ALBERT

Description: In the present study, molecular pathologies of 8 patients from unrelated 4 consanguineous and one non-consanguineous families were characterized; 4 novel and 1 previously reported homozygous mutations were identified in the P5N-1 gene. 1) A frameshift 700–701InsA mutation in exon 9 was identified in a 40 years old patient who has been followed as hereditary spherocytosis (HS) for many years because of marked spherocytes and highly increased osmotic fragility. He underwent splenectomy (11yr) and cholecystectomy (26yr). Afterwards, Hb level stayed between 8.5–11g/dl. Co-existence of Gilbert syndrome 7/7 TATA repeat genotype accounts for very high serum bilirubin level (18–28mg/dl). 2) A splicing mutation (IVS8+1 G〉A) was found in a 30 years old patient who was followed as HS due to associated spherocytosis and slightly increased osmotic fragility. She received blood transfusions regularly, underwent splenectomy and cholecystectomy (14yr). Afterwards Hb stayed at 9.6–10.5 g/dl. Co-existence of Gilbert 7/7 genotype accounts for the high bilirubin (15mg/dl). 3) A splicing (IVS7+1 G〉A) and a silent (T275C in exon 6) mutations were detected in 2 siblings. Despite non-consanguinity, identification of two homozygous mutations indicated the presence of a common ancestor. The girl received transfusion several times, underwent splenectomy and cholecystectomy(18yr). The boy, diagnosed in family survey (2yr) and underwent splenectomy (10yr), currently has highly increased osmotic fragility. Both had few acanthocytic spherocytes. After splenectomy Hb stayed around 11 gr/dl, bilirubin around 5–6 gr/dl in both. 4) A missense T220C mutation in exon 5 (C74R) was identified in 24 and 21 years old sisters. The elder had iron deficiency anemia and the younger diagnosed in a family survey had retinitis pigmentosa. Hb were around 10g/dl, bilirubin around 2.5–6 mg/dl, few acontocytic spherocytes observed in both. 5) A nonsense T543G mutation (Y181X) in exon 8 was detected in 14 and 7 years old brothers. The mutation was reported previously in another Turkish family, however, haplotype analysis failed to indicate founder effect. The older required blood transfusions while hospitalized several times due to recurrent attacks of cerebral infarcts. Hb fluctuated between 6.5–10 g/dl, bilirubin was around 3 mg/dl. He underwent splenectomy recently (17yr). The younger had iron deficiency anemia, Hb was between 7.2–9.9 gr/dl. Few aconthocytic spherocytes were observed in both. All mutations were screened in at least 100 individuals from a healthy Turkish population and no mutation was detected. In conclusion: 1- The mutation spectrum of P5N-1 gene is quite heterogeneous in Turkish population (7 different mutations in 9 unrelated families so far). 2- P5N-1 deficiency is classified as non-spherocytic hemolytic anemia, however, all patients of this study more or less had spherocyte in the peripheral blood smear. In addition, some patients even had increased erythrocyte osmotic fragility emerging as confounding factor in diagnosis. Taken together, there is a reason to suggest that the possibility of P5N-1 deficiency should be considered in at least some of the patients diagnosed as HS. 3- The co-existence of Gilbert disease could modify the clinical presentation of the disease. This study was supported by Hacettepe University Research Fund (02G116).

Description: Defects in erythrocyte ankyrin are the most common cause of typical, dominant hereditary spherocytosis (HS). Detection of ankyrin gene mutations has been complicated by allelic heterogeneity, large gene size, frequent de novo mutations, and associated mRNA instability. Using denaturing high-performance liquid chromatography (DHPLC)–based mutation detection, a mutation in the splice acceptor of exon 17 was discovered in a Turkish family. Reticulocyte RNA and functional minigene splicing assays in heterologous cells revealed that this mutation was associated with a complex pattern of aberrant splicing, suggesting that removal of intron 16 is important for ordered ankyrin mRNA splicing. As predicted by clinical, laboratory, and biochemical studies, the parents were heterozygous and the proband was homozygous for this mutation. These data indicate that DHPLC offers a highly sensitive, economic, and rapid method for mutation detection and, unlike previously suggested, homozygosity for a mutation associated with dominant ankyrin-linked HS may be compatible with life.

Description: Electrophoresis of red cell hexokinase in agarose electrophoresis revealed two major (1 and 2) and two minor (3 and 4) bands. Platelet and leukocyte hexokinase patterns differed from those of red cells. There was a strong band 1, but considerably faster bands termed 5, 6 and 7 were also observed which were sensitive to changes in glucose concentration. The presence of contaminating leukocytes can significantly alter the electrophoretic pattern of "red cell" hexokinase activity. Bands 2, 3 and 4 of red cells appeared to be synthesized independently of band 1 and absence of band 1 did not effect either red cell metabolism or survival. Absence of bands 2, 3 and 4 may be associated with hemolytic anemia, decreased erythrocyte hexokinase activity and decreased erythrocyte glycolysis. Young red cells had increased activity of all bands, particularly band 2. No influence of hemoglobin type on hexokinase patterns was observed, nor was there any selective influence of cell storage, medium glucose, or 2-mercaptoethanol on individual bands. None of the various isoenzyme patterns were associated with abnormal hexokinase kinetics.

Description: Ataxia telangiectasia (AT) and Fanconi anemia (FA) are rare, clinically distinct autosomal recessive disorders characterized by genomic instability and predisposition to cancer. In recent years, it was demonstrated that ATM and FA genes function in parallel and interrelate in DNA damage response pathways to maintain genomic integrity and cell viability. To date, no case or a family with these two genomic instability disorders with proven genetic defects, clinical and laboratory features has been reported. In this study, we characterize the molecular pathologies of a prototype family exceptionally harboring both AT and FA patients. Among the 5 ofsprings of a consanguineous parents of first degree cousin marriage, 1 male was diagnosed as AT, 1 male and 1 female as FA, 1 male and 1 female appeared to have none of these two disorders. Family history revealed that a 1.5 years old female child was deceased, and the mother had 1 early abortion and 1 intrauterine death at the 8th months of pregnancy. During the follow up period of 5 years, the patient with AT and the male patient with FA were lost due to the complications of the related disorders. Linkage analysis of DNA samples of the family members for FA led to the assignment of the family to complementation group A. The mutation analysis of 43 exons of FANCA gene revealed novel homozygous co-deletions of 10 (1361–1370del) and 1 (1374delC) nucleotides at the 5′ end of exon 15 leading to 3 aminoacid deletions and frameshift. Screening of the family members for the mutation revealed that 2 FA patients were homozygous and the parents and the patient with AT were heterozygous for the mutation while other 2 siblings were non-carriers for FA. The mutation was not detected among 200 chromosomes from normal population. On the other hand, linkage analysis of the family for AT with the intra ATM gene microsatellite markers showed that AT patient was homozygous while other family members were heterozygous for the common haplotype observed in parents. In this patient, sequencing of the 62 coding exons of the ATM gene led to the identification of a homozygous C to T substitution (8977C〉T) at exon 61. This alteration resulted in replacement of Arginin with termination at codon 2993 (R2993X) leading to 63 aminoacid truncation of the ATM protein. Consistent with the linkage results, all the other family members including 2 FA patients were heterozygous for the mutation. The results of this study showed that the state of homozygosity for AT with a coexistent heterozygosity for FA or homozygosity for FA with a coexistent heterozygosity for AT are compatible with life. This observation indicates that even in the presence of null mutations, such combinations do not alter phenotypic expression of the homozygous states of neither AT nor FA. However, in this family with 5 living siblings, absence of any case coinciding homozygous mutations for both disorders may indicate that such possibilities are not compatible with life. Even though there is no laboratory evidence due to absence of fetal study, presence of maternal history of an abortion and an intrauterine death may be considered as evidences in supporting assumption above.

Description: Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive immune dysregulation disorder associated with Perforin, Munc13-4 and Syntaxin 11 genes. The mutations in the Perforin gene are the most common cause of the disease. Among these mutations, the role of the Alanin91Valin (A91V) alteration in the pathogenesis of the disease has long been controversial. Even though this alteration can be considered as a polymorphism based on its high frequency in normal population (〉3.7%) and homozygous existence in some healthy individuals, it is also considered as a genetic risk factor depending on its much higher frequency observed in FHL patients (22.7%) and compound heterozygous existence with other disease causing mutations in the Perforin gene in some FHL patients. Contrary to the previous publications concerning with the co-existence of heterozygous A91V with homozygous mutations in other described FHL genes, there has been no reports on homozygous co-existence of A91V up until this communication where we present the interesting results of a study which shed light not only on the role of A91V in development of FHL, but also on the etiopathogenesis of genetic diseases in general. The subject of the study was a 12 year old female patient who was a product of a first degree consanguineous marriage. Initial diagnosis was lymphomatoid granulomatosis due to the presence of symptoms associated solely with central nervous system. The correct diagnosis could be made 1 year later upon development of systemic findings of FHL. There was no history of similar disorder in the family. Linkage analysis in the family revealed homozygosity for both Perforin and Munc13-4 genes in the patient and for only Munc13-4 gene in one of the asymptomatic sibling who was heterozygous for Perforin gene. Syntaxin 11 gene was excluded in this analysis. Detected merely in the patient was a homozygous A91V substitution (272C〉T) in the sequencing of the Perforin gene. Sequencing of the complete coding (32 exons) and the flanking sequences, on the other hand, led to the identification of a homozygous three nucleotide in-frame deletion (2135-2137delTCG) in exon 23 of Munc13-4 gene. This novel mutation resulted in the replacement of nonpolar two aminoacids (Ile-Gly) at positions 712-713 with a polar single aminoacid (Ser). It is plausible that the substitution of highly conserved two aminoacids, especially one (Ile) playing important role in the stability of proteins, with a hydrophilic one would alter the three dimensional structure and the stability of the protein, and would lead to FHL. Ironically, however, an asymptomatic sibling who is currently 22 year old was also homozygous for the mutation. This finding led to the assumption that the Munc13-4 mutation alone may not be sufficient for the development of the disease, but may be a genetic risk factor requiring co-existence of additional homozygous genetic risk factor situated in another FHL gene. If this is the case, it is reasonable to state that homozigosity for A91V in Perforin as well as homozygosity for the 2135-2137del mutation in Munc13-4 is a strong genetic susceptibility factor contributing significantly to the pathogenesis of the disease when they are co-existed. However, this notion could be valid as long as the sibling with homozygous Munc13-4 mutation stays asymptomatic. On the other hand, late onset and atypical presentation in the propositus may indicate that the homozygous co-existence of both alterations is not associated with serious clinical course of the disease as far as the presenting age of the disease is concerned.

Description: Pyrimidine 5′ nucleotidase-I (P5N-I) deficiency is a rare autosomal recessive disorder associated with hemolytic anemia, marked basophilic stippling, and accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. Recently, the structure and location of the P5N-I gene have been published. This paper presents the results of a study characterizing the molecular pathologies of P5N-I deficiency in a total of 6 Turkish patients from 4 unrelated families of consanguineous marriages. Mutation analysis in the P5N-I gene led to the identification of 3 novel mutations in these patients. In 4 patients from 2 families, a homozygous insertion of double G at position 743 was detected in exon 9 (743-744insGG), leading to premature termination of translation 23 bp downstream. In one family, a homozygous T to G transition at position 543 (543T〉G) in exon 8 resulted in the replacement of tyrosine (Tyr) with a stop codon (Tyr181Stop). In another family, a homozygous insertion of a single A in exon 7 (384-385insA) created a stop signal at the codon nearby. In all families, the parents were heterozygous for the relevant mutations. None of these changes was detected in 200 chromosomes from a healthy Turkish population. These mutations were not correlated with any particular phenotype.