Madhumita Roy Chowdhury

Prenatal diagnosis in hemophilia A using factor VIII gene polymorphism—Indian experience

The heterogeneous nature of the mutations, the size, and the complexity of the factor VIII gene makes direct mutation analysis in hemophilia A families in India an option that is not very feasible and practical. Thus, carrier screening and prenatal diagnosis of hemophilia A often depends on haplotype analysis using restriction fragment length polymorphisms (RFLP) and short tandem repeat (STR) markers to track the defective factor VIII gene within a family. The main objective of this present study was to assess the utility of using polymerase chain reaction (PCR)-based five polymorphic markers: four intragenic Hind III, Bcl I, intron 13, and intron 22 STRs and one extragenic marker St14 in prenatal diagnosis. Forty-one chorionic villus samples (CVS) were studied from 41 families with a history of hemophilia A. PCR and RFLP were used for screening. Intron 22 STR showed the highest informativeness (60.9%), followed by Hind III (51.2%), Bcl I (46.3%), & intron 13 STR (51.2%); the extragenic marker St14 (VNTR) was informative in 46.3% of families. Linkage analysis, with the combined use of these five PCR-based polymorphic markers, gives good informativeness of 87.8% in the Indian population. Of the 41 CVS tested, 21 were found to be male fetuses and of these 13 were found likely to be affected with hemophilia A. Only in 12.2% of the families were none of the markers informative.

Fragile X screening for FRAXA and FRAXE mutations using PCR based studies: Results of a five year study

Background: Fragile X syndrome is the most common cause of inherited X-linked mental retardation. It is due to a mutation in a gene on X chromosome leading to hyper-expansion of a trinucleotide repeat sequence. The two most common Fragile sites with clinical significance are FRAXA at Xq27.3 comprising CGG repeat and a more distal FRAXE associated with amplification of a GCC repeat, located at Xq28. The frequency of occurrence of Fragile X syndrome is estimated to be 1/4000 male births. Screening of referrals for the mutations associated with the Fragile X syndrome constitutes a significant workload in many genetic laboratories. Aims: The aim of the present study was to establish the use of PCR based simple and rapid method of initial screening of samples, so that only a minority of samples tested positive with the above methods need to be screened by Southern blotting which is more time consuming and involves use of radioactive material. materials and Methods: Study includes 294 patients with mental retardation. DNA extracted from blood was used for simultaneous amplification of the triplet repeat sequences at the FRAXA and FRAXE loci. Secondly samples from females were analyzed for heterozygosity of normal FRAXA allele. For confirmation of the presence of an expanded FRAXA allele in all the male positive cases, Southern blot hybridization was carried out. PCR based assay was done to detect methylation of the CpG island upstream of the FMR-1 gene. Results: Out of the 294 cases 23 (7.8%) were found to be having full mutation (FM) for FRAXA (21 males, 1 female & 1 male with mosaic FM/PM) and 13 females as having premutation (PM). All these 36 cases were confirmed by Southern blotting using appropriate probes. Among the females the heterozygosity for FRAXA allele was found to be 46%. Conclusion: Non-radioactive PCR methods are efficient and rapid test for intial screening of samples for the presence of FRAXA and FRAXE mutations. Since a large majority of referrals do not have Fragile X, this economical and reliable method reduces the number of samples needing Southern blotting.

Application of chromosomal microarrays in the evaluation of intellectual disability/global developmental delay patients - A study from a tertiary care genetic centre in India.

Intellectual disability (ID)/Global developmental delay (GDD) is a diverse group of disorders in terms of cognitive and non-cognitive functions and can occur with or without associated co-morbidities. It affects 1-3% of individuals globally and in at least 30-50% of cases the etiology remains unexplained. The widespread use of chromosomal microarray analysis (CMA) in a clinical setting has allowed the identification of submicroscopic copy number variations (CNVs), throughout the genome, associated with neurodevelopmental phenotypes including ID/GDD. In this study we investigated the utility of CMA in the detection of CNVs in 106 patients with unexplained ID/DD, dysmorphism with or without multiple congenital anomalies (MCA). CMA study was carried out using Agilent 8×60K chips and Illumina Human CytoSNP-12 chips. Pathogenic CNVs were found in 15 (14.2%) patients. In these patients, CNVs on single chromosome were detected in 10 patients while 5 patients showed co-occurrence CNVs on two chromosomes. The size of these CNVs ranged between 322kb to 13Mb. The yield of pathogenic CNVs was similar for both mild and severe ID/GDD cases. One patient described in this paper is considered to harbour a likely pathogenic CNV with deletion in 17q22 region. Only few cases have been described in literature for 17q22 deletion and patient reported here was found to have an atypical deletion in 17q22 region (Case 90). This study re-affirms the view point that CMA is a powerful diagnostic tool in the evaluation of idiopathic ID/GDD patients irrespective of the degree of severity. Identifying pathogenic CNVs helps in counseling and prenatal diagnosis if desired.

Inherited 5p deletion syndrome due to paternal balanced translocation: Phenotypic heterogeneity due to duplication of 8q and 12p

5p deletion syndrome or Cri du Chat syndrome is a autosomal deletion syndrome, caused by the de novo deletion of chromosome 5p in the majority of the cases. Clinical features include developmental delay, microcephaly, subtle facial dysmorphism and high-pitched cry. With the advent of newer techniques such as multiplex ligation-dependent probe amplification, rapid diagnosis is possible and chromosomal microarray helps in accurate delineation of the breakpoints. In this study, we characterized probands from two Indian families who had duplication of another chromosome in addition to deletion of 5p region. In the first family, two females of 3 and 5 yr of age had deletion of 5p15.33p15.2 (14.7 Mb) and duplication of 8q24.21q24.3 (15.4 Mb). Proband in the second family was a 2-year-old female and had deletion of 5p15.33p14.3 (22.55 Mb) along with duplication of 12p13.33p13.31 (7.7 Mb). In both the families, father was balanced translocation carrier of the chromosomes involved. Patients in family 1 had overwhelming features of 5p deletion while patient in family 2, besides having features of 5p deletion, showed many features of 12p duplications. Prenatal diagnosis was possible in both the families. To the best of our knowledge, this is the first detailed molecular cytogenetic analysis and prenatal diagnosis report of 5p deletion syndrome from India.

Novel missense mutation in the coagulation factor IX catalytic domain associated with severe haemophilia B – Factor IXDelhi

Summary.  Factor IX is a vitamin K‐dependent serine protease, which exists as a zymogen in the blood. On activation to factor IXa, by factor XIa or tissue factor–factor VIIa complex, it forms tenase complex with factor VIIIa, in the presence of Ca2+. This tenase complex enzymatically converts factor X to factor Xa, thereby bringing about the coagulation cascade. Mutations in factor IX gene have been shown to cause haemophilia B, which is inherited as an X‐linked recessive disorder. Herein we report a novel missense mutation at the nucleotide position 30829‐T > A in the exon 8 of factor IX gene. This transversion leads to the substitution of histidine 236 to glutamine. This resulting abnormal protein has been named factor IXDelhi. Molecular modelling was performed to predict the molecular pathology of this mutation. We predict that this change in the catalytic domain may affect the surface loop that accommodates Ca2+, thereby leading to severe bleeding disorder.

Application of whole exome sequencing in elucidating the phenotype and genotype spectrum of junctional epidermolysis bullosa: A preliminary experience of a tertiary care centre in India

BACKGROUND Junctional epidermolysis bullosa (JEB) is a diverse group of genodermatoses associated with extreme skin fragility. Despite several well-characterized genetic studies, molecular diagnosis of this heterogeneous group is still challenging. Recent advances in the field of genomics have seen the successful implementation of whole exome sequencing (WES) as a fast and efficient diagnostic strategy in several genodermatoses. OBJECTIVE In view of the scarcity and need of molecular studies for JEB in India, we sought to explore the potential of WES in understanding the mutational spectrum of this rare, in certain subtypes lethal, sub-group of EB. METHODS WES was performed using genomic DNA from each case of EB, followed by massively parallel sequencing. Resulting reads were mapped to the human reference genome hg19. Sanger sequencing subsequently confirmed the potentially pathogenic mutations. RESULTS Overall, four unrelated families (6 patients) of JEB with a highly variable clinical presentation including a rare case of LOC syndrome were studied. WES revealed 4 variations in 3 genes (LAMA3, LAMB3 and COL17A1) that are implicated in JEB. None of the variations were recurrent. In addition we proposed the probable molecular consequence of a missense mutation on the structure-function relationship of lamininβ3 protein through computational modeling studies. CONCLUSIONS Being the first report documenting the phenotype-genotype correlations of JEB patients from India, our preliminary experience with WES is clearly encouraging and serves as a nidus for future large-scale molecular studies to actively identify and understand JEB patients in Indian population.

Role of Cytogenetics and Molecular Genetics in Human Health and Medicine

Abstract The complexities of cytogenetics and molecular genetics have been described in easy-to-understand language. The basics of cytogenetics and molecular genetics have been explained, and examples have been used to demonstrate a correlation in a number of human diseases.

Validation of Polymerase Chain Reaction–Based Assay to Detect Actual Number of CGG Repeats in FMR1 Gene in Indian Fragile X Syndrome Patients

Molecular genetic testing for fragile X (FX) is complicated due to the large variation in the size of CGG expansion. The aim of this study was to apply this new technique using AmplideX FMR1 PCR assay, which is considered a better diagnostic tool for detecting expanded alleles in Indian population. The primary objective was to identify the carrier status of females and to correlate the instability of premutation alleles in females with the repeat sizes. 24 children with FX based on rapid PCR and 29 female relatives of these patients were included. Out of the 29 females screened, those whose child (or children) was affected by FX, were all premutation carriers confirming their role in transmission. The smallest PM allele that expanded into FM in the next generation was 78 repeats and the smallest PM allele detected was 63 repeats, and when transmitted from mother to offspring remained in the premutation range. In 4 families, the repeat size of the allele reduced from PM to normal repeat numbers in their daughters and in 1 case to borderline PM range. Thus, apart from the reduced turnaround time, this PCR based assay offers advantage by its sensitivity to detect CGG repeats in the intermediate region and lower range of premutation alleles. It also provides added information of AGG interruptions, which may have an impact on the counseling of women with intermediate and PM alleles.

Frequency of primary mutations of leber's hereditary optic neuropathy patients in North Indian population

Purpose: Lebers hereditary optic neuropathy (LHON) is an inherited optic neuropathy characterized by subacute painless vision loss. The majority of LHON is caused due to one of the three primary mutations in the mitochondrial DNA (m.G3460A, m.G11778A, and m.T14484C). The frequency of these mutations differs in different populations. The purpose of this study is to observe the frequency of three common primary mutations in the North Indian population. Methods: Forty LHON patients within the age group of 10–50 years underwent molecular testing for primary mutations. For two patients, testing for mother and other siblings was also carried out, using bidirectional sequencing. Results: A total of 11 out of 40 (27.5%) patients were found to be carrying m.G11778A mutation. Siblings of two probands were also positive for the same mutation. In one family, two primary mutations (m.G11778A and m.T14484C) were found in the proband and in the mother as well. Conclusion: In this study, 27.5% mutation was detected in North Indian LHON families. These results suggest that m.G11778A mutation is more frequent in this population. The results of the present study are compatible with studies of an Asian population and Northern European population.

Identification of a novel homozygous mutation in transmembrane channel like 1 (TMC1) gene, one of the second-tier hearing loss genes after GJB2 in India

Background & objectives: Hearing impairment is a common and heterogeneous sensory disorder in humans. Among about 90 genes, which are known to be associated with hearing impairment, mutations in the GJB2 (gap junction protein beta 2) gene are the most prevalent in individuals with hereditary hearing loss. Contribution of the other deafness-causing genes is relatively poorly understood. Here, we present our findings on two families with transmembrane channel like 1 (TMC1) gene variants of the 47 families with nonsyndromic hearing loss (NSHL) studied. Methods: Forty seven families including 26 consanguineous families with at least two hearing impaired children and one normal hearing child and 21 non-consanguineous families having at least three hearing impaired children and one normal hearing child were enrolled for this study. Genetic linkage studies were carried out in 41 families that were GJB2 (Connexin 26) negative. Seven polymorphic short tandem repeat markers at the DFNB7/11 locus were studied employing fluorescently labelled markers. Results: A novel homozygous missense mutation c.1283C>A (p.Ala428Asp) was identified co-segregating with hearing loss. This change results in substitution of a highly conserved polar alanine to a charged aspartic acid and is predicted to be deleterious. In addition, a previously reported nonsense mutation, p.R34X in TMC1, was found. Interpretation & conclusions: While mutations in TMC1 are not as common a cause of NSHL as those in GJB2, TMC1 should be considered for diagnostic investigations in cases of NSHL in GJB2-negative families.