Mahrukh H. Zargar

High prevalence of factor V Leiden and prothrombin G20101A mutations in Kashmiri patients with venous thromboembolism

AIM The genetic variants of the factor V (G1691A), prothrombin (G20210A) and MTHFR (C677T) genes have been widely implicated as inherited risk factors for developing venous thrombosis. This study was undertaken to reveal the frequency of these mutations in Kashmiri patients with venous thromboembolism. METHODOLOGY A case-control study was designed with 250 VTE patients and 250 healthy controls. The mutations were analysed using ARMS-PCR and PCR-RFLP approach. RESULT The factor V Leiden G1691A mutation was found in 17/250 (6.8%) VTE patients and prothrombin G20210A mutation was found in 7/250 (2.8%) VTE patients while no mutation was found in any of the healthy controls. Both the mutations were found to be significantly associated with the increased risk of VTE (p = 0.0001 and 0.0150 respectively) while no association of VTE risk with MTHFR C677T polymorphism was found (p = 0.53). CONCLUSION The increased frequency of factor V Leiden G1691A and prothrombin G20210A mutation in VTE patients indicates a significant role of these mutations in the development of VTE in our population. We therefore suggest the routine screening of these two mutations as thrombophilic markers in Kashmiri patients with venous thromboembolism.

Clastogen-Induced Chromosomal Breakage Analysis of Suspected Fanconis Anemia Cases of Kashmir, North India

Clastogen induced chromosome breakage analysis is widely used for the differential diagnosis of Fanconis anemia. Mitomycin-C (MMC) induced chromosome fragility test was performed on the cultured lymphocytes of 50 children with clinical suspicion of Fanconis anemia. According to the results of the MMC test, the patients were divided into two subgroups: FA displaying typical sensitivity to MMC and non FA. The present study revealed 7(14%) of examined patients to have a FA cellular phenotype with increased MMC-induced chromosome fragility. The percentage of MMC-induced aberrant cells was increased more than 36 times in FA patients (Mean=67.14%) when compared to non FA patients (Mean=1.82). The number of MMC-induced breaks/cells was more than 09 times higher in FA patients (Mean=2.42 breaks/cell) when compared to non FA patients (Mean=0.25 breaks/cells). Our results indicate that the clastogen induced sensitivity test is a reliable in vitro method for verification of the FA cellular phenotype. The study being the first of its kind from Kashmir (North India) lays the basis for further studies on patients of this region with a clinical suspicion of FA.

Identification of Unique Pattern of CFTR Gene Mutations in Cystic Fibrosis in an Ethnic Kashmiri Population (North India)

Background: Cystic Fibrosis (CF) one of the most common severe autosomal recessive disorders is caused by mutations in CFTR gene. The mutation distributions vary widely between different geographical and ethnic groups. In view of ethnic nature of Kashmiri population (North India), we aim at looking for the 3 common mutations Δ508, 3849+10 kb, C>T and W1282X in CF suspected cases. Method: The mutations were evaluated in 150 highly suspected children with CF, proven by clinical features. ARMS-PCR was used for mutation detection of Δ508 and W1282X while as 3849+10 kb, C>T was assessed by indigenously developed ARMS-PCR and results were confirmed by RFLP. Results: Of the 150 suspected CF cases, one of the three mutations was found in 60 out of the 300 alleles genotyped. Δ508 mutation was found in 36 of 150 (24%) cases, 3849+10 kb, C>T in 24 of 150(16%) cases while as no mutation was observed in W1282X. Interestingly 08 of 09 samples with normal sweat chloride were detected positive for 3849+10 kb, C>T mutation. Conclusion: In this report, frequency of the Δ508 mutation in Kashmiri children with CF is less as compared to the Western Countries. Interestingly, we identified 3849+10 kb, C>T mutation as unique in population under study with much higher frequency as compared to rest of the world. Further we found intron 19, 3849+10 kb, C>T mutation serves as marker in those CF cases having sweat chloride negative.

De novo Xp terminal deletion in a triple X female with recurrent spontaneous abortions: a case report

Trisomy X (47,XXX) is a human sex chromosome aneuploidy in which females have an extra X chromosome, compared to the 46,XX karyotype in typical females. Females with 47,XXX karyotypes have been reported to have varied phenotypic changes that include features like tall stature, epicanthal folds, hypotonia and clinodactyly. Seizures, renal and genitourinary abnormalities, and premature ovarian failure are also few associated findings. However, puberty, sexual development and fertility are usually normal in trisomy X females (Tartaglia et al. 2010). The case presented in this study had two consecutive spontaneous pregnancy losses though she was devoid of any intrauterine malformation as revealed by her ultrasonography report. Her immunological profile was absolutely normal. Apparently, the female was not having any kind of facial deformity or syndromic features. Cytogenetic evaluation of the female revealed 47(XXX) karyotype with del(Xp21→Xpter) which was possibly the only discernible cause of the recurrent spontaneous abortions. Besides, karyotype analysis of the other family members revealed no structural or numerical abnormality of chromosomes. Cytogenetic evaluation of her husband revealed 46,XY karyotype. To our knowledge, till date a triple X female having recurrent spontaneous abortions with del(Xp21→Xpter) has not been reported. Trisomy X is a condition caused by the presence of an extra X chromosome in females. It is the most common chromosomal abnormality in females, occurring with an incidence of approximately 1 in 1000 female births. As some individuals are only mildly affected or asymptomatic, it is estimated that only 10% of individuals with trisomy X are actually diagnosed (Tartaglia et al. 2010). Marked facial

Variations in the inhibin gene in Kashmiri women with primary ovarian insufficiency

Abstract Inhibin is a glycoprotein produced by granulosa cells and its main function is the negative feedback control of follicle stimulating hormone (FSH) which has an important role in folliculogenesis. Mutation in the INHα gene leading to decreased bioactive inhibin has been associated with primary ovarian insufficiency (POI). The aim of this study was to investigate the role of variations in the INHα gene in increasing the susceptibility to POI in Kashmiri women. INHα c.769G > A mutation was analysed in 100 POI cases and 100 controls using PCR-RFLP and agarose gel electrophoresis. The INHα c.769G > A mutation was found in 10% of POI cases with 8% having heterozygous mutation and 2% having a homozygous mutation. The frequency of mutation in healthy controls was zero. Statistically, a very significant association was found between INHα c.769G > A mutation and the occurrence of POI (p = 0.0015). Moreover, the mutation was also significantly associated with high levels of FSH in POI patients (p < 0.0001). Given the significant association of INHα c.769G > A mutation with the increased FSH levels and POI in Kashmiri population, we suggest this mutation can be used to identify POI variants for screening of women susceptible to POI before the disease onset and can further facilitate putative therapy for such patients.

Molecular Identification of Intron 2 Splice Mutation and 8bp Deletion in CYP21 Gene for Congenital Adrenal Hyperplasia (CAH) Patients in Kashmir (North India)

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by alteration in CYP21 gene which ultimately leads to 21-hydroxylase deficiency. The present study aimed at evaluation of 2 common mutations viz, Intron 2 Splice (INT2S) mutation and 8 bp deletions in exon 3 of CYP21 gene and to establish their frequencies in Kashmir population (North India). The mutations were tested by Amplification Refractory Mutation System-PCR (ARMS-PCR) in 50 cases of CAH, proven by clinical features and raised 17-hydroxy progesterone (17OHP) levels. The results revealed that 15(30%) cases had INT2S mutation while as 8 bp deletion was not detected in any patient. In INT2S mutation, 7 cases were homozygous with I2-G genotype and 8 cases were heterozygous. The frequency of AG heterozygotes was found in 5 cases and CG heterozygote genotype was found in 3 cases. CAH patients with ambiguous genitalia were seen to harbor most of the INT2S mutations with I2-G in 3 cases and CG heterozygotes in 2 cases. In non-consanguineous group of patients, 4 homozygous I2-G mutations and 4 were I2- GC heterozygotes were detected in comparison to 3 and 1 in consanguineous patients respectively. Our study confirms that INT2S mutations but not 8 bp deletions exist in CYP21 gene in CAH patients in Kashmir population.

Occurrence of Chromosomal Alterations in Recurrent Spontaneous Abortion Couples: A Case-Only Study from Kashmir, North India

The present study was proposed to unveil the incidence and pattern of chromosomal abnormalities in recurrent spontaneous abortion couples of Kashmir, North India. A total of 71 couples within the age group of 24 to 42 years and having history of two or more recurrent spontaneous abortions were included in the study. Peripheral blood lymphocyte cultures were set for each subject according to standard protocol and chromosomal analysis was carried out on well spread metaphases by the help of Cytovision software Version 3.9. The incidence of chromosomal abnormalities in spontaneous abortion couples of this region was found to be 7.75% that include numerical (1.40%) as well as structural (7.75%) chromosomal abnormalities. Both males (2.11%) and females (5.63%) possessed chromosomal aberrations that comprised balanced translocations (4.22%), duplications (0.70%), deletions (0.70%) and inversions (2.11%). Besides, We report three unique balanced translocations viz., t(1;3)(q24.3;p25)(1 case); t(6,16)(p11;q23)(1 case) and t(7;14)(p13;q12)(2 cases). that have not been found elsewhere in the literature. We conclude from the present study that chromosomal alterations do occur as an etiology in the RSA couples of Kashmir and their incidence is consistent with many reports around the world. The precise molecular characterization of the unique breakpoint regions reported in our study could help in identification of new genes involved in recurrent spontaneous abortions. The study being the first of its kind in this part of the world forms the basis for further studies of the couples of this region with recurrent spontaneous abortions.