have identified associations betweengenetic variation in the Toll-like receptor genes and allergic disorders such as asthma andallergic rhinitis. The present study aim to search for genetic variation associated with allergicrhinitis in the Toll-like receptor genes. Methods: A first association analysis genotyped 73 SNPs in 182 cases and 378 controls from a Swedishpopulation. Based on these results an additional 24 SNPs were analyzed in one Swedishpopulation with 352 cases and 709 controls and one Chinese population with 948 cases and580 controls. Results: The first association analysis identified 4 allergic rhinitis-associated SNPs in the TLR7-TLR8gene region. Subsequent analysis of 24 SNPs from this region identified 7 and 5 significantSNPs from the Swedish and Chinese populations, respectively. The corresponding riskassociatedhaplotypes are significant after Bonferroni correction and are the most commonhaplotypes in both populations. The associations are primarily detected in females in theSwedish population, whereas it is seen in males in the Chinese population. Furtherindependent support for the involvement of this region in allergic rhinitis was obtained fromquantitative skin prick test data generated in both populations. Conclusions: Haplotypes in the TLR7-TLR8 gene region were associated with allergic rhinitis in oneSwedish and one Chinese population. Since this region has earlier been associated withasthma and allergic rhinitis in a Danish linkage study this speaks strongly in favour of thisregion being truly involved in the development of this disease.

Description: Background: Balkan Endemic Nephropathy (BEN) is late-onset kidney disease thought to arise from chronic exposure to aristolochic acid, a phytotoxin that contaminates wheat supplies in rural areas of Eastern Europe. It has recently been demonstrated that humans are capable of perceiving aristolochic acid at concentrations below 40 nM as the result of high-affinity interactions with the TAS2R43 bitter taste receptor. Further, TAS2R43 harbors high-frequency loss-of-function mutations resulting in 50-fold variability in perception. This suggests that genetic variation in TAS2R43 might affect susceptibility to BEN, with individuals carrying functional forms of the receptor being protected by an ability to detect tainted foods. Methods: To determine whether genetic variation in TAS2R43 predicts BEN susceptibility, we examined genotype-phenotype associations in a case--control study. A cohort of 88 affected and 99 control subjects from western Bulgaria were genotyped with respect to two key missense variants and a polymorphic whole-gene deletion of TAS2R43 (W35S, H212R, and wt/Delta), which are known to affect taste sensitivity to aristolochic acid. Tests for association between haplotypes and BEN status were then performed. Results: Three major TAS2R43 haplotypes observed in previous studies (TAS2R43-W35/H212, -S35/R212 and --Delta) were present at high frequencies (0.17, 0.36, and 0.47 respectively) in our sample, and a significant association between genotype and BEN status was present (P = 0.020; odds ratio 1.18). However, contrary to expectation, BEN was positively associated with TAS2R43-W35/H212, a highly responsive allele previously shown to confer elevated bitter sensitivity to aristolochic acid, which should drive aversion but might also affect absorption, altering toxin activation. Conclusions: Our findings are at strong odds with the prediction that carriers of functional alleles of TAS2R43 are protected from BEN by an ability to detect and avoid aristolochic acid exposure. Evidence for a positive association between high-sensitivity alleles and BEN status suggests instead that possession of toxin-responsive receptor variants may paradoxically increase vulnerability, possibly by shifting attractive responses associated with low-intensity bitter sensations. The broad-spectrum tuning of the ~25-member TAS2R family as a whole toward xenobiotics points to a potentially far-reaching relevance of bitter responses to exposure-related disease in both individuals and populations.

Description: Background: Tandem mass spectrometry (MS/MS) analysis is a powerful tool for newborn screening, and many rare inborn errors of metabolism are currently screened using MS/MS. However, the sensitivity of MS/MS screening for several inborn errors, including citrin deficiency (screened by citrulline level) and a carnitine uptake defect (CUD, screened by free carnitine level), is not satisfactory. This study was conducted to determine whether a second-tier molecular test could improve the sensitivity of citrin deficiency and CUD detection without increasing the false-positive rate. Methods: Three mutations in the SLC25A13 gene (for citrin deficiency) and one mutation in the SLC22A5 gene (for CUD) were analyzed in newborns who demonstrated an inconclusive primary screening result (with levels between the screening and diagnostic cutoffs). Results: The results revealed that 314 of 46 699 newborns received a second-tier test for citrin deficiency, and two patients were identified; 206 of 30 237 newborns received a second-tier testing for CUD, and one patient was identified. No patients were identified using the diagnostic cutoffs. Although the incidences for citrin deficiency (1:23 350) and CUD (1:30 000) detected by screening are still lower than the incidences calculated from the mutation carrier rates, the second-tier molecular test increases the sensitivity of newborn screening for citrin deficiency and CUD without increasing the false-positive rate. Conclusions: Utilizing a molecular second-tier test for citrin deficiency and carnitine transporter deficiency is feasible.

Description: IntroductionDiabetes mellitus type 2 (DM2) is highly associated with increased risk for chronic kidney disease (CKD), end stage renal disease (ESRD) and cardiovascular morbidity. Epidemiological and genetic studies generate hypotheses for innovative strategies in DM2 management by unravelling novel mechanisms of diabetes complications, which is essential for future intervention trials. We have thus initiated the DIAbetes COhoRtE study (DIACORE). Methods: DIACORE is a prospective cohort study aiming to recruit 6000 patients of self-reported Caucasian ethnicity with prevalent DM2 for at least 10 years of follow-up. Study visits are performed in University-based recruiting clinics in Germany using standard operating procedures. All prevalent DM2 patients in outpatient clinics surrounding the recruiting centers are invited to participate. At baseline and at each 2-year follow-up examination, patients are subjected to a core phenotyping protocol. This includes a standardized online questionnaire and physical examination to determine incident micro- and macrovascular DM2 complications, malignancy and hospitalization, with a primary focus on renal events. Confirmatory outcome information is requested from patient records. Blood samples are obtained for a centrally analyzed standard laboratory panel and for biobanking of aliquots of serum, plasma, urine, mRNA and DNA for future scientific use. A subset of the cohort is subjected to extended phenotyping, e.g. sleep apnea screening, skin autofluorescence measurement, non-mydriatic retinal photography and non-invasive determination of arterial stiffness. Results: By September 2012, 2050 patients have been recruited. Conclusion: DIACORE will enable the prospective evaluation of factors involved in DM2 complication pathogenesis using high-throughput technologies in biosamples and genetic epidemiological studies.

Description: Background: Serum 25-hydroxyvitamin D3 (Vitamin D) insufficiency and single-nucleotide polymorphisms (SNPs) on its receptor, Vitamin D receptor (VDR), have been reported to be involved in melanoma susceptibility in populations mostly from northern countries.ObjectiveTo investigate 25-hydroxyvitamin D3 levels and VDR SNPs in melanoma patients from sunny area of Barcelona, two studies were carried out. The first study evaluated the levels of Vitamin D at time of melanoma diagnosis and the second one analyzed the association between VDR genetic variants and risk of having a high nevus number, the strongest phenotypic risk factor for melanoma. Methods: The levels of 25-hydroxyvitamin D3 in 81 melanoma patients at diagnosis were measured. In a second group of melanoma patients, including 150 with low and 113 with high nevus number, 11 VDR SNPs were analyzed for their association with nevus number. Results: In the first study, 68% of patients had insufficient levels of 25-hydroxyvitamin D3 (

Description: Background: Atherosclerosis is the primary cause of coronary heart disease (CHD), preceding the onset of cardiovascular disease by decades in most cases. Here we examine the association between single nucleotide polymorphisms (SNPs) integrated on Metabochip and coronary artery calcification (CAC), a valid risk factor for CHD, in an unselected, population-based German cohort. Methods: The Metabochip is a custom iSELECT array containing 〉195,000 SNPs that was designed to support large-scale follow-up of putative associations for metabolic and cardiovascular-associated traits. We used generalized linear regression models to explore the impact of Metabochip SNPs on quantitative CAC in 4,329 participants. Results: The 9p21 variant, rs1537373, was most strongly associated (Beta = 0.30; 95% confidence interval (CI) = 0.21-0.39; p = 4.05x10-11) with quantitative CAC. The second strongest association with CAC was with rs9349379 in the phosphatase and actin regulator 1 gene, PHACTR1, (Beta = 0.30; 95%CI = 0.22-0.40; p = 4.67x10-11). Both SNPs remained nominally significant in dichotomized analyses for the presence of any CAC (odds ratiors1537373 (OR) = 1.19; 95%CI = 1.07-1.31; p = 0.001 and ORrs9349379 = 1.26; 95%CI = 1.14-1.40); p = 1.5x10-5). Fine mapping of the 9p21 and PHACTR1 gene region revealed several other SNPs that were strongly associated with CAC. Conclusion: We demonstrate that SNPs near 9p21 and in PHACTR1 that have previously been shown to be associated with CHD are strongly associated with CAC in the Heinz Nixdorf Recall Study cohort. Our findings suggest that the 9p21 and 6q24 loci might be involved in cardiac outcome via promoting development of atherosclerosis in the coronary arteries.

Description: Background: The incidence of Alzheimer's disease, particularly in developing countries, is expected to increase exponentially as the population ages. Continuing research in this area is essential in order to better understand this disease and develop strategies for treatment and prevention. Genome-wide association studies have identified several loci as genetic risk factors of AD aside from apolipoprotein E such as bridging integrator (BIN1), clusterin (CLU), ATP-binding cassette sub-family A member 7 (ABCA7), complement receptor 1 (CR1) and phosphatidylinositol binding clathrin assembly protein (PICALM). However genetic research in developing countries is often limited by lack of funding and expertise. This study therefore developed and validated a simple, cost effective polymerase chain reaction based technique to determine these single nucleotide polymorphisms. Methods: An allele-specific PCR method was developed to detect single nucleotide polymorphisms of BIN1 rs744373, CLU rs11136000, ABCA7 rs3764650, CR1 rs3818361 and PICALM rs3851179 in human DNA samples. Allele-specific primers were designed by using appropriate software to permit the PCR amplification only if the nucleotide at the 3'-end of the primer complemented the base at the wild-type or variant-type DNA sample. The primers were then searched for uniqueness using the Basic Local Alignment Search Tool search engine. Results: The assay was tested on a hundred samples and accurately detected the homozygous wild-type, homozygous variant-type and heterozygous of each SNP. Validation was by direct DNA sequencing. Conclusion: This method will enable researchers to carry out genetic polymorphism studies for genetic risk factors associated with late-onset Alzheimer's disease (BIN1, CLU, ABCA7, CR1 and PICALM) without the use of expensive instrumentation and reagents.

Description: Background: The proximal chromosome 15q is prone to unequal crossover, leading to rearrangements. Although 15q11q13 duplications are common in patients with developmental delays and mental impairment, 15q aneusomies resulting in greater or equal to 4 copies of 15q11q13 are rare and no pentasomy 15q11q13 has been reported in the literature. Thus far, all reported high copy number 15q11q13 cases are from the West populations and no such study in Chinese patients have been documented. Dosage-response pattern of high copy number 15q11q13 on clinical presentations is still a subject for further study.Case presentationIn this study, we characterized two Han Chinese patients with high copy number 15q11q13. Using chromosome banding, high resolution SNP-based cytogenomic array, fluorescence in situ hybridization, and PCR-based microsatellite analysis, we identified two patients with tetrasomy 15q11q13 and pentasomy 15q11q13. Both 15q11q13 aneusomies resulted from a maternally inherited supernumerary marker chromosome 15, and each was composed of two different sized 15q11q13 segments covering the Prader-Willi/Angelman critical region: one being about 10 Mb with breakpoints at BP1 and BP5 regions on 15q11 and 15q13, respectively, and another about 8 Mb in size with breakpoints at BP1 and BP4 regions on 15q. Both patients presented with similar clinical features that included neurodevelopmental delays, mental impairment, speech and autistic behavior, and mild dysmorphism. The patient with pentasomy 15q11q13 was more severely affected than the patient with tetrasomy 15q11q13. Low birth weight was noted in patient with pentasomy 15q11q13. Conclusions: To the best of our knowledge, this is the first case of pentasomy 15q11q13 and the first study of high copy number 15q11q13 in Han Chinese patients. Our findings demonstrate that patients with tetrasomy and pentasomy of chromosome 15q11q13 share similar spectrum of phenotypes reported in other high copy number 15q11q13 patients in the West, and positive correlation between 15q11q13 copy number and degree of severity of clinical phenotypes. Low birth weight observed in the pentasomy 15q11q13 patient was not reported in other patients with high copy number 15q11q13. Additional studies would be necessary to further characterize high copy number 15q11q13 aneusomies.

Description: Background: A previous meta-analysis reported a positive association between an insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme gene (ACE) and the risk of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Here, we updated this meta-analysis and additionally assessed the association of this polymorphism with ALI/ARDS mortality. Methods: We searched electronic databases through October 2011 for the terms "angiotensin-converting enzyme gene", "acute lung injury", and "acute respiratory distress syndrome," and reviewed all studies that reported the relationship of the I/D polymorphism in ACE with ALI/ARDS in humans. Seven studies met the inclusion criteria, comprising 532 ALI/ARDS patients, 3032 healthy controls, and 1432 patients without ALI/ARDS. We used three genetic models: the allele, dominant, and recessive models. Results: The ACE I/D polymorphism was not associated with susceptibility to ALI/ARDS for any genetic model. However, the ACE I/D polymorphism was associated with the mortality risk of ALI/ARDS in Asian subjects (Pallele 〈 0.0001, Pdominant = 0.001, P recessive = 0.002). This finding remained significant after correction for multiple comparisons. Conclusions: There is a possible association between the ACE I/D polymorphism genotype and the mortality risk of ALI/ARDS in Asians.Akihisa Matsuda and Taro Kishi These authors participated equally in this work.

Description: Background: 5,10-methylenetetrahydrofolate reductase (MTHFR) variants, C677T and A1298C, have been reported to be associated with decreased risk of acute lymphoblastic leukemia (ALL). However, results derived from individually underpowered studies are conflicting. We carried out an updated meta-analysis on the association between MTHFR polymorphisms and ALL risk. Methods: Relevant publications were searched through PUBMED and EMBASE databases. The associations between MTHFR C677T and A1298C polymorphisms and the risk of ALL were evaluated by odds ratios (ORs). The heterogeneity and publication bias were estimated. Meta-regression analysis was performed to evaluate the potential sources of heterogeneity. Results: C677T polymorphism was associated with a reduced risk of ALL (allele contrast: ORRE = 0.91, 95% CI: 0.83-0.99). Subgroup analysis showed MTHFR C677T variant was associated with decreased susceptibility to ALL in children and Caucasians. Meta-regression showed the logOR for the association between T allele and ALL increased as sex ratio (M/F) in the case group increased (P = 0.01). Regarding A1298C polymorphism, no significant association was observed (allele contrast: ORRE = 1.01, 95% CI: 0.91-1.11). There was no publication bias for C677T or A1298C polymorphism. Conclusions: The present meta-analysis suggests that the C677T polymorphism, not A1298C, in MTHFR gene is associated with a decreased risk of ALL, particularly among children and Caucasians subjects. Our findings suggest that the influence of the C677T polymorphism on ALL susceptibility is modified by sex ratio in cases (M/F). Since folate intake may be a possible confounding factor, including this factor in future prospective studies is warranted. Further meta-analysis studies should be at least stratified for folate levels and gender to give more powerful and informative results.

Description: Background: In recent genetic association studies, common variants including rs12917707 in the UMOD locus have shown strong evidence of association with eGFR, prevalent and incident chronic kidney disease and uromodulin urinary concentration in general population cohorts. The association of rs12917707 with end-stage renal disease (ESRD) in a recent case-control study was only nominally significant. Methods: To investigate whether rs12917707 associates with ESRD, graft failure (GF) and urinary uromodulin levels in an independent cohort, we genotyped 1142 ESRD patients receiving a renal transplantation and 1184 kidney donors as controls. After transplantation, 1066 renal transplant recipients were followed up for GF. Urinary uromodulin concentration was measured at median [IQR] 4.2 [2.2-6.1] yrs after kidney transplantation. Results: The rs12917707 minor allele showed association with lower risk of ESRD (OR 0.89 [0.76-1.03], p = 0.04) consistent in effect size and direction with the previous report (Boger et al, PLoS Genet 2011). Meta-analysis of these findings showed significant association of rs12917707 with ESRD (OR 0.91 [0.85-98], p = 0.008). In contrast, rs12917707 was not associated with incidence of GF. Urinary uromodulin concentration was lower in recipient-carriers of the donor rs12917707 minor allele as compared to non-carriers, again consistent with previous observations in general population cohorts. Conclusions: Our study thus corroborates earlier evidence and independently confirms the association between UMOD and ESRD.

Description: Background: Autistic spectrum disorders (ASDs) are a family of neurodevelopmental disorders with strong genetic components. Recent studies have shown that copy number variations in dosage sensitive genes can contribute significantly to these disorders. One such gene is the transcription factor MECP2, whose loss of function in females results in Rett syndrome, while its duplication in males results in developmental delay and autism.Case presentationHere, we identified a Chinese family with two brothers both inheriting a 2.2 Mb MECP2-containing duplication (151,369,305 -- 153,589,577) from their mother. In addition, both brothers also had a 213.7 kb duplication on Chromosome 2, inherited from their father. The older brother also carried a 48.4 kb duplication on Chromosome 2 inherited from the mother, and a 8.2 kb deletion at 11q13.5 inherited from the father. Based on the published literature, MECP2 is the most autism-associated gene among the identified CNVs. Consistently, the boys displayed clinical features in common with other patients carrying MECP2 duplications, including intellectual disability, autism, lack of speech, slight hypotonia and unsteadiness of movement. They also had slight dysmorphic features including a depressed nose bridge, large ears and midface hypoplasia. Interestingly, they did not exhibit other clinical features commonly observed in American-European patients with MECP2 duplication, including recurrent respiratory infections and epilepsy. Conclusions: To our knowledge, this is the first identification and characterization of Chinese Han patients with MECP2-containing duplications. Further cases are required to determine if the above described clinical differences are due to individual variations or related to the genetic background of the patients.

Description: Background: Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer syndrome caused bya loss of function of the APC gene. Large deletions in APC are a common cause of FAP;despite the existence of a variety of gene dosage detection methodologies, most are laborintensive and time and resource consuming. Methods: We describe a new duplex qPCR method for gene dosage analysis based on thecoamplification of a target and a reference gene in a SYBR Green reaction, followed by acomparison of the ratio between the target and the reference peaks of the melting curve forthe test (patient) and control samples. The reliability of the described duplex qPCR wasvalidated for several genes (APC, HPRT1, ATM, PTEN and BRCA1). Results: Using this novel gene dosage method, we have identified an APC gene deletion in a FAPpatient undergoing genetic testing. Comparative genomic hybridization based on microarrays(aCGH) was used to confirm and map the extent of the deletion, revealing a 5.2 MBrearrangement (5q21.3-q22.3) encompassing the entire APC and 19 additional genes. Conclusion: The novel assay accurately detected losses and gains of one copy of the target sequences,representing a reliable and flexible alternative to other gene dosage techniques. In addition,we described a FAP patient harboring a gross deletion at 5q21.3-q22.3 with an unusualphenotype of the absence of mental impairment and dysmorphic features.

Description: Background: von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome in which the patientsdevelop retinal and central nervous system hemangioblastomas, pheochromocytomas andclear-cell renal tumors. The autosomal dominant disease is caused by mutations in the VHLgene. Methods: VHL mutational analysis was carried out by sequencing of the coding sequence and bymultiplex ligation-dependent probe amplification analysis. The functional consequence of thevariants was investigated using in silico prediction tools. Results: A total of 289 probands suspected of having VHL syndrome have been screened formutations in the VHL gene. Twenty-six different VHL mutations were identified in 36families including one in-frame duplication, two frame-shift mutations, four nonsensemutations, twelve missense mutations, three intronic mutations and four large genomic rearrangements. Three of these mutations (c.319 C 〉 T, c.342_343dupGGT andc.520_521dupAA) were novel. Conclusions: In this study we report the VHL germ-line mutations found in Danish families. We foundthree novel VHL mutations where two were classified as pathogenic and the latter wasclassified as a variant of unknown significance. Together, our findings contribute to theinterpretation of the potential pathogenicity of VHL germ-line mutations.

Description: Background: Germline mutations of BRCA1/2 are associated with hereditary breast and ovarian cancer.Recent data suggests excess mortality in mutation carriers beyond that conferred byneoplasia, and recent in vivo and in vitro studies suggest a modulatory role for BRCAproteins in endothelial and cardiomyocyte function. We therefore tested the association ofBRCA2 variants with clinical cardiovascular disease (CVD). Methods: Using data from 1,170 individuals included in two multi-ethnic population-based studies(SHARE and SHARE-AP), the association between BRCA2 variants and CVD wasevaluated. 15 SNPs in BRCA2 with minor allele frequencies (MAF) 〉 0.01 had beenpreviously genotyped using the cardiovascular gene-centric 50 k SNP array. 115 individuals(9.8%) reported a CVD event, defined as myocardial infarction (MI), angina, silent MI,stroke, and angioplasty or coronary artery bypass surgery. Analyses were adjusted for ageand sex. The SNPs rs11571836 and rs1799943 were subsequently genotyped using theMassARRAY platform in 1,045 cases of incident MI and 1,135 controls from the SouthAsian subset of an international case-control study of acute MI (INTERHEART), andrs11571836 was imputed in 4,686 cases and 4500 controls from the Pakistan Risk ofMyocardial Infarction Study (PROMIS). Results: Two BRCA2 SNPs, rs11571836 and rs1799943, both located in untranslated regions, wereassociated with lower risk of CVD (OR 0.47 p = 0.01 and OR 0.56 p = 0.03 respectively) inthe SHARE studies. Analysis by specific ethnicities demonstrated an association with CVDfor both SNPs in Aboriginal People, and for rs11571836 only in South Asians. No associationwas observed in the European and Chinese subgroups. A non-significant trend towards anassociation between rs11571836 and lower risk of MI was observed in South Asians fromINTERHEART [OR = 0.87 (95% CI: 0.75-1.01) p = 0.068], but was not evident in PROMIS[OR = 0.96 (95% CI: 0.90-1.03) p = 0.230]. Meta-analysis of both case-control studiesresulted in a combined OR of 0.94 (95% CI: 0.89-1.004, p = 0.06). Conclusions: Although there was an association between two SNPs in BRCA2 and CVD in a multi-ethnicpopulation, these results were not replicated in two South Asian case-control studies ofincident MI. Future studies exploring the association between BRCA variants andcardiovascular disorders are needed to clarify the role, if any, for BRCA variants in CVDpathogenesis.

Description: Background: CpG island aberrant methylation is shown to be an important mechanism in gene silencing.The important role of IL-17 in inflammatory response to H. pylori colonization has beenindicated. We investigated the influence of IL17A polymorphisms, -197 G 〉 A (rs2275913)and *1249 C 〉 T (rs3748067), on the methylation of DAPK and CDH1. Methods: Gastric mucosal samples were obtained from 401 subjects without malignancies. Methylationstatus of gene was determined by MSP. The genotyping of IL17A was performed by PCRSSCP. Results: Methylations of DAPK and CDH1 were seen in 196 and 149 of all 401 subjects, respectively.Overall, *1249 T carrier was associated with a decreased risk for DAPK methylation,whereas -197 G 〉 A was not. In the subjects older than 60 years old, *1249 T carrier wasmore strongly associated with gene methylation and -197 A carrier tended to be associatedwith an increased risk for CDH1 methylation. When evaluating by inflammation promotinghaplotype (-197 mutant carrier with *1249 homozygote), this haplotype had a more stronglyincreased risk for both DAPK and CDH1 methylations in comparatively older subjects. Bothatrophy and metaplasia scores were significantly increased with age in -197 A carrier or*1249 cm3 homozygote, whereas were not in -197 GG homozygote or *1249 T carrier. PGI/II ratio was more significantly decreased in -197 A carrier than in GG homozygote underinfluence of H. pylori infection. Conclusions: In -197 A allele carrier with *1249 CC homozygote, the methylations of both DAPK andCDH1 may be increased gradually, but more rapidly than the other genotypes, with age andaltered gastric mucosal structure induced by H. pylori infection.

Description: Background: Renalase (gene name RNLS), a recently discovered enzyme with monoamine oxidase activity, is implicated in the degradation of catecholamines. Recent studies delineate a possible role of this enzyme in blood pressure (BP) maintenance and cardiac protection and two single nucleotide polymorphisms, RNLS rs2576178 A〉G and rs2296545 C〉G have been associated with hypertension. The latter SNP leads to a non synonymous Asp to Glu substitution deleting a flavin adenine dinucleotide (FAD) binding site with possible impaired functionality. We tested the hypothesis that these polymorphisms could affect BP levels, hypertension prevalence, and risk of incident cardiovascular events in middle-aged Swedes. Methods: The polymorphisms were genotyped in 5696 participants of the population-based Cardiovascular Cohort of the "Malmo Diet and Cancer" (MDC-CC). The incidence of cardiovascular events (coronary events [n=408], strokes [n=330], heart failure [n=190] and atrial fibrillation/flutter [n=406]) was monitored for an average of approximately 15 years of follow-up. Results: Both before and after adjustment for sex, age and BMI the polymorphisms did not show any effect on BP level and hypertension prevalence. Before and after adjustment for major cardiovascular risk factors, the hazard ratio for cardiac and cerebrovascular events was not significantly different in carriers of different genotypes. A significant interaction was found between the rs2296545 C〉G and age with respect to BP/hypertension. Conclusions: Our data do not support a major role for these RNLS polymorphisms in determining BP level and incident events at population level. The positive interaction with age suggest that the effect of the rs2296545 C〉G polymorphism, if any, could vary between different ages.

Description: Background: Chronic kidney disease progression has been linked to pro-inflammatory cytokines andmarkers of inflammation. These markers are also elevated in end-stage renal disease (ESRD),which constitutes a serious public health problem.ObjectiveTo investigate whether single nucleotide polymorphisms (SNPs) located in genes related toimmune and inflammatory processes, could be associated with ESRD development.Design and methodsA retrospective case-control study was carried out on 276 patients with ESRD and 288control subjects. Forty-eight SNPs were genotyped via SNPlex platform. Logistic regressionwas used to assess the relationship between each single polymorphism and the developmentof ESRD. Results: Four polymorphisms showed association with ESRD: rs1801275 in the interleukin 4 receptor(IL4R) gene (OR: 0.66 (95%CI = 0.46-0.95); p = 0.025; overdominant model), rs4586 inchemokine (C-C motif) ligand 2 (CCL2) gene (OR: 0.70 (95%CI = 0.54-0.90); p = 0.005;additive model), rs301640 located in an intergenic binding site for signal transducer andactivator of transcription 4 (STAT4) (OR: 1.82 (95%CI = 1.17-2.83); p = 0.006; additivemodel) and rs7830 in the nitric oxide synthase 3 (NOS3) gene (OR: 1.31 (95%CI = 1.01-1.71); p = 0.043; additive model). After adjusting for multiple testing, results lostsignificance. Conclusion: Our preliminary data suggest that four genetic polymorphisms located in genes related toinflammation and immune processes could help to predict the risk of developing ESRD.

Description: Background: Left ventricular hypertrabeculation/noncompaction (LVHT) is a cardiac abnormality ofunknown etiology which has been described in children as well as in adults with and withoutchromosomal aberrations. LVHT has been reported in association with various cardiac andextracardiac abnormalities like epilepsy and facial dysmorphism.Case presentationA unique combination of LVHT, atrial septal defect, pulmonary valve stenosis, aorticstenosis, epilepsy and minor facial anomalies is presented in a 5.5 years old girl. Microarraybasedgenomic hybridization (array-CGH) detected six previously not described copy numbervariants (CNVs) inherited from a clinically unaffected father and minimally affected mother,thus, most likely, not clinically significant but rare benign variants. Conclusions: Despite this complex phenotype de novo microdeletions or microduplications were notdetected by array CGH. Further investigations, such as whole exome sequencing, couldreveal point mutations and small indels as the possible cause.

Description: Background: Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine, implicated in the development and progression of allergic diseases. Recent studies have demonstrated significantly increased expression and synthesis of TSLPin nasal mucosa of patients with allergic rhinitis (AR), compared with nonallergic control subjects. Also, there is significant correlation between the level of TSLP mRNA and symptom severity in AR patients. In this study, we investigated whether polymorphisms in the TSLP gene were associated with increased risk of AR in the Chinese population. Methods: In a candidate gene association study, we tested 11 single nucleotide polymorphisms (SNPs) in the TSLP gene in 368 AR and 325 control adult Han Chinese subjects from Beijing. The 11 SNPs were selected from the Chinese HapMap genotyping dataset to ensure complete genetic coverage. AR was established by questionnaire and clinical examination, and blood was drawn from all subjects for DNA extraction. The PLINK software package was used to perform statistical testing. Results: In the single-locus analysis of AR risk, no significant differences in allele and genotype frequencies were found between AR and control subjects. Further logistic regression analyses adjusted for age and gender also failed to reveal significant associations between AR and the selected SNPs. Similarly, analysis stratified by gender, and haplotype or diplotype did not reveal any association with AR risk. Conclusion: Although TSLP presents itself as a good candidate for contributing to allergy, this study failed to find an association between specific SNPs in the TSLP gene and AR susceptibility in the Han Chinese population.

Description: Background: In this study we aimed to quantify tumor suppressor gene (TSG) promoter methylationdensities levels in primary neuroblastoma tumors and cell lines. A subset of these TSGs isassociated with a CpG island methylator phenotype (CIMP) in other tumor types. Methods: The study panel consisted of 38 primary tumors, 7 established cell lines and 4 healthyreferences. Promoter methylation was determined by bisulphate Pyrosequencing for 14TSGs; and LINE-1 repeat element methylation was used as an indicator of global methylationlevels. Results: Overall mean TSG Z-scores were significantly increased in cases with adverse outcome, butwere unrelated to global LINE-1 methylation. CIMP with hypermethylation of three or moregene promoters was observed in 6/38 tumors and 7/7 cell lines. Hypermethylation of one ormore TSG (comprising TSGs BLU, CASP8, DCR2, CDH1, RASSF1A and RASSF2) wasevident in 30/38 tumors. By contrast only very low levels of promoter methylation wererecorded for APC, DAPK1, NORE1A, P14, P16, TP73, PTEN and RARB. Similarinvolvements of methylation instability were revealed between cell line models andneuroblastoma tumors. Separate analysis of two proposed CASP8 regulatory regions revealedfrequent and significant involvement of CpG sites between exon 4 and 5, but modestinvolvement of the exon 1 region.Conclusions/significanceThe results highlight the involvement of TSG methylation instability in neuroblastomatumors and cell lines using quantitative methods, support the use of DNA methylationanalyses as a prognostic tool for this tumor type, and underscore the relevance of developingdemethylating therapies for its treatment.

Description: Background: Concomitant primary cutaneous melanoma in monozygotic twins has been reported in only two pairs but in neither of them genetic analysis was performed. Two high-penetrance susceptibility genes, CDKN2A and CDK4 and one low-penetrance gene, MC1R, are well-defined genetic risk factors for melanoma. MITF has been recently identified as a novel intermediate risk melanoma-predisposing gene.Case presentationWe describe the extraordinary occurrence of a primary cutaneous invasive melanoma in two 44-year-old identical, female twins, on the same body site within 30 days of each other and report for the first time the genetic analysis of melanoma susceptibility genes in both twins. Data on characteristics of the twins were collected through a standardized questionnaire and skin examination. Exons 1alpha, 1beta, 2 and 3 of CDKN2A, exon 2 of CDK4, the entire open reading frame of MC1R and the recently described MITF c.952 G 〉 A (p.Glu318Lys) variant were investigated by direct sequencing. Sequencing analysis of the high-penetrance susceptibility genes showed no changes in CDKN2A and in exon 2 of the CDK4 gene. Both patients were heterozygous for the same CDKN2A UTR c.*29C 〉 G variant. Interestingly, the same two heterozygous variants of the MC1R were identified in both twins: the c.451C 〉 T (p.Arg151Cys) and the c.456C 〉 A (p.Tyr152*) variants. Neither patient showed the c.952 G 〉 A (p.Glu318Lys) substitution in the MITF gene. Conclusions: Identification of two high-risk MC1R variants in our identical twins in the absence of CDKN2A and CDK4 mutations highlights the contribution of low penetrance genes, such as MC1R, in melanoma susceptibility.

Description: Background: Hepatic steatosis in HCV patients has been postulated as a risk factor associated with a higher frequency of fibrosis and cirrhosis. A single genetic variant, PNPLA3 I148M, has been widely associated with increased hepatic steatosis. Previous studies of the PNPLA3 I148M sequence variant in HCV infected individuals have reported an association between this variant and prevalence of steatosis, fibrosis, and cirrhosis. To evaluate the impact of PNPLA3 I148M variant on metabolic traits and treatment response in HCV genotype 2 and 3 infected patients. Methods: Three hundred and eighty-two treatment naive HCV genotype 2 or 3 infected patients were included in a phase III, open label, randomized, multicenter, investigator-initiated trial (the NORDynamIC study), in which pretreatment liver biopsies were mandatory. PNPLA3I148M genotyping was performed in a total of 359 Caucasian patients. Results: In HCV genotype 2 infected patients carrying the PNPLA3 148 M allele, there was significantly increased insulin resistance (P = 0.023) and lower viral load (P = 0.005) at baseline as well as the first seven days of antiviral treatment. These results were not observed in HCV genotype 3 infected patients. Conclusions: Our results suggest a possible association between the PNPLA3 148 M allele and insulin resistance as well as baseline viral load in HCV genotype 2, but not in genotype 3.

Description: Background: Dysequilibrium syndrome is a genetically heterogeneous condition that combines autosomal recessive, nonprogressive cerebellar ataxia with mental retardation. The condition has been classified into cerebellar ataxia, mental retardation and disequilibrium syndrome types 1 (CAMRQ1), 2 (CAMRQ2) and 3 (CAMRQ3) and attributed to mutations in VLDLR, CA8 and WDR81 genes, respectively. Quadrupedal locomotion in this syndrome has been reported in association with mutations in all three genes. Methods: SNP mapping and candidate gene sequencing in one consanguineous Omani family from the United Arab Emirates with cerebellar hypoplasia, moderate mental retardation, delayed ambulation and truncal ataxia was used to identify the mutation. In a second unrelated consanguineous Omani family, massively parallel exonic sequencing was used. Results: We identified a homozygous missense mutation (c.2117 G 〉 T, p.C706F) in the VLDLR gene in both families on a shared affected haplotype block.This is the first reported homozygous missense mutation in VLDLR and it occurs in a highly conserved residue and predicted to be damaging to protein function. Conclusions: We have delineated the phenotype associated with dysequilibrium syndrome in two Omani families and identified the first homozygous missense pathogenic mutation in VLDLR gene with likely founder effect in the southwestern part of the Arabian Peninsula.

Description: Background: Diagnostic analysis of patients with developmental disorders has improved over recent years largely due to the use of microarray technology. Array methods that facilitate copy number analysis have enabled the diagnosis of up to 20 % more patients with previously normal karyotyping results. A substantial number of patients remain undiagnosed, however.Methods and ResultsUsing the Genome-Wide Human SNP array 6.0, we analyzed 35 patients with a developmental disorder of unknown cause and normal array comparative genomic hybridization (array CGH) results, in order to characterize previously undefined genomic aberrations. We detected no seemingly pathogenic copy number aberrations. Most of the vast amount of data produced by the array was polymorphic and non-informative. Filtering of this data, based on copy number variant (CNV) population frequencies as well as phenotypically relevant genes, enabled pinpointing regions of allelic homozygosity that included candidate genes correlating to the phenotypic features in four patients, but results could not be confirmed. Conclusions: In this study, the use of an ultra high-resolution SNP array did not contribute to further diagnose patients with developmental disorders of unknown cause. The statistical power of these results is limited by the small size of the patient cohort, and interpretation of these negative results can only be applied to the patients studied here. We present the results of our study and the recurrence of clustered allelic homozygosity present in this material, as detected by the SNP 6.0 array.

Description: Background: Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial. Methods: Eighteen females showing a mosaic pattern of dystrophin expression on muscle biopsy were recruited and classified as symptomatic (7) or asymptomatic (11), based on the presence or absence of muscle weakness. The causative DMD gene mutations were identified in all cases, and the X-inactivation pattern was assessed in muscle DNA. Transcriptional analysis in muscles was performed in all females, and relative quantification of wild-type and mutated transcripts was also performed in 9 carriers. Dystrophin protein was quantified by immunoblotting in 2 females. Results: The study highlighted a lack of relationship between dystrophic phenotype and X-inactivation pattern in females; skewed X-inactivation was found in 2 out of 6 symptomatic carriers and in 5 out of 11 asymptomatic carriers. All females were characterized by biallelic transcription, but no association was found between X-inactivation pattern and allele transcriptional balancing. Either a prevalence of wild-type transcript or equal proportions of wild-type and mutated RNAs was observed in both symptomatic and asymptomatic females. Moreover, very similar levels of total and wild-type transcripts were identified in the two groups of carriers. Conclusions: This is the first study deeply exploring the DMD transcriptional behaviour in a cohort of female carriers. Notably, no relationship between X-inactivation pattern and transcriptional behaviour of DMD gene was observed, suggesting that the two mechanisms are regulated independently. Moreover, neither the total DMD transcript level, nor the relative proportion of the wild-type transcript do correlate with the symptomatic phenotype.

Description: Background: Plasma level of total homocysteine (tHcy) is negatively correlated with kidney function ingeneral population. However, the causal mechanism of this correlation is poorly understood.The purpose of this study is to investigate the association of methylenetetrahydrofolatereductase (MTHFR) C677T gene polymorphism, which is a major genetic determinant of theplasma tHcy level, with estimated glomerular filtration rate (eGFR) in Chinese. Methods: A total of 18 814 hypertensive patients (6 914 males, 11 900 females) were included in thestudy. Results: Association between the eGFR and MTHFR C677T genotype was examined by sex-specificregression analyses. In males, TT genotype was associated with 1.37 ml/min/1.73 m2decrease in eGFR (p = 0.004) and with an increased risk (OR = 1.32, p = 0.008) for the lowestquintile of eGFR after adjusting for age, BMI, and blood pressures. However, suchassociation was not observed in females (p 〉 0.05). This association suggests MTHFR C677Tpolymorphism may play a role in the regulation of eGFR in males. Conclusions: MTHFR 677 T is a risk allele for decreased kidney function in Chinese males, implicatingthis gene in the pathogenesis of chronic kidney disease (CKD).

Description: Background: Natriuretic peptides (NPs) are peptide hormones that exert their biological actions by bindingto three types of cell surface natriuretic peptide receptors (NPRs). The receptor NPR-Bbinding C-type natriuretic peptide (CNP) acts locally as a paracrine and/or autocrine regulatorin a wide variety of tissues. Mutations in the gene NPR2 have been shown to causeacromesomelic dysplasia-type Maroteaux (AMDM), an autosomal recessive skeletaldisproportionate dwarfism disorder in humans. Methods: In the study, presented here, genotyping of six consanguineous families of Pakistani originwith AMDM was carried out using polymorphic microsatellite markers, which are closelylinked to the gene NPR2 on chromosome 9p21-p12. To screen for mutations in the geneNPR2, all of its coding exons and splice junction sites were PCR amplified from genomicDNA of affected and unaffected individuals of the families and sequenced. Results: Sequence analysis of the gene NPR2 identified a novel missence mutation (p.T907M) in fivefamilies, and a splice donor site mutation c.2986 + 2 T 〉 G in the other family. Conclusion: We have described two novel mutations in the gene NPR2. The presence of the samemutation (p.T907M) and haplotype in five families (A, B, C, D, E) is suggestive of a foundereffect.

Description: Background: The enzymes of the cytochrome P450 family (CYPs) play an important role in the metabolism of a great variety of anticancer agents; therefore, polymorphisms in genes encoding for metabolizing enzymes and drugs transporters can affect drug efficacy and toxicity. Methods: The genetic polymorphisms of cytochrome P450 were studied in 395 patients with breast cancer by RLFP analysis. Results: Here, we studied the association of functionally significant variant alleles of CYP3A4, CYP3A5, CYP2B6, CYP2C8, CYP2C9 and CYP2C19 with the clinical response to neoadjuvant chemotherapy in breast cancer patients. A significant correlation was observed between the CYP2C9*2 polymorphism and chemotherapy resistance (OR¿=¿4.64; CI 95%¿=¿1.01 ¿ 20.91), as well as between CYP2C9*2 heterozygotes and chemotherapy resistance in women with nodal forms of breast cancer and a cancer hereditary load (OR¿=¿15.50; CI 95%¿=¿1.08 ¿ 826.12) when the potential combined effects were examined. No significant association between chemotherapy resistance and the other examined genotypes and the potential combined clinical and tumour-related parameters were discovered. Conclusion: In conclusion, CYP2C9*2 was associated with neoadjuvant chemotherapy resistance (OR¿=¿4.64; CI 95%¿=¿1.01 ¿ 20.91) in the population of interest.

Description: Background: A recent genome-wide association study (GWAS) using chronic HBV (hepatitis B virus) carriers with and without hepatocellular carcinoma (HCC) in five independent Chinese populations found that one SNP (rs17401966) in KIF1B was associated with susceptibility to HCC. In the present study, a total of 580 HBV-derived HCC cases and 1351 individuals with chronic hepatitis B (CHB) or asymptomatic carrier (ASC) were used for replication studies in order to evaluate the reported association with HBV-derived HCC in other East Asian populations. Results: We did not detect any associations between rs17401966 and HCC in the Japanese cohorts (replication 1: OR = 1.09, 95% CI = 0.82-1.43; replication 2: OR = 0.79, 95% CI = 0.54-1.15), in the Korean cohort (replication 3: OR = 0.95, 95% CI = 0.66-1.36), or in the Hong Kong Chinese cohort (replication 4: OR = 1.17, 95% CI = 0.79-1.75). Meta-analysis using these cohorts also did not show any associations with P = 0.97. Conclusions: None of the replication cohorts showed associations between rs17401966 and HBV-derived HCC. This may be due to differences in the genetic diversity among the Japanese, Korean and Chinese populations. Other reasons could be the high complexity of multivariate interactions between the genomic information and the phenotype that is manifesting. A much wider range of investigations is needed in order to elucidate the differences in HCC susceptibility among these Asian populations.

Description: Background: Genetic variants are likely to contribute to a portion of prostate cancer risk. Full elucidationof the genetic etiology of prostate cancer is difficult because of incomplete penetrance andgenetic and phenotypic heterogeneity. Current evidence suggests that genetic linkage toprostate cancer has been found on several chromosomes including the X; however,identification of causative genes has been elusive. Methods: Parametric and non-parametric linkage analyses were performed using 26 microsatellitemarkers in each of 11 groups of multiple-case prostate cancer families from the InternationalConsortium for Prostate Cancer Genetics (ICPCG). Meta-analyses of the resultant familyspecificlinkage statistics across the entire 1,323 families and in several predefined subsetswere then performed. Results: Meta-analyses of linkage statistics resulted in a maximum parametric heterogeneity lod score(HLOD) of 1.28, and an allele-sharing lod score (LOD) of 2.0 in favor of linkage to Xq27-q28 at 138 cM. In subset analyses, families with average age at onset less than 65 yearsexhibited a maximum HLOD of 1.8 (at 138 cM) versus a maximum regional HLOD of only0.32 in families with average age at onset of 65 years or older. Surprisingly, the subset offamilies with only 2-3 affected men and some evidence of male-to-male transmission ofprostate cancer gave the strongest evidence of linkage to the region (HLOD = 3.24, 134 cM).For this subset, the HLOD was slightly increased (HLOD = 3.47 at 134 cM) when familiesused in the original published report of linkage to Xq27-28 were excluded. Conclusions: Although there was not strong support for linkage to the Xq27-28 region in the complete setof families, the subset of families with earlier age at onset exhibited more evidence of linkagethan families with later onset of disease. A subset of families with 2-3 affected individualsand with some evidence of male to male disease transmission showed stronger linkagesignals. Our results suggest that the genetic basis for prostate cancer in our families is muchmore complex than a single susceptibility locus on the X chromosome, and that futureexplorations of the Xq27-28 region should focus on the subset of families identified here withthe strongest evidence of linkage to this region.

Description: Background: The neuronal ceroid lipofuscinoses (NCLs, or Batten disease) comprise the most commonMendelian form of childhood-onset neurodegeneration, but the functions of the knownunderlying gene products remain poorly understood. The clinical heterogeneity of thesedisorders may shed light on genetic interactors that modify disease onset and progression.Case presentationWe describe a proband with congenital hypotonia and an atypical form of infantile-onset,biopsy-proven NCL. Pathologic and molecular work-up of this patient identified CLN5mutations as well as a mutation-previously described as incompletely penetrant or a variantof unknown significance-in POLG1, a nuclear gene essential for maintenance ofmitochondrial DNA (mtDNA) copy number. The congenital presentation of this patient is farearlier than that described for either CLN5 patients or affected carriers of the POLG1 variant(c.1550 G 〉 T, p.Gly517Val). Assessment of relative mtDNA copy number and mitochondrialmembrane potential in the proband and control subjects suggested a pathogenic effect of thePOLG1 change as well as a possible functional interaction with CLN5 mutations. Conclusions: These findings suggest that an incompletely penetrant variant in POLG1 may modify theclinical phenotype in a case of CLN5 and are consistent with emerging evidence ofinteractions between NCL-related genes and mitochondrial physiology.

Description: Background: Multiple investigators have established the feasibility of using buccal brush samples togenotype single nucleotide polymorphisms (SNPs) with high-density genome-widemicroarrays, but there is currently no consensus on the accuracy of copy number variants(CNVs) inferred from these data. Regardless of the source of DNA, it is more difficult todetect CNVs than to genotype SNPs using these microarrays, and it therefore remains anopen question whether buccal brush samples provide enough high-quality DNA for thispurpose. Methods: To demonstrate the quality of CNV calls generated from DNA extracted from buccalsamples, compared to calls generated from blood samples, we evaluated the concordance ofcalls from individuals who provided both sample types. The Illumina Human660W-QuadBeadChip was used to determine SNPs and CNVs of 39 Arkansas participants in the NationalBirth Defects Prevention Study (NBDPS), including 16 mother-infant dyads, who providedboth whole blood and buccal brush DNA samples. Results: We observed a 99.9% concordance rate of SNP calls in the 39 blood-buccal pairs. From thesame dataset, we performed a similar analysis of CNVs. Each of the 78 samples wasindependently segmented into regions of like copy number using the Optimal Segmentationalgorithm of Golden Helix SNP & Variation Suite 7.Across 640,663 loci on 22 autosomal chromosomes, segment-mean log R ratios had anaverage correlation of 0.899 between blood-buccal pairs of samples from the same individual,while the average correlation between all possible blood-buccal pairs of samples fromunrelated individuals was 0.318. An independent analysis using the QuantiSNP algorithmproduced average correlations of 0.943 between blood-buccal pairs from the same individualversus 0.332 between samples from unrelated individuals.Segment-mean log R ratios had an average correlation of 0.539 between mother-offspringdyads of buccal samples, which was not statistically significantly different than the averagecorrelation of 0.526 between mother-offspring dyads of blood samples (p=0.302). Conclusions: We observed performance from the subject-collected mail-in buccal brush samplescomparable to that of blood. These results show that such DNA samples can be used forgenome-wide scans of both SNPs and CNVs, and that high rates of CNV concordance wereachieved whether using a change-point-based algorithm or one based on a hidden Markovmodel (HMM).

Description: Background: Huntington disease (HD) is caused by an expanded CAG repeat in the HD gene. Although thelength of the CAG repeat strongly correlates with the age-at-onset (AAO), AAO in HDindividuals may differ dramatically in spite of similar expanded CAG repeat lengths.Additional genetic or environmental factors are thought to influence the disease onset.Several modifier genes have been discovered so far but they do not fully explain thevariability of AAO in HD. To potentially identify a novel genetic modifier, we analyzedsingle nucleotide polymorphisms (SNPs) in the kalirin (KALRN) gene. Kalirin is a proteincrucially involved in spine plasticity and its interaction with huntingtin-associated protein-1(HAP-1) and a potential protein dysfunction might contribute to spine pathogenesis in HD. Methods: The selected SNPs were genotyped by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) and association of SNPs with AAO was investigated with theframework of linear models in an analysis of variance and covariance. Results: Eleven SNPs in the kalirin gene were examined in an association study in European HDpatients. The ten coding SNPs under investigation were monomorphic, whereas SNPrs10934657 in the promoter region showed a minor allele frequency 〉1%. An analysis ofcovariance together with the influence of the expanded HD allele was applied in 680 HDpatients. SNP rs10934657 did not affect the AAO of the examined HD population. Conclusions: The results did not reveal an association between the analyzed kalirin polymorphisms and theAAO in HD. However, it does not exclude other SNPs of the kalirin gene as susceptiblegenetic modifiers.

Description: Background: We used a gene - based replication strategy to test the reproducibility of prior acute lung injury candidate gene associations. Methods: We phenotyped 474 patients from a prospective severe trauma cohort study for ALI in the first 5 days following trauma. Genomic DNA from subjects' blood was genotyped using the IBC chip, a multiplex SNP array. Results were filtered for 25 candidate genes present on the IBC platform using prespecified literature search criteria. For each gene, we grouped SNPs according to haplotype blocks and tested the joint effect of all SNPs on susceptibility to ALI using the SNP-set kernel association test. Results were compared to single SNP analysis of the candidate SNPs. Analyses were separate for genetically determined ancestry (African or European). Results: We identified 4 genes in African ancestry and 2 in European ancestry trauma subjects which replicated their associations with ALI. Ours is the first replication of IL6, IL10, IRAK3, and VEGFA associations in non-European populations with ALI. Only one gene - VEGFA - demonstrated association with ALI in both ancestries, with distinct haplotype blocks in each ancestry driving the association. We also report the association between trauma-associated ALI and NFKBIA in European ancestry subjects. Conclusions: Prior ALI genetic associations are reproducible and replicate in a trauma cohort. Kernel - based SNP-set analysis is a more powerful method to detect ALI association than single SNP analysis, and thus may be more useful for replication testing. Further, gene-based replication can extend candidate gene associations to diverse ethnicities.

Description: Background: While some factors of breast morphology, such as density, are directly implicated in breast cancer, therelationship between breast size and cancer is less clear. Breast size is moderately heritable, yet the geneticvariants leading to differences in breast size have not been identified. Methods: To investigate the genetic factors underlying breast size, we conducted a genome-wide association study(GWAS) of self-reported bra cup size, controlling for age, genetic ancestry, breast surgeries, pregnancyhistory and bra band size, in a cohort of 16,175 women of European ancestry. Results: We identified seven single-nucleotide polymorphisms (SNPs) significantly associated with breast size(p 〈 5 . 10-8): rs7816345 near ZNF703, rs4849887 and (independently) rs17625845 flanking INHBB,rs12173570 near ESR1, rs7089814 in ZNF365, rs12371778 near PTHLH, and rs62314947 near AREG. Twoof these seven SNPs are in linkage disequilibrium (LD) with SNPs associated with breast cancer (those near1ESR1 and PTHLH), and a third (ZNF365) is near, but not in LD with, a breast cancer SNP. The other threeloci (ZNF703, INHBB, and AREG) have strong links to breast cancer, estrogen regulation, and breastdevelopment. Conclusions: These results provide insight into the genetic factors underlying normal breast development and show thatsome of these factors are shared with breast cancer. While these results do not directly support any possibleepidemiological relationships between breast size and cancer, this study may contribute to a betterunderstanding of the subtle interactions between breast morphology and breast cancer risk.

Description: Background: Cornelia de Lange syndrome (CdLS) is a dominantly inherited disorder characterized byfacial dysmorphism, growth and cognitive impairment, limb malformations and multipleorgan involvement. Mutations in NIPBL gene account for about 60% of patients with CdLS.This gene encodes a key regulator of the Cohesin complex, which controls sister chromatidsegregation during both mitosis and meiosis. Turner syndrome (TS) results from the partial orcomplete absence of one of the X chromosomes, usually associated with congenitallymphedema, short stature, and gonadal dysgenesis.Case presentationHere we report a four-year-old female with CdLS due to a frameshift mutation in the NIPBLgene (c.1445_1448delGAGA), who also had a tissue-specific mosaic 45,X/46,XX karyotype.The patient showed a severe form of CdLS with craniofacial dysmorphism, pre- and postnatalgrowth delay, cardiovascular abnormalities, hirsutism and severe psychomotorretardation with behavioural problems. She also presented with minor clinical featuresconsistent with TS, including peripheral lymphedema and webbed neck. The NIPBL mutationwas present in the two tissues analysed from different embryonic origins (peripheral bloodlymphocytes and oral mucosa epithelial cells). However, the percentage of cells withmonosomy X was low and variable in tissues. These findings indicate that, ontogenically, theNIPBL mutation may have appeared before the mosaic monosomy X. Conclusions: The coexistence in several patients of these two rare disorders raises the issue of whetherthere is indeed a cause-effect association. The detailed clinical descriptions indicatepredominant CdLS phenotypes, although additional TS manifestations may appear inadolescence.

Description: Background: Genetic variations in TGFB1 gene have been studied in relation to coronary heart disease (CHD) risk, but the results were inconsistent. Methods: We performed a systematic review of published studies on the potential role of TGFB1 genetic variation in CHD risk. Articles that reported for the association of TGFB1 genetic variants with CHD as primary outcome were searched via Medline and HuGE Navigator through July 2011. The reference lists from included articles were also reviewed. Results: Data were available from 4 studies involving 1777 cases and 7172 controls for rs1800468, 7 studies involving 5935 cases and 10677 controls for rs1800469, 7 studies involving 6634 cases and 9620 controls for rs1982073, 5 studies involving 5452 cases and 9999 controls for rs1800471, and 4 studies involving 5143 cases and 4229 controls for rs1800472. The pooled odds ratios (ORs) for CHD among minor T allele carriers of rs1800469, minor C allele carriers of rs1982073, and minor C allele carriers of rs1800471 versus homozygous major allele carriers was 1.14 (95% confidence interval [CI]: 1.05-1.24), 1.18 (95% CI: 1.04-1.35), and 1.16 (95% CI: 1.02-1.32), respectively. No substantial heterogeneity for ORs was detected among the included Caucasian populations for all SNPs. However, for rs1800471, the statistical significance disappeared after adjusting for potential publication bias. No significant association was found between rs1800468 and rs1800472 variants and CHD risk. Conclusion: Two genetic variants (rs1800469 and rs1982073) in TGFbeta1 are associated with CHD risk, minor allele carriers having a 15% increased risk.

Description: Background: Insulin like growth factor 2 (IGF2) is an imprinted gene, which has an important role in fetalgrowth as established in mice models. IGF2 is downregulated through hypomethylation of adifferentially methylated region (DMR) in Silver Russell syndrome (SRS), characterised bygrowth restriction. We have previously reported that severe pre- and post-natal growthrestriction associated with insulin resistance and precocious pubarche in a woman withoutbody asymmetry or other SRS features resulted from a balanced translocation affecting theregulation of her IGF2 gene expression. We hypothesised that severe pre- and post-natalgrowth restriction associated with insulin resistance and precocious pubarche in the absenceof SRS are also caused by downregulation of IGF2 through hypomethylation, gene mutationor structural chromosomal abnormalities. Methods: We performed routine karyotyping, IGF2 gene sequencing and investigated DNAmethylation of the IGF2 differentially methylated region (DMR)0 and H19 DMR usingpyrosequencing, in four women selected for very low birth weight (

Description: Background: Telomere length, an indicator of ageing and longevity, has been correlated with several biomarkers of cardiometabolic disease in both Arab children and adults. It is not known,however, whether or not telomere length is a highly conserved inheritable trait in thishomogeneous cohort, where age-related diseases are highly prevalent. As such, the aim of this study was to address the inheritability of telomere length in Saudi families and the impactof cardiometabolic disease biomarkers on telomere length. Methods: A total of 119 randomly selected Saudi families (123 adults and 131 children) were included in this cross-sectional study. Anthropometrics were obtained and fasting blood samples weretaken for routine analyses of fasting glucose and lipid profile. Leukocyte telomere length was determined using quantitative real time PCR. Results: Telomere length was highly heritable as assessed by a parent-offspring regression [h2 = 0.64 (p = 0.0006)]. Telomere length was modestly associated with BMI (R2 0.07; p-value 0.0087),total cholesterol (R2 0.08; p-value 0.0033), and LDL-cholesterol (R2 0.15; p-value 3 x 10-5) after adjustments for gender, age and age within generation. Conclusion: The high heritability of telomere length in Arab families, and the associations of telomere length with various cardiometabolic parameters suggest heritable genetic fetal and/or epigenetic influences on the early predisposition of Arab children to age-related diseases and accelerated ageing.

Description: Background: To date, evaluation of the association of the ABO blood group and breast cancer has yieldedmixed results. SNP rs505922, located within the first intron of the ABO gene, has beenassociated with the adenocarcinoma subtype of pancreatic cancer. To evaluate the associationbetween genetic variation in the ABO blood group and risk of breast cancer, rs505922 wasgenotyped in 629 Caucasian women with invasive breast cancer, representing a variety ofclinical and pathological tumor types. Methods: Genomic DNA was isolated from blood. TaqMan SNP assay C_2253769_10 was used todetermine genotypes for each patient at rs505922. Statistical analysis was performed usingchi-square analysis using a P-value

Description: Background: Recent genome-wide association studies have identified a single nucleotide polymorphismwithin the last intron of MAP2K5 associated with a higher body mass index (BMI) in adults.MAP2K5 is a component of the MAPK-family intracellular signaling pathways, respondingto extracellular growth factors such as brain derived neurotrophic factor (BDNF) and nervegrowth factor (NGF). In this study, we examined the association of this variant in two cohortsof children from Sweden and Greece. Methods: We examine the association of rs2241423 to BMI in a cohort of 474 Swedish childrenadmitted for treatment of childhood obesity and 519 children matched for gender, ethnicityand socioeconomic background from the Stockholm area, as well as a cross-sectional cohortof 2308 Greek school children (Healthy Growth Study). Children were genotyped using apredesigned TaqMan polymorphism assay. Logistic regression was used to test for anassociation of rs2241423 to obesity in the cohort of Swedish children. Linear regression wasused to test for an association of rs2241423 to BMI z-score and phenotypic in the cohort ofGreek children. Models were adjusted for age and gender. In the cohort of Greek children themodel was also adjusted for stage of pubertal development. Results: The minor allele of rs2241423, allele A, was associated with a protective effect againstobesity in the cohort of Swedish children (p = 0.029, OR = 0.79 (95% CI: 0.64-0.98)), andwith a lower BMI z-score in the cohort of Greek children (p = 0.028, beta = 0.092). Noassociation to phenotypic measurements of body fat distribution could be observed in ourstudy. Conclusions: rs2241423 was associated with BMI and obesity in two independent European cohortssuggesting a role for MAP2K5 in early weight regulation.

Description: Background: Ficolin-2 coded by FCN2 gene is a soluble serum protein and an innate immune recognitionelement of the complement system. FCN2 gene polymorphisms reveal distinct geographicalpatterns and are documented to alter serum ficolin levels and modulate disease susceptibility. Methods: We employed a real-time PCR based on Fluorescence Resonance Energy Transfer (FRET)method to genotype four functional SNPs including -986 G 〉 A (#rs3124952), -602 G 〉 A(#rs3124953), -4A 〉 G (#rs17514136) and +6424 G 〉 T (#rs7851696) in the ficolin-2(FCN2) gene. We characterized the FCN2 variants in individuals representing Brazilian (n =176), Nigerian (n = 180), Vietnamese (n = 172) and European Caucasian ethnicity (n = 165). Results: We observed that the genotype distribution of three functional SNP variants (986 G 〉 A, -602 G 〉 A and -4A 〉 G) differ significantly between the populations investigated (p

Description: Background: MicroRNAs (miRNAs) are post-transcriptional regulators involved in numerous biologicalprocesses including the pathogenesis of Alzheimer's disease (AD). A key gene of AD,ADAM10, controls the proteolytic processing of APP and the formation of the amyloidplaques and is known to be regulated by miRNA in hepatic cancer cell lines. To predictmiRNAs regulating ADAM10 expression concerning AD, we developed a computationalapproach. Methods: MiRNA binding sites in the human ADAM10 3' untranslated region were predicted using theRNA22, RNAhybrid and miRanda programs and ranked by specific selection criteria withrespect to AD such as differential regulation in AD patients and tissue-specific expression.Furthermore, target genes of miR-103, miR-107 and miR-1306 were derived from six publiclyavailable miRNA target site prediction databases. Only target genes predicted in at least fourout of six databases in the case of miR-103 and miR-107 were compared to genes listed in theAlzGene database including genes possibly involved in AD. In addition, the target geneswere used for Gene Ontology analysis and literature mining. Finally, we used a luciferaseassay to verify the potential effect of these three miRNAs on ADAM10 3' UTR in SH-SY5Ycells. Results: Eleven miRNAs were selected, which have evolutionary conserved binding sites. Three ofthem (miR-103, miR-107, miR-1306) were further analysed as they are linked to AD and moststrictly conserved between different species. Predicted target genes of miR-103 (p-value =0.0065) and miR-107 (p-value = 0.0009) showed significant overlap with the AlzGenedatabase except for miR-1306. Interactions between miR-103 and miR-107 to genes wererevealed playing a role in processes leading to AD. ADAM10 expression in the reporter assaywas reduced by miR-1306 (28%), miR-103 (45%) and miR-107 (52%). Conclusions: Our approach shows the requirement of incorporating specific, disease-associated selectioncriteria into the prediction process to reduce the amount of false positive predictions. Insummary, our method identified three miRNAs strongly suggested to be involved in AD,which possibly regulate ADAM10 expression and hence offer possibilities for thedevelopment of therapeutic treatments of AD.

Description: Background: Hyperhomocysteinemia as a consequence of the MTHFR 677 C 〉 T variant is associated withcardiovascular disease and stroke. Another factor than can potentially contribute to thesedisorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G 〉 Tand eNOS 786 T 〉 C variants that make an individual more susceptible to endothelialdysfunction. A number of genotyping methods have been developed to investigate these variants.However, simultaneous detection methods using polymerase chain reaction-restriction fragmentlength polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplexPCR-RFLP method for the simultaneous detection of MTHFR 677 C 〉 T and eNOS +894 G 〉 Tand eNOS 786 T 〉 C variants was developed. A total of 114 healthy Malay subjects wererecruited. The MTHFR 677 C 〉 T and eNOS +894 G 〉 T and eNOS 786 T 〉 C variants weregenotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well assnpBLAST. Allele frequencies of MTHFR 677 C 〉 T and eNOS +894 G 〉 T and eNOS 786 T 〉C were calculated using the Hardy Weinberg equation. Methods: The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primerpair was designed using Primer 3 Software version 0.4.0 and validated against the BLASTdatabase. The primer specificity, functionality and annealing temperature were tested usinguniplex PCR methods that were later combined into a single multiplex PCR. RestrictionFragment Length Polymorphism (RFLP) was performed in three separate tubes followed byagarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. Results: The allele frequencies for MTHFR 677 C 〉 T were 0.89 (C allele) and 0.11 (T allele); for eNOS+894 G 〉 T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS 786 T〉 C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele). Conclusions: Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used tosuccessfully genotype subjects for the MTHFR 677 C 〉 T and eNOS +894 G 〉 T and eNOS786 T 〉 C variants simultaneously with 100 % concordance from DNA sequencing data. Thismethod can be routinely used for rapid investigation of the MTHFR 677 C 〉 T and eNOS +894G 〉 T and eNOS 786 T 〉 C variants.

Description: Background: Previous studies have examined the associations between polymorphisms of adiponectin gene(ADIPOQ) and cardiovascular disease (CVD), but those studies have been inconclusive. Theaim of this study was to access the relationship between three single nucleotidepolymorphisms (SNPs), +45 T 〉 G (rs2241766), +276 G 〉 T (rs1501299) and -11377 C 〉 G(rs266729) in ADIPOQ and CVD. Methods: A comprehensive search was conducted to identify all studies on the association of ADIPOQgene polymorphisms with CVD risk. The fixed and random effect pooled measures (i.e. oddsratio (OR) and 95% confidence interval (CI)) were calculated in the meta-analysis.Heterogeneity among studies was evaluated using Q test and the I2. Publication bias wasestimated using modified Egger's linear regression test. Results: Thirty-seven studies concerning the associations between the three polymorphisms ofADIPOQ gene and CVD risk were enrolled in this meta-analysis, including 6,398 cases and10,829 controls for rs2241766, 8,392 cases and 18,730 controls for rs1501299 and 7,835cases and 14,023 controls for rs266729. The three SNPs were significantly associated withCVD, yielding pooled ORs of 1.22 (95%CI: 1.07, 1.39; P = 0.004), 0.90 (95%CI: 0.83, 0.97;P = 0.007) and 1.09(95%CI: 1.01, 1.17; P = 0.032) for rs2241766, rs1501299 and rs266729,respectively. Rs2241766 and rs1501299 were significantly associated with coronary heartdisease (CHD), yielding pooled ORs of 1.29 (95%CI: 1.09, 1.52; P = 0.004) and 0.89(95%CI: 0.81, 0.99; P = 0.025), respectively. The pooled OR for rs266729 and CHD was1.09 (95%CI: 0.99, 1.19; P = 0.090). Significant between-study heterogeneity was found inour meta-analysis. Evidence of publication bias was observed in the meta-analysis. Conclusions: The present meta-analysis showed that the associations between rs2241766, rs1501299 andrs266729 in the ADIPOQ and CVD were significant but weak. High quality studies are stillneeded to confirm the associations, especially for rs2241766.

Description: Background: Familial hypercholesterolemia (FH) is a human monogenic disease induced by a variety of mutations with striking genetic diversity. Despite this variability recurrent mutations occur in each population studied, which allows both elucidating prevalent mutations and developing DNA diagnostic tools for the disease. Recent research of FH in St. Petersburg, Moscow and Novosibirsk (major cities in Russia) demonstrates that each megapolis has its own FH mutation spectrum sharing only small part of mutations with other populations in Russia and Europe. In order to optimize molecular-genetic diagnostic protocols for FH in Russia we studied mutation spectrum in other regions including Petrozavodsk, a smaller town in relatively close proximity to St. Petersburg. Methods: The principal method was automated detection of single-strand conformation polymorphism followed by direct PCR amplified DNA sequencing. Results: Twelve different mutations of the low density lipoprotein (LDL) receptor gene were detected in the Petrozavodsk sample (80 patients). Out of these twelve mutations, seven have never been described before (c.192_201delinsGGACTTCA, c. 195_196insT, c. 618 T 〉 G, c. 1340C 〉 G, c. 1686_1693delinsT, c. 1936C 〉 A, c. 2191delG). Other five mutations (c. 58G 〉 A, c. 925_931del, c. 1194C 〉 T, c. 1532 T 〉 C, c. 1920C 〉 T) were previously characterized elsewhere. All new mutations are considered to be a probable cause of the FH in their carriers. Direct evidence of the neutral character of c.58G 〉 A or p. (Gly20Arg) is provided for the first time. Each pathogenic mutation was a trait of its own unique pedigree and so far has not been found in other patients. Conclusions: Strikingly, out of twelve mutations characterized in the Petrozavodsk sample only one mutation, c. 925_931del, has previously been found in patients from St. Petersburg and Finland (most closely located studied populations), suggesting some common roots in origin of these populations in the past or limited gene exchange between them nowadays. No recurrent mutations were detected.

Description: Background: Noonan syndrome is an autosomal dominant developmental disorder with a high phenotypic variability, which shares clinical features with other rare conditions, including LEOPARD syndrome, cardiofaciocutaneous syndrome, Noonan-like syndrome with loose anagen hair, and Costello syndrome. This group of related disorders, so-called RASopathies, is caused by germline mutations in distinct genes encoding for components of the RAS-MAPK signalling pathway. Due to high number of genes associated with these disorders, standard diagnostic testing requires expensive and time consuming approaches using Sanger sequencing. In this study we show how targeted Next Generation Sequencing (NGS) technique can enable accurate, faster and cost-effective diagnosis of RASopathies. Methods: In this study we used a validation set of 10 patients (6 positive controls previously characterized by Sanger-sequencing and 4 negative controls) to assess the analytical sensitivity and specificity of the targeted NGS. As second step, a training set of 80 enrolled patients with a clinical suspect of RASopathies has been tested. Targeted NGS has been successfully applied over 92% of the regions of interest, including exons for the following genes: PTPN11, SOS1, RAF1, BRAF, HRAS, KRAS, NRAS, SHOC, MAP2K1, MAP2K2, CBL. Results: All expected variants in patients belonging to the validation set have been identified by targeted NGS providing a detection rate of 100%. Furthermore, all the newly detected mutations in patients from the training set have been confirmed by Sanger sequencing. Absence of any false negative event has been excluded by testing some of the negative patients, randomly selected, with Sanger sequencing. Conclusion: Here we show how molecular testing of RASopathies by targeted NGS could allow an early and accurate diagnosis for all enrolled patients, enabling a prompt diagnosis especially for those patients with mild, non-specific or atypical features, in whom the detection of the causative mutation usually requires prolonged diagnostic timings when using standard routine. This approach strongly improved genetic counselling and clinical management.

Description: Background: Charcot-Marie-Tooth disease (CMT) is a heterogeneous disorder of the peripheral nervous system. So far, mutations in hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), beta subunit (HADHB) gene exhibit three distinctive phenotypes: severe neonatal presentation with cardiomyopathy, hepatic form with recurrent hypoketotic hypoglycemia, and later-onset axonal sensory neuropathy with episodic myoglobinuria. Methods: To identify the causative and characterize clinical features of a Korean family with motor and sensory neuropathies, whole exome study (WES), histopathologic study of distal sural nerve, and lower limb MRIs were performed. Results: WES revealed that a compound heterozygous mutation in HADHB is the causative of the present patients. The patients exhibited an early-onset axonal sensorimotor neuropathy without episodic myoglobinuria, and showed typical clinical and electrophysiological features of CMT including predominant distal muscle weakness and atrophy. Histopathologic findings of sural nerve were compatible with an axonal CMT neuropathy. Furthermore, they didn't exhibit any other symptoms of the previously reported HADHB patients. Conclusions: These data implicate that mutation in HADHB gene can also cause early-onset axonal CMT instead of typical manifestations in mitochondrial trifunctional protein (MTP) deficiency. Therefore, this study is the first report of a new subtype of autosomal recessive axonal CMT by a compound heterozygous mutation in HADHB, and will expand the clinical and genetic spectrum of HADHB.

Description: Background: Cerebral palsy (CP) is a group of nonprogressive disorders of movement and posture caused by abnormal development of, or damage to, motor control centers of the brain. A single nucleotide polymorphism (SNP), rs1800795, in the promoter region of the interleukin-6 (IL6) gene has been implicated in the pathogenesis of CP by mediating IL-6 protein levels in amniotic fluid and cord plasma and within brain lesions. This SNP has been associated with other neurological, vascular, and malignant processes as well, often as part of a haplotype block. Methods: To refine the regional genetic association with CP, we sequenced (Sanger) the IL6 gene and part of the promoter region in 250 infants with CP and 305 controls. Results: We identified a haplotype of 7 SNPs that includes rs1800795. In a recessive model of inheritance, the variant haplotype conferred greater risk (OR = 4.3, CI = [2.0-10.1], p = 0.00007) than did the lone variant at rs1800795 (OR = 2.5, CI = [1.4-4.6], p = 0.002). The risk haplotype contains one SNP (rs2069845, CI = [1.2-4.3], OR = 2.3, p = 0.009) that disrupts a methylation site. Conclusions: The risk haplotype identified in this study overlaps with previously identified haplotypes that include additional promoter SNPs. A risk haplotype at the IL6 gene likely confers risk to CP, and perhaps other diseases, via a multi-factorial mechanism.

Description: Background: SRD5A3 is responsible for SRD5A3-CDG, a type of congenital disorder of glycosylation, and mutations have been reported in 15 children. All the mutations are recessive and truncating.Case presentationWe present 2 brothers at the age of 38 and 40 years with an initial diagnosis of cerebellar ataxia. We found the candidate disease loci via linkage analysis using data from single nucleotide polymorphism genome scans and homozygous truncating mutation SRD5A3 p.W19X, which was previously reported in 3 unrelated children, by exome sequencing.Clinical investigations included physical and ocular examinations and blood tests. Severe ocular involvement with retinal bone spicule pigmentation and optic atrophy are the most prominent disabling clinical features of the disease. The serum transferrin isoelectric focusing (TIEF) pattern is abnormal in the patient investigated. Conclusion: Our patients are older, with later onset and milder clinical phenotypes than all patients with SRD5A3-CDG reported so far. They also have atypical ocular findings and variable phenotypes. Our findings widen the spectrum of phenotypes resulting from SRD5A3 mutations and the clinical variability of SRD5A3-CDG, and suggest screening for SRD5A3 mutations in new patients with at least a few of the clinical symptoms of SRD5A3-CDG.

Description: Background: Genome-wide association studies have revealed an association between several loci in the nicotinic acetylcholine receptor gene cluster CHRNA5-A3-B4 and daily cigarette consumption. Recent studies have sought to refine this phenotype, and have shown that a locus within this cluster, marked primarily by rs1051730 and rs16969968, is also associated with levels of cotinine, the primary metabolite of nicotine. This association remains after adjustment for self-reported smoking, which suggests that even amongst people who smoke the same number of cigarettes there is still genetically-influenced variation in nicotine consumption. This is likely to be due to differences in smoking topography, that is, how a cigarette is smoked (e.g., volume of smoke inhaled per puff, number of puffs taken per cigarette). The aim of this study is to determine potential mediation of the relationship between the rs1051730 locus and cotinine levels by smoking topography. Methods: Adopting a recall-by-genotype design, we will recruit 200 adults from the Avon Longitudinal Study of Parents and Children on the basis of minor or major homozygote status at rs1051730 (100 in each genotype group). All participants will be current, daily smokers. Our primary study outcome measures will be measures of smoking topography: total volume of smoke (ml) inhaled per cigarette, total volume of smoke (ml) inhaled over of the course of one day, and salivary cotinine level (ng/ml).DiscussionThis study will extend our understanding of the biological basis of inter-individual variability in heaviness of smoking, and therefore in exposure to smoking-related toxins. The novel recall-by-genotype approach we will use is efficient, maximising statistical power, and enables the collection of extremely precise phenotypic data that are impractical to collect in a larger sample. The methods described within this protocol also hold the potential for wider application in the field of molecular genetics.

Description: Background: Congenital cataract is a Mendelian disorder that frequently causes blindness in infants. To date, various cataract-associated loci have been mapped; more than 30 genes have been identified by linkage analysis. However, the pathogenic loci in some affected families are still unknown, and new research strategies are needed. In this study, we used linkage-exome combinational analysis to further investigate the pedigree of a four-generation Chinese family with autosomal dominant coralliform cataract. Methods: We combined whole exome sequencing and linkage analysis to identify the causative mutation. The exome capture and next-generation sequencing were used to sequence the protein-coding regions in the genome of the proband to identify rare mutations, which were further screened for candidate mutations in linkage regions. Candidate mutations were independently verified for co-segregation in the whole pedigree using Sanger sequencing. Results: We identified a C to A transversion at nucleotide position c.70 in exon 2 of CRYGD, a cataract-associated gene. This mutation resulted in a threonine substitution for proline at amino acid residue 24. Conclusions: We identified a missense P24T mutation in CRYGD that was responsible for coralliform cataract in our studied family. Our findings suggest that the combination of exome sequencing and linkage analysis is a powerful tool for identifying Mendelian disease mutations that might be missed by the classic linkage analysis strategy.

Description: Background: Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to its cognate transfer RNA and therefore plays an essential role in protein biosynthesis. Methods: We used exome sequencing, aminoacylation assays, homology modeling, and immune-isolation of transfected MARS to identify and characterize mutations in the methionyl-tRNA synthetase gene (MARS) in an infant with an unexplained multi-organ phenotype. Results: We identified compound heterozygous mutations (F370L and I523T) in highly conserved regions of MARS. The parents were each heterozygous for one of the mutations. Aminoacylation assays documented that the F370L and I523T MARS mutants had 18 +/- 6% and 16 +/- 6%, respectively, of wild-type activity. Homology modeling of the human MARS sequence with the structure of E. coli MARS showed that the F370L and I523T mutations are in close proximity to each other, with residue I523 located in the methionine binding pocket. We found that the F370L and I523T mutations did not affect the association of MARS with the multisynthetase complex. Conclusion: This infant expands the catalogue of inherited human diseases caused by mutations in aminoacyl-tRNA synthetase genes.

Description: Background: Several studies in type 2 diabetes patients have shown significant associations between the SOD2 gene Val16Ala polymorphism and albuminuria, but this association has not been explored in the Mexican population. Methods: We evaluated the association between the SOD2 gene Val16Ala polymorphism (rs4880) and macroalbuminuria in a sample of 994 unrelated Mexican type 2 diabetes patients. The study included 119 subjects with urinary albumin 〉300mg/dL and 875 subjects with urinary albumin

Description: Background: Several studies analyzed the associations of Vitamin D receptor (VDR) polymorphisms with urolithiasis risk in different ethnic groups. However, the results were inconclusive. To evaluate a more precise estimation of the relationship, a meta-analysis was performed. Methods: Pubmed, EMBASE, Wanfang Database, China National Knowledge Infrastructure (CNKI) and Weipu Database were searched. Data were extracted independently by two investigators. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations. Results: Twenty-three case--control studies were included in this meta-analysis. Significant associations between ApaI, BsmI, FokI, and TaqI polymorphisms and urolithiasis risk were observed. However, sensitivity analyses for BsmI and FokI polymorphisms indicated that the results were not reliable and credible. In addition, there was a significant association of the ApaI-TaqI haplotype with urolithiasis risk. Conclusions: This meta-analysis suggested that ApaI and TaqI polymorphisms in VDR gene were associated with urolithiasis risk.

Description: Background: Sickle cell anemia (SCA) presents a complex pathophysiology which can be affected by a number of modifying factors, including genetic and biochemical ones. In Brazil, there have been no studies verifying betaS-haplotypes effect on oxidative stress parameters. This study evaluated betaS-haplotypes and Hb F levels effects on oxidative stress markers and their relationship with hydroxyurea (HU) treatment in SCA patients. Methods: The studied group was composed by 28 SCA patients. Thirteen of these patients were treated with HU and 15 of them were not. We used molecular methodology (PCR-RFLP) for hemoglobin S genotype confirmation and haplotypes identification. Biochemical parameters were measured using spectrophotometric methods (Thiobarbituric-acid-reactive substances and Trolox equivalent antioxidant capacity levels, catalase and GST activities) and plasma glutathione levels by High-performance liquid chromatography coupled to electrochemical detection. Results: We found the highest frequency of Bantu haplotype (48.2%) which was followed by Benin (32.1%). We observed also the presence of Cameroon haplotype, rare in Brazilian population and 19.7% of atypical haplotypes. The protective Hb F effect was confirmed in SCA patients because these patients showed an increase in Hb F levels that resulted in a 41.3% decrease on the lipid peroxidation levels (r =-0.74, p=0.01). Other biochemical parameters have not shown differential expression according to patient's haplotypes. Bantu haplotype presence was related to the highest lipid peroxidation levels in patients (p 〈 0,01), but it also conferred a differential response to HU treatment, raising Hb F levels in 52.6% (p = 0.03) when compared with the group with the same molecular profile without HU usage. Conclusions: SCA patients with Bantu haplotype showed the worst oxidative status. however these patients also demonstrated a better response to the treatment with HU. Such treatment seems to have presented a "haplotype-dependent" pharmacological effect.

Description: Background: IL-33, an IL-1-like cytokine, is a ligand for IL1RL1, which is an important effector molecule of type 2 T helper responses. Although IL-33/IL1RL1 interaction has been suggested to be important in the development of atherosclerosis, genetic influences of the polymorphisms of IL33 in human ischemic stroke are unclear. The aim of this study was to examine whether the single nucleotide polymorphisms in IL33 are associated with ischemic stroke in Northern Chinese population. Methods: We used a nested case--control study involving 90 ischemic stroke patients and 270 age-matched, sex-matched and blood pressure-matched non-ischemic stroke controls from a rural population and determined the genotypes of four polymorphisms (rs1929992, rs10975519, rs4742170, rs16924159) in IL33 by Snapshot SNP genotyping assays to assess any links with ischemic stroke. Results: Univariate analysis showed two single nucleotide polymorphisms (rs1929992, rs4742170) in IL33 were associated with ischemic stroke in additive, dominant, and recessive model. Binary Logistic Regression shows that rs4742170 variation is the most important factor associated with ischemic stroke (adjusted odds ratio (OR) = 1.880, 95% confidence interval (CI) = 1.316-2.686 in an additive model; OR = 2.091, CI = 1.249-3.498 in a dominant model; OR = 2.623, CI = 1.366-5.036 in a recessive model). Conclusion: In this sample of patients, genetic variation of rs4742170 in IL33 is significantly associated with the developing of ischemic stroke.