Identifying and predicting the pathogenic effects of a novel variant inducing severe early onset MMA: a bioinformatics approach.

BACKGROUND: Methylmalonic acidemia (MMA) is a rare metabolic disorder resulting from functional defects in methylmalonyl-CoA mutase. Mutations in the MMAB gene are responsible for the cblB type of vitamin B12-responsive MMA. RESULTS: This study used Whole-exome sequencing (WES), Sanger sequencing, linkage analysis, and in-silico evaluation of the variants' effect on protein structure and function to confirm their pathogenicity in a 2-day-old neonate presenting an early-onset metabolic crisis and death. WES revealed a homozygous missense variant on chromosome 12, the NM_052845.4 (MMAB):c.557G > A, p.Arg186Gln, in exon 7, a highly conserved and hot spot region for pathogenic variants. After being confirmed by Sanger sequencing, the wild-type and mutant proteins' structure and function were modeled and examined using in-silico bioinformatics tools and compared to the variant NM_052845.4 (MMAB):c.556C > T, p.Arg186Trp, a known pathogenic variant at the same position. Comprehensive bioinformatics analysis showed a significant reduction in the stability of variants and changes in protein-protein and ligand-protein interactions. Interestingly, the variant c.557G > A, p.Arg186Gln depicted more variations in the secondary structure and less binding to the ATP and B12 ligands compared to the c.556C > T, p.Arg186Trp, the known pathogenic variant. CONCLUSION: This study succeeded in expanding the variant spectra of the MMAB, forasmuch as the variant c.557G > A, p.Arg186Gln is suggested as a pathogenic variant and the cause of severe MMA and neonatal death. These results benefit the prenatal diagnosis of MMA in the subsequent pregnancies and carrier screening of the family members. Furthermore, as an auxiliary technique, homology modeling and protein structure and function evaluations could provide geneticists with a more accurate interpretation of variants' pathogenicity.

Characterization and haplotype study of 6 novel STR markers related to the KCNQ1 gene in heterogeneous cardiovascular disorders in the Iranian population

Background/aim: The KCNQ1 gene has a significant role in long QT syndrome, Jervell and Lange-Nielsen syndrome, familial atrial fibrillation, and short QT syndrome. Analyzing such heterogeneous disorders, six novel short tandem repeat (STR) markers around the KCNQ1 gene were found and evaluated in a healthy population, and other statistical traits of the markers were detected. Materials and methods: Using Tandem Repeats Finder (TRF) and Sequence-Based Estimation of Repeat Variability (SERV) software, STR markers were detected with valid tetra- and pentanucleotide repeats. The markers were investigated for a total of 60 unrelated Iranian healthy individuals and analyzed using GenAlEx 6.502 and Cervus 3.0.7. Results: A total of 77 haplotypes was detected, of which 25 haplotypes were unique and the others occurred at least two times. The number of haplotypes per locus ranged from 7 to 18 with the highest frequency of 69.2%, and the observed heterozygosity was calculated as 0.589. The power of discrimination ranged from 0.70 to 0.96. Five of the markers meet Hardy­Weinberg equilibrium. Conclusion: A novel panel of STR markers was described with high allele heterozygosity and segregation in every locus, which may lead to faster and more credible recognition of the disease-inducing KCNQ1 gene and allow it to be used for human identity testing and prenatal diagnosis.

Identification of a Novel KCNQ1 Frameshift Mutation and Review of the Literature among Iranian Long QT Families

Background: Long QT syndrome (LQTS) is characterized by the prolongation of QT interval, which results in syncope and sudden cardiac death in young people. KCNQ1 is the most common gene responsible for this syndrome. Methods: Molecular investigation was performed by DNA Sanger sequencing in Iranian families with a history of syncope. In silico examinations were performed for predicting the pathogenicity of the novel variant. Results: A novel homozygous KCNQ1 frameshift mutation, c.1426_1429delATGC (M476Pfs*4), was identified, and then the current literatures of five patients were reviewed regarding the LQTS. Conclusion: The novel frameshift mutation has been reported for the first time among the Iranian population. Our finding along with the case series study of LQTS patients illustrates the importance of genetic and case series in precise detection of the frequency of LQTS carriers.

Molecular Basis of α-Thalassemia in Iran

Alpha-thalassemia (α-thal) is probably the most prevalent monogenic condition in the world. Deletions are the most common types of mutations in α-thal, followed by point mutations and small insertion/deletion. In the context of national screening program for prevention of thalassemia and hemoglobinopathies in Iran, α-thal carriers have come to more attention. Therefore, the frequency and distribution of α-globin mutations in various regions of the country have been studied in recent years. A comprehensive search was performed in PubMed, Scopus, and national databases for finding reports on mutation detection in α-thal carriers and HbH disease with Iranian origin. The mutation data of 10849 α-thal carriers showed that -α3.7 and α-5NT were the most common deletional and nondeletional mutations, respectively. In HbH disease cases, the -α3.7/--MED was the most prevalent genotype. Overall, 42 different mutations have been identified in α-globin cluster reflecting the high heterogeneity of the mutations in Iranian populations.

Molecular Characterization of δ-Thalassemia in Iran.

δ-Thalassemia (δ-thal) (OMIM #142000) resulting from mutations on the HBD gene usually has no clinical consequences. However, it may cause the misdiagnosis of ß-thalassemia (ß-thal) carriers by lowering the Hb A2 level to the normal range. Therefore, a study for δ-thal should be considered as a step in the detection of at-risk couple in our region. The aim of the present study was to characterize the mutations of the HBD gene in ß-thal carriers with normal Hb A2 levels, and also in normal individuals with Hb A2 of less than 2.0%. Four ß-thal carriers with normal Hb A2 and 39 individuals with Hb A2 of less than 2.0% were enrolled. Genomic DNA was extracted by the salting out method and the HBD gene was investigated by polymerase chain reaction (PCR) and direct DNA sequencing. Hb A2-Yialousa (HBD: c.82 G > T) was the most common variant found in the HBD gene, but the following mutations were also found: Hb A2-NYU (HBD: c.39 T > A), Hb A2-Coburg (HBD: c.350 G > A), Hb A2-Etolia (HBD: c.257 T > C), Hb A2-Fitzroy (HBD: c.428 C > A) and the δ-IVS-I-5 (G > T) (HBD: c.92 + 5 G > T). One case was a compound heterozygote for δ-IVS-I-5/Hb A2-Fitzroy. The results of this single center study suggest that the mutations in the HBD gene in the Iranian population are heterogeneous and should be considered in genetic counseling of families.

Prenatal diagnosis of ß-thalassemia in twin pregnancies in Iran.

OBJECTIVE: Prenatal diagnosis of ß-thalassemia carrier couples has helped to prevent bearing affected children. Among 177 couples referred to our laboratory for prenatal diagnosis, 14 mothers had twin pregnancies.  METHODS: By using direct and indirect methods, we determined their mutations and linkage analysis using polymorphic markers (restriction fragment length polymorphism [RFLP]).  RESULTS:  It was shown that in five families both fetuses were heterozygote carriers. In another five families, one fetus was normal and the other one was carrier. In two families, one fetus was affected and the other one was heterozygous carrier; in one case one fetus was affected and the other one was homozygote normal. In the last family both fetuses were homozygote normal. If all fetuses were fraternal then one would expect to see seven homozygote normal and the same number affected, and 14 carriers. CONCLUSION: Our results indicated that at least in cases where both fetuses had identical genotypes, then they may be identical twins. Molecular testing indeed showed that in three cases the twins were identical. Another point is that in three cases, one of the twin fetuses was affected and the other one was either normal or heterozygote in which only the affected fetuses were aborted by the specialist.

The carrier frequency of α-globin gene triplication in an Iranian population with normal or borderline hematological parameters.

The -α(3.7) rightward deletion is the most frequent α-globin mutation worldwide, while frequencies of the ααα(anti 3.7) triplication are only sporadically known. Carriers of the ααα(anti 3.7) triplication show no clinical symptoms or significant hematological changes, but co-inheritance with ß-thalassemia (ß-thal) has been reported to worsen the clinical and hematological features of the patient as well as the trait. We have screened the α-globin gene rearrangements of 280 individuals with normal hematological indices and 117 persons with borderline hematological parameters. We used multiplex polymerase chain reaction (m-PCR) and multiplex ligation-dependent probe amplification (MLPA) technology to detect triplications and quadruplications. Only the ααα(anti 3.7) triplication was observed. The carrier frequency in the first group was 2.14% and in the second group 1.7%. No phenotype aggravation was noticed in two carriers of ß-thal and the ααα(anti 3.7) triplication, while a mild ß-thalassemia intermedia (ß-TI) was observed in a ß-thal carrier with six α-globin genes. Due to the high consanguinity in the country, homozygosity for the ααα(anti 3.7) triplication and for other rearrangements can be expected. Therefore, an accurate determination of the frequencies and a routine control for these mutations is essential for a correct genotype-phenotype prediction during genetic counseling for ß-thal.

First report on the co-inheritance of beta-globin IVS-I-5 (G-->C) thalassemia with delta globin CD12 {Asn-->Lys (AAT-->AAA)}HbA2-NYU in Iran.

BACKGROUND: Co-inheritance of ß- and δ-globin mutations in Iran is not uncommon. This situation may interfere with correct diagnosis and genetic counseling of α- and ß-thalassemia in screening programs. Here we report the co-inheritance of ß- and δ-globin gene mutations in an individual with microcytosis, hypochromia and a normal hemoglobin A2 (HbA2) level. METHODS: Genomic DNA extraction, amplification refractory mutation system (ARMS) polymerase chain reaction and direct DNA sequencing of δ- and ß-globin genes were exploited for detection of the mutations in these two genes in an individual with low hematological indices and normal HbA2. RESULTS: ARMS-PCR technique revealed the ß(+) IVSI-5 (G to C) mutation and direct DNA sequencing of the δ-globin gene detected a previously reported delta codon 12 (AAT-->AAA) HbA2-NYU. This study reports HbA2-NYU in association with the ß IVSI-5 (G to C) mutation in Iran. DISCUSSION: This report emphasizes that normal HbA2 expression in a ß-goblin carrier is due to mutation in the δ-globin gene and may cause misdiagnosis of thalassemia.

Co-inheritance of hemoglobin D and ß-thalassemia traits in three Iranian families: clinical relevance.

Here we report the result of three cases referred to our lab that had a combination of ß-thalassemia and hemoglobin D (Hb D) traits. These individuals had no symptoms of profound anemia and hematological indices were similar to that of a ß-thalassemia heterozygote. In all three cases, the Hb D level was elevated and no HbA was detected electrophoretically. The electrophoresis pattern suggested that all cases were homozygotes for Hb D. PCR followed by digestion with EcoRI and sequencing of the ß-globin gene confirmed the presence of Cd 121 GAA>CAA in the heterozygous form with another ß-globin mutation. In all cases, the mutations in the ß-globin gene were detected by ARMS PCR technique and they were either IVSII-I or IVSI-5. Hematological studies of the family members showed that thalassemia which caused the mutations and Hb D were in the trans position.

Identification of a novel δ-globin gene mutation in an Iranian family.

δ-Thalassemia (δ-thal) has no clinical symptoms, but its coinheritance with ß-thal may cause misdiagnosis, especially in countries with a high prevalence of ß-thal where prevention programs have been implemented. The molecular basis of most ß-thal syndromes have been defined, while the spectrum of mutations causing δ-thal have not been well characterized. A couple was referred to us for thalassemia molecular screening. Since she had rather low values of Hb A2 and normal Hb F, her δ-globin gene was amplified and directly sequenced. We found two different mutations on her δ-globin genes: HBD: c.92+5G>T/HBD:c.428C>A. The c.92+5G>T mutation has not been previously reported. Two different mutations in trans may explain the reduced Hb A2 level.

A novel polymorphism causes a different restriction pattern by RsaI in the beta-globin gene cluster: application in prenatal diagnosis.

beta-Thalassemia (beta-thal) is a major health problem in Iran and the incidence of carriers is around 3-4%. The disease is caused by heterogeneous mutations in the beta-globin gene and is characterized by hypochromic microcytic anemia. The human beta-globin complex spans a region of 70 kb and contains over 20 restriction fragment length polymorphisms (RFLPs). At least nine RFLP markers including RsaI/beta in the beta-globin gene cluster have been routinely exploited for prenatal diagnosis. Here, we report a novel polymorphism upstream of the beta-globin gene characterized by RsaI digestion. Sequencing of a fragment containing this area showed a nucleotide change (T>C) at position -223 upstream of the beta-globin gene. This change could interfere with precise interpretation of the RsaI digestion pattern in linkage analysis and prenatal diagnosis of beta-thal.