ALBERT

Beschreibung: Dyskeratosis congenita (DC) is a rare inherited form of bone marrow failure (BMF) caused by mutations in telomere maintaining genes including TERC and TERT. Here we studied the prevalence of TERC and TERT gene mutations and of telomere shortening in an unselected population of patients with BMF at our medical center and in a selected group of patients referred from outside institutions. Less than 5% of patients with BMF had pathogenic mutations in TERC or TERT. In patients with BMF, pathogenic TERC or TERT gene mutations were invariably associated with marked telomere shortening (≪ 1st percentile) in peripheral blood mononuclear cells (PBMCs). In asymptomatic family members, however, telomere length was not a reliable predictor for the presence or absence of a TERC or TERT gene mutation. Telomere shortening was not pathognomonic of DC, as approximately 30% of patients with BMF due to other causes had PBMC telomere lengths at the 1st percentile or lower. We conclude that in the setting of BMF, measurement of telomere length is a sensitive but nonspecific screening method for DC. In the absence of BMF, telomere length measurements should be interpreted with caution.

Beschreibung: Diamond-Blackfan anemia (DBA), a congenital bone marrow failure syndrome, is characterized by red blood cell aplasia, macrocytic anemia, clinical heterogeneity, and increased risk of malignancy. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital anomalies, present in ~30–47% of patients. The disease is associated with mutations in six ribosomal protein (RP) genes, S19, S24, S17, L35A, L5, and L11, in about 40–45% of patients. To continue our large scale screen of RP genes in a DBA population, we sequenced 12 RP genes, S15, L36, L31, L37A, S7, S27A, S14, S23, L3, L23, S17, and L27A in our DBA patient cohort of 200 families. We identified the second known mutation in RPS17 and possible pathogenic single mutations in four more RP genes, S7, L36, S15, and S27A. These are a donor splice site mutation (intron 2) in RPS7, a deletion of two nucleotides causing frameshift in RPS17 and RPL36, and two missense changes in RPS15 and RPS27A. Northern blot analysis demonstrated that lymphoblastoid cells from the patient with RPS7 mutation displayed higher levels of 45S and 30S pre-rRNAs compared to normal cells, similar to results in HeLa cells with siRNA-based knock-down of RPS7. There is a strong defect in 5′-ETS processing, resulting in accumulation of 45S and 30S pre-rRNAs, and a strong drop of levels of the 41S, 21S and 18S-E intermediates, whereas the amount of precursors to the large ribosomal subunit RNAs were unchanged. These results suggest that mutation of RPS7 in this DBA patient directly affects maturation of pre-rRNA. In addition, review of available medical data of 20 patients with mutations in RPL5 revealed that majority of them (14/20) have physical malformations including craniofacial, thumb and heart anomalies. Similarly, 12/18 patients with RPL11 mutations presented with physical malformations, while among 76 reported DBA patients with RPS19 mutations, only 35 (46%) had physical abnormalities. Remarkably, 9 of 14 patients with RPL5 mutations and physical abnormalities have cleft lip/ palate or cleft soft palate, isolated or in combination with other facial malformations and/or with other physical abnormalities such as heart or thumb anomalies. In contrast, none of 12 patients with RPL11 mutations and malformations have craniofacial abnormalities (p=0.007, Fisher’s exact test [FET]). Moreover, none of the 35 reported patients with RPS19 mutations and malformations presented with cleft lip and/or palate (p=9.745×10−7for RPL5 vs RPS19, FET). We conclude craniofacial clefting is associated with mutations in RPL5. In addition, 8/20 patients with mutated RPL5 and 8/18 patients with mutated RPL11 have thumb abnormalities, compared to only 9% of patients with RPS19 mutations. Moreover, congenital heart defects were found more often among patients with RPL5 mutations (5/20) compared with RPL11 (3/18) and RPS19 (4/76) (p=0.017 for RPL5 vs RPS19, FET). Strikingly, the majority (11/20) of patients with RPL5 mutations presented with multiple, severe abnormalities, including craniofacial, heart and/or thumb malformations. In contrast, patients with RPL11 and RPS19 mutations who presented with multiple physical abnormalities were uncommon, three patients out of 18, and 16 out of 76, respectively (p=0.02 for RPL5 vs RPL11 and p=0.0047 for RPL5 vs RPS19, FET). In summary, we identified single mutations in four genes as well as the second mutation in RPS17, suggesting that sequence changes in RPS7, RPS17, RPL36, RPS15, and RPS27A are rare events in DBA. Mutations in RPL5 are associated with multiple physical abnormalities including craniofacial, thumbs and heart anomalies, while thumb malformations are predominantly present in patients carrying mutations in RPL11.

Beschreibung: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure (BMF) syndrome. The classical features of DC include nail dystrophy, abnormal skin pigmentation, and mucosal leukoplakia. The diagnosis of DC can be difficult. Originally, the diagnosis was based on the presence of the classical mucocutaneous features. However, the identification of four genes responsible for DC (DKC1, TERC, TERT, and NOP10) showed that these mucocutaneous features are only present in a proportion of patients with DC. Additionally, screening for mutations in the affected genes is expensive and is negative in about 50% of patients with classical features of DC. The products of the genes mutated in DC are the components of the telomerase ribonucleoprotein complex, which is essential for telomere maintenance. Therefore it has been postulated that DC is a disease arising from excessive telomere shortening. Here we examined whether the measurement of telomeres could be used as a screening test to identify individuals with DC. For this purpose we examined telomere length in peripheral blood mononuclear cells from 169 patients who presented with bone marrow failure including 17 patients with DC, diagnosed by the presence of classical cutaneous features or the identification of mutations in DKC1, TERC or TERT, 28 patients with paroxysmal nocturnal hemoglobinuria, 25 patients with Diamond Blackfan anemia, 5 patients with Shwachman-Diamond syndrome, 8 patients with myelodysplastic syndrome, and 74 patients with aplastic anemia of unknown cause classified as idiopathic aplastic anemia. In addition we measured telomere length in 12 patients with idiopathic pulmonary fibrosis and in 45 individuals with a de novo deletion of chromosome 5p including the TERT gene. Their telomere lengths were compared with those of 202 age-matched healthy controls. Moreover, mutations were screened in the genes associated with DC. In cases where a mutation was identified, telomere length and mutations were also examined in all the family members. Our results show that all patients with DC and bone marrow failure have very short telomeres far below the first percentile of healthy controls. Not all mutation carriers, including some carriers of apparently dominant mutations, have very short telomeres. What is more, very short telomeres could be found in healthy individuals in these families, some of whom were not mutation carriers. These findings indicate that in patients with BMF the measurement of telomere length is a sensitive screening method for DC, whether very short telomeres in this setting are also specific for DC remains to be determined. However, in contrast to a previous study, we find that telomere length does not always identify mutation carriers in the families of DC.

Beschreibung: Diamond-Blackfan anemia (DBA), a form of congenital red cell aplasia with marked clinical heterogeneity and increased risk of malignancy, has been associated with mutations in ribosomal protein (RP) gene RPS19 in 25% of probands and in RPS24 or RPS17 in ∼2% of patients. Thus, DBA appears to be a disorder of ribosome synthesis. To test the hypothesis that mutations in other RP genes may also cause DBA, we carried out direct sequencing of candidate RP genes. Genomic DNA samples from 96 unrelated DBA probands (14 familial and 82 sporadic cases) without RPS19 or RPS24 mutations were screened for mutations in RPS3a, RPS13, and RPS16 (previous studies revealed that RPs S19, S24, S3a, S13, and S16 are involved in binding of eIF-2 to the 40S subunit); RP genes L18, L13A, L36, L28, L18A, L40, S5, S9, S11, and S28 (located on chromosome 19); and RP genes, L5, L11, L22, S8, and S27 (on chromosome 1). PCR primers were designed to amplify the coding exons and intron/exon boundaries. We found multiple mutations in two RP genes, L5 and L11. Subsequently we sequenced these two genes in 42 additional DNA samples from DBA probands. In total, we screened 5′UTR, promoter and coding regions, and exon/intron boundaries of RPL5 and RPL11 in 138 DBA unrelated probands. We identified 14 mutations in RPL5 in 138 probands (∼10%), 13 of which are nonsense mutations, deletions or insertions of 1–5 nucleotides causing frameshift and premature termination. One missense mutation, 418G〉A, results in a G140S substitution. We found nine mutations in RPL11 in138 DBA probands (6.5%), including five acceptor or donor splice site mutations (introns 1–4) and four deletions or insertions of 1–4 nucleotides causing frameshifts (codons 32-120). None of these sequence changes were found on the NCBI (http://www.ncbi.nlm.nih.gov/SNP/) or the HapMap (http://www.hapmap.org/) SNP lists. Both genes, as well as RPL23 have recently been demonstrated by others to activate the p53 tumor suppressor protein by inhibiting MDM2-mediated p53 ubiquitination and degradation. Moreover, knockdown of any of these genes by siRNAs markedly reduced p53 induction by the ribosomal biogenesis stressor, actinomycin-D. These findings suggest that DBA patients with mutated L5 and L11 proteins may have inadequate p53 pathway activation and (consistent with clinical observations) be at increased risk for neoplasia. We are currently investigating the role of RPL5 and RPL11 mutations in ribosomal biogenesis and in the p53-mediated cell cycle arrest and apoptosis in DBA patients.

Beschreibung: Background: Diamond Blackfan anemia (DBA) is a rare disorder characterized by red cell aplasia, congenital anomalies and a predisposition to cancer. Corticosteroids and red cell transfusions are the mainstays of therapy. Hematopoietic stem cell transplantation is curative in DBA, however its role in these patients is controversial. Purpose: The purpose of this report is to review the outcome and treatment-related morbidity for HLA matched sibling versus alternative donor SCT for DBA patients enrolled in the Diamond Blackfan Anemia Registry (DBAR). Methods: The DBAR is a comprehensive database of patients with DBA who are enrolled after informed consent is obtained. The patients, their families, and their physicians complete a detailed questionnaire. A review of medical records and telephone interviews are performed to complete and clarify the information provided. Results: As of May 1, 2005, 420 patients have been enrolled in the DBAR, 36 of which have met the criteria for the diagnosis of DBA and have undergone an allogeneic HLA-matched sibling or alternative donor SCT. The median age at SCT for all patients was 7 years 10 months; 7 years 4 months versus 9 years 8 months for the 21 HLA-matched sibling and 15 alternative donor SCT, respectively. The major indication for SCT was transfusion dependence. In addition, two patients developed severe aplastic anemia and one significant thrombocytopenia. The majority of HLA-matched sibling transplants were done using a non-irradiation-containing conditioning regimen. One patient undergoing an HLA-matched umbilical cord SCT using a non-myeloablative conditioning regimen is engrafted but too early to evaluate. The majority of alternative donor transplants were performed using total body irradiation. One unrelated bone marrow transplant recipient also underwent non-myeloablative conditioning and is fully engrafted one year post-transplant. Sixteen of the 21 HLA-matched sibling donor transplants are alive and well, 4 of the failures occurring in patients older than 10 years of age (11, 12, 12 and 22 years) from interstitial pneumonia of unknown etiology, uncharacterized infection, veno-occlusive disease (VOD) of the liver and, in a heavily iron-overloaded patient with hepatitis C, adenovirus pneumonia, respectively. A 21 month old died from VOD. Of the 15 alternative donor SCT, one patient received bone marrow from a 3/6-mismatched sibling; another patient received a 5/6-mismatched parental bone marrow; 8 received unrelated bone marrow; 4 unrelated cord blood; and 1 unrelated peripheral blood stem cells. Three of these 15 patients are alive. The survival for allogeneic sibling versus alternative donor SCT is 72.7% ± 10.7% versus 19.1% ± 11.9% at greater than 5 years from SCT (p=0.01) (excluding a patient with osteogenic sarcoma diagnosed after SCT) or 17.1% ± 10.8% (including the osteogenic sarcoma patient, p=0.012). For patients under 10 years of age, the survival for allogeneic sibling versus alternative donor SCT is 92.3% ± 7.4% and 16.7% ± 14.8%, respectively, at greater than 4 years from SCT (p=0.01). Conclusions: The best outcomes for stem cell transplantation in DBA patients occur when performed prior to the age of 10 years, using HLA-matched sibling donors. Given the poor survival with alternative donor SCT, these transplants should be reserved only for those DBA patients with other complications requiring transplantation, i.e. aplastic anemia, red cell allosensitization or leukemia.

Beschreibung: Because of the variable and often protean manifestations of chronic GVHD, its diagnosis is difficult and based solely on clinical features. As part of a Children’s Oncology Group multi-institutional clinical trial (ASCT0031 - a randomized study of hydroxychloroquine plus standard therapy for children with newly-diagnosed extensive chronic GVHD), we prospectively evaluated laboratory markers that have been previously reported in single-institution studies to be associated with chronic GVHD. Fifty-four patients were enrolled: median age 12 (1–21) yrs: 37M/17F. The diagnosis of extensive chronic GVHD was made at a median of 210 days (range 82–914) following transplant. Eighteen of 45(40%) patients had eosinophilia (absolute eosinophil count (AEC)≥500/μL). Twelve of 43 (28%) patients had an anti-nuclear antibody (ANA) titer ≥1:80 and 9 of these (21%) had a titer ≥1:160. Finally, 11 of 48 (23%) patients had an elevated IgG level. At presentation, 67% of patients had one or more of eosinophilia, ANA≥1:80, and hyper-IgG. A Th-2 predominance is suggested by eosinophilia (↑ IL-5) and hyper-IgG and positive ANA (↑ IL-4). Of note, of 9 patients with sclerodermatous chronic GVHD, 6 had hyper-IgG and 5 had eosinophilia. These results support a Th2 predominance in a major subset of children presenting with chronic GVHD. These results emphasize the importance of including evaluation of eosinophil count, ANA, and IgG level as tests supportive of the diagnosis of chronic GVHD in children.

Beschreibung: Abstract 175 Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by anemia usually presenting during infancy or in early childhood, birth defects, and increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart and urinary system defects, that are present in ∼30–50% of patients. To date, DBA has been associated with mutations in seven ribosomal protein (RP) genes, S19, S24, S17, L35A, L5, L11, and S7 in about ∼43% of patients. To complete our large scale screen of RP genes in a DBA population, we sequenced 49 ribosomal protein genes in our DBA patient cohort of 117 probands. Together with our previous efforts of screening 29 RP genes, this completes the analysis of all known RP genes (excluding RPS4Y on the Y chromosome). Here we report probable mutations in four more RP genes, RPS10, RPS26, RPL19, and RPL26, and variants of unknown significance in six more, RPL9, RPL14, RPL23A, RPL7, RPL35, and RPL3. RPS10 and RPS26 are now confirmed DBA genes as we identified five mutations in RPS10 in five probands and nine mutations in RPS26 in 12 probands. We found single small frameshifting deletions in RPL19 and RPL26 in single DBA families. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knock-down of RPS10 or RPS26 expression with siRNAs, which indicate that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins. This brings to 11, the total number of RP genes mutated in ∼54% of patients with DBA, with nine more genes harboring variants of unknown significance requiring further study. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, congenital abnormalities, and cancer predisposition. Small ribosomal subunit genes RPS19, RPS24, and RPS17 are mutated in approximately one-third of patients. We used a candidate gene strategy combining high-resolution genomic mapping and gene expression microarray in the analysis of 2 DBA patients with chromosome 3q deletions to identify RPL35A as a potential DBA gene. Sequence analysis of a cohort of DBA probands confirmed involvement RPL35A in DBA. shRNA inhibition shows that Rpl35a is essential for maturation of 28S and 5.8S rRNAs, 60S subunit biogenesis, normal proliferation, and cell survival. Analysis of pre-rRNA processing in primary DBA lymphoblastoid cell lines demonstrated similar alterations of large ribosomal subunit rRNA in both RPL35A-mutated and some RPL35A wild-type patients, suggesting additional large ribosomal subunit gene defects are likely present in some cases of DBA. These data demonstrate that alterations of large ribosomal subunit proteins cause DBA and support the hypothesis that DBA is primarily the result of altered ribosomal function. The results also establish that haploinsufficiency of large ribosomal subunit proteins contributes to bone marrow failure and potentially cancer predisposition.