Genetic analysis of familial hypercholesterolemia in Asian Indians: A single-center study.

BACKGROUND: Familial hypercholesterolemia (FH), an autosomal codominant disorder characterized by very high low-density lipoprotein cholesterol, is strongly associated with premature coronary artery disease. OBJECTIVES: Molecular landscape of FH in Asian Indians is not well studied, although this ethnic group comprises a large proportion of the world population. Knowledge of mutations in these groups is useful for identifying persons affected with FH, saving their lives, and cascade screening in their relatives. METHODS: Potential cases of FH (n = 100) were identified by criteria adapted for the Indian population from Dutch Lipid Clinic Network criteria. Pathogenic variants were analyzed in LDLR, APOB 100 (exons 26 and 29), PCSK9, and APOE genes using Sanger sequencing and multiplex ligation-dependent probe amplification technique. Cases in whom there were no pathogenic variants were tested by next-generation sequencing using a targeted panel of genes. RESULTS: Thirty-eight pathogenic variants were identified in 47 of 100 unrelated probands. Of these variants, 33 were identified in LDLR, 3 in APOB, and 2 in PCSK9 genes. Ten pathogenic variants were novel. Mutations were detected in 91.4% of those subjects classified as definite, 40% as probable, and in 18.8% as possible FH cases based on modified Dutch Lipid Clinic Network criteria. A likely founder mutation in intron 10 (c.1587-1G>A) of LDLR gene was observed in 6 North Indian families. The conventional pathogenic variants in APOB and PCSK9 genes and those previously reported in LDLR gene among Asian Indians were not detected in this cohort. CONCLUSION: This study demonstrates genetic heterogeneity of FH in India. The variants observed were different from those described in Western populations. Next-generation sequencing technology helped identify new mutations in APOB gene, suggesting that in less-studied populations, it is better to sequence the whole gene rather than test for specific mutations.

Noninvasive prenatal testing (NIPT) detects variant of Turner syndrome not detectable by fluorescent in situ hybridization.

Introduction: Noninvasive prenatal testing (NIPT) is a reliable screening method for fetal aneuploidy detection of trisomy 18, 13, 21 along with few sex chromosome abnormalities monosomy X, XXX, XXY (Klinefelter), XYY (Jacob) syndromes and certain microdeletions which include cri-du-chat, DiGeorge, 1p36, Angelman, and Prader-Willi syndromes in comparison to the available screening methods. Prenatal screening of Turners syndrome is possible by ultrasound in certain conditions only. Recently benefits of early detection and treatment of Turners syndrome has been emphasized, enforcing on accurate and early screening prenatally.Case details: The current case emphasizes on the reliability of NIPT testing which comes with an advantage of early screening. A 24-year-old primi gravida was referred for NIPT as she tested for high risk on biochemical screening. The Panorama™ NIPT results showed low risk for trisomies, 21, 18, and 13 but high risk of monosomy X and was advised confirmatory amniocentesis. The fluorescence in situ hybridization (FISH) report revealed no numerical abnormality detected for any of the five chromosomes tested. On receiving this discordant report, the sample was rerun for NIPT, to rule out any laboratory-related issues. The result obtained on a rerun was consistent with the first report and showed monosomy X again. The karyotype report was available three weeks later and a rare variant of Turners syndrome was identified.Discussion: Panorama™ NIPT considers single nucleotide polymorphisms spread across the chromosomes for analysis, different variants of aneuploidy can be picked up in comparison to FISH, similar to the current case wherein it could not as it was a centromeric probe. Reported first case of X chromosome variant detected by NIPT confirmed by karyotyping, missed by FISH.

KCNQ1 mutations associated with Jervell and Lange-Nielsen syndrome and autosomal recessive Romano-Ward syndrome in India-expanding the spectrum of long QT syndrome type 1.

Long QT syndrome type 1 (LQT1) is the most common type of all Long QT syndromes (LQTS) and occurs due to mutations in KCNQ1. Biallelic mutations with deafness is called Jervell and Lange-Nielsen syndrome (JLNS) and without deafness is autosomal recessive Romano-Ward syndrome (AR RWS). In this prospective study, we report biallelic mutations in KCNQ1 in Indian patients with LQT1 syndrome. Forty patients with a clinical diagnosis of LQT1 syndrome were referred for molecular testing. Of these, 18 were excluded from the analysis as they did not fulfill the inclusion criteria of broad T wave ECG pattern of the study. Direct sequencing of KCNQ1 was performed in 22 unrelated probands, parents and at-risk family members. Mutations were identified in 17 patients, of which seven had heterozygous mutations and were excluded in this analysis. Biallelic mutations were identified in 10 patients. Five of 10 patients did not have deafness and were categorized as AR RWS, the rest being JLNS. Eight mutations identified in this study have not been reported in the literature and predicted to be pathogenic by in silico analysis. We hypothesize that the homozygous biallelic mutations identified in 67% of families was due to endogamous marriages in the absence of consanguinity. This study presents biallelic gene mutations in KCNQ1 in Asian Indian patients with AR JLNS and RWS. It adds to the scant worldwide literature of mutation studies in AR RWS. © 2016 Wiley Periodicals, Inc.

Molecular Diagnosis of Hereditary Fructose Intolerance: Founder Mutation in a Community from India.

Hereditary fructose intolerance (HFI) is a difficult-to-confirm diagnosis, requiring either invasive liver biopsy-enzyme assay or potentially hazardous fructose challenge test or expensive molecular genetic analysis. Therefore, worldwide there has been a trend towards finding "common mutations" in distinct ethnic groups to simplify the process of diagnosis. The nonspecific presentation of the disease often leads to diagnostic confusion with other metabolic liver disorders such as glycogenoses, galactosemia, and tyrosinemia. This leads to much delay in diagnosis with consequent harm to the patient.We report mutations in the ALDOB gene, from eleven Indian patients, seven of whom belong to the Agarwal community. Six patients from the Agarwal community and two non-Agarwal patients harbored one novel mutation, c.324+1G>A (five homozygous and one heterozygous), in the ALDOB gene. Haplotyping performed in families confirmed a founder effect. The community has been known to harbor founder mutations in other genes such as the MLC1, PANK2, and CAPN3 genes, thus providing another evidence for a founder effect in the community in case of HFI. This may pave the path for a simpler and quicker test at least for this community in India. In addition to the founder mutation, we report four other novel mutations, c.112+1delG, c.380-1G>A, c.677G>A, and c.689delA, and a previously reported mutation, c.1013C>T, in the cohort from India.

Molecular evaluation of a novel missense mutation & an insertional truncating mutation in SUMF1 gene.

BACKGROUND & OBJECTIVES: Multiple suphphatase deficiency (MSD) is an autosomal recessive disorder affecting the post translational activation of all enzymes of the sulphatase family. To date, approximately 30 different mutations have been identified in the causative gene, sulfatase modifying factor 1 (SUMF1). We describe here the mutation analysis of a case of MSD. METHODS: The proband was a four year old boy with developmental delay followed by neuroregression. He had coarse facies, appendicular hypertonia, truncal ataxia and ichthyosis limited to both lower limbs. Radiographs showed dysostosis multiplex. Clinical suspicion of MSD was confirmed by enzyme analysis of four enzymes of the sulphatase group. RESULTS: The patient was compound heterozygote for a c.451A>G (p.K151E) substitution in exon 3 and a single base insertion mutation (c.690_691 InsT) in exon 5 in the SUMF1 gene. The bioinformatic analysis of the missense mutation revealed no apparent effect on the overall structure. However, the mutated 151-amino acid residue was found to be adjacent to the substrate binding and the active site residues, thereby affecting the substrate binding and/or catalytic activity, resulting in almost complete loss of enzyme function. CONCLUSIONS: The two mutations identified in the present case were novel. This is perhaps the first report of an insertion mutation in SUMF1 causing premature truncation of the protein.

Investigation of Calpain 10 (rs2975760) gene polymorphism in Asian Indians with Gestational Diabetes Mellitus.

BACKGROUND: Type 2 Diabetes Mellitus (T2DM) and Gestational Diabetes Mellitus (GDM) are part of a heterogeneous and complex metabolic group of disorders that share common pathophysiological circumstances, including ß-cell dysfunction and insulin resistance. The protein Calpain 10 (CAPN10) plays a role in glucose metabolism, pancreatic ß-cell insulin secretion, and thermogenesis. OBJECTIVE: Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based genotyping of CAPN10 (rs2975760) polymorphism was carried out in T2DM and GDM with suitable controls for each of the pathologies from the same population. Genomic DNA was isolated from 787 participants, including 250 cases of T2DM, 287 pregnant women, of which 137 were identified as having GDM and the remaining 150 were confirmed as non-GDM, and 250 healthy control volunteers, and association analysis was carried out for genotypes and alleles. RESULTS: In the present study, T2DM was compared with healthy controls and was not found to be associated with the CAPN10 C allele (odds ratio, OR: 1.09; 95% CI = 0.8011-1.484; p = 0.5821). GDM also did not show any association when compared with non-GDM (OR: 1.124; 95% CI = 0.7585-1.667; p = 0.5606) respectively. CONCLUSION: Our study suggests that the CAPN10 (rs2975760) polymorphism scrutinized in this study is not associated with T2DM and GDM.

Mitochondrial D310 instability in Asian Indian breast cancer patients.

Somatic mutations in mitochondrial DNA (mtDNA) have been demonstrated in various tumors. Mitochondrial D-loop is a non-coding region in the mitochondrial genome, which has essential transcription and replication elements, and alterations in this region may affect both these processes. The D-loop has a poly-C tract (PCT) located between 303 and 315 nucleotides known as D310, which has been identified as a frequent hot spot mutation region in human neoplasia. In the present study, 77 pairs of breast tumor and adjacent non-tumorous tissue samples were analyzed by polymerase chain reaction-single-strand conformational polymorphism, restriction fragment length polymorphism, and sequencing to evaluate the frequency of D310 (PCT) mutations and its association with clinicopathologic parameters of breast cancer. Alterations were detected in 25 of 77 (32.5 %) breast cancer samples; these included 7/25 (28 %) cases with heteroplasmy. This is the first study from Asian Indian breast cancer (BC) patients indicating a relatively high frequency of D310 mutations, suggesting that mtDNA instability at D310 may be a common characteristic of BC. However, 66.7 % of the alterations were observed in stage II BC, indicating that this may be a more important change for early progression of the disease rather than its initiation.

Association of PON1 and APOA5 gene polymorphisms in a cohort of Indian patients having coronary artery disease with and without type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is a major cause of coronary artery disease (CAD) and is responsible for a great deal of morbidity and mortality in Asian Indians. Several gene polymorphisms have been associated with CAD and T2DM in different ethnic groups. This study will give an insight about the association of two selected candidate gene polymorphisms; paraoxonase1 (PON1) Q192R and apolipoprotein A5 (APOA5) -1131T>C were assessed in a cohort of South Indian patients having CAD with and without T2DM. Polymerase chain reaction-based genotyping of PON1 Q192R (rs662) and APOA5-1131T>C (rs662799) polymorphism was carried out in 520 individuals, including 250 CAD patients (160 with T2DM and 90 without T2DM), 150 T2DM patients with no identified CAD, and 120 normal healthy sex- and age-matched individuals as controls. The PON1 192RR genotype and R allele frequency were elevated in both CAD and T2DM patients when compared with controls; however, only CAD patients with T2DM showed a statistical significance (p=0.023; OR=1.49; 95% CI: 1.04-2.12) when compared with controls. The APOA5-1131CC genotype and C allele also showed a significant association between the CAD+T2DM patients when compared with CAD without T2DM and healthy controls (p=0.012; OR=1.71; 95% CI: 1.0-2.67). An additive interaction between the PON1 RR and APOA5 TC genotypes was identified between the T2DM and CAD patients (p=0.028 and 0.0382, respectively). PON1 and APOA5 polymorphisms may serve as biomarkers in the South Indian population to identify T2DM patients who are at risk of developing CAD.

Molecular pathogenesis of endometriosis; Toll-like receptor-4 A896G (D299G) polymorphism: a novel explanation.

BACKGROUND: Endometriosis is a distressing gynecological disorder. Toll-like receptor 4 (TLR4) is specific for recognition of the molecular pattern of gram-negative bacteria. TLR4 is present on the surface of endometrial cells. Their role in the molecular pathogenesis of endometriosis is postulated through a novel explanation. OBJECTIVE: To study the TLR4 A896G polymorphism in cases of endometriosis and age- and sex-matched healthy controls and evaluate its role in the molecular pathogenesis of endometriosis. DESIGN: Case-control study, involving patients from four gynecological centers from Hyderabad Samples: The study was carried out on 400 women who include 200 surgically confirmed cases of endometriosis and 200 healthy women volunteers in whom endometriosis was excluded by the standard diagnostic criteria. RESULTS: An association of G allele, GG, and AG genotype of TLR4 A896G polymorphism was seen in cases of endometriosis. "G" allele was found to be significantly associated with endometriosis (odds ratio=4.4827; 95% confidence interval: 2.2829-8.8021; χ(2) p<0.0001). An eightfold increase of endometriosis risk was seen in women who carry GG genotype, whereas AA genotype can be considered as protective in our population. CONCLUSION: TLR4 A896G polymorphism (rs4986790) is a functional polymorphism resulting in hypo-responsiveness of the receptor, thus resulting in peritoneal inflammation. The molecular microenvironment becomes favorable for the endometrial cells to adhere to the peritonium, thereby resulting in the initiation of endometriosis.

Regulation of IGF2 transcript and protein expression by altered methylation in breast cancer.

PURPOSE: Breast Cancer is one of the leading causes of cancer deaths among women worldwide. The role of epigenetics as a distinct mechanism to alter gene expression in a tissue-specific manner has emerged as an important mechanism in the pathophysiology of cancer. Present study was carried out to assess the role of methylation in regulating transcription and protein expression of Insulin-like growth factor 2 (IGF2), an oncogene with parental imprinting. METHODS: Paraffin-embedded archival breast tumor and adjacent normal tissue samples were used for carrying out PCR-based methylation assay, genomic PCR, immunohistochemistry and Real-Time Reverse transcriptase PCR. RESULTS: A significant loss of methylation in exon 9 CpG cluster of IGF2 in breast tumor tissues was observed when compared to normal tissue (P < 0.0001). Expression of IGF2 by immunohistochemistry exhibited a mean twofold increase correlating with the hypomethylation of this specific CpG. Real-Time RT PCR showed increased transcripts in the tumor tissue supporting the IHC and methylation results. A total of 33% of tumor samples heterozygous for the ApaI IGF2 polymorphism exhibited biallelic IGF2 expression due to loss of imprinting; this was not seen in any of the normal breast tissues. CONCLUSIONS: Altered methylation of exonic CpG plays an important role in the enhanced transcription/expression of IGF2 in breast tumors. Methylation analysis of exon 9 CpG can be used as a biomarker for upregulation of IGF2 in breast tumor tissue and maybe developed as a diagnostic test in future.

Association of methylene tetrahydrofolate reductase C677T genotype with type 2 diabetes mellitus patients with and without renal complications.

Diabetes is gradually getting the status of a global epidemic, with India projected as the capital of type 2 diabetes mellitus (T2DM). Nephropathy is an important complication of diabetes and a major cause of end-stage renal disease. Studies from different parts of the world have given controversial results regarding the association of methylene tetrahydrofolate reductase (MTHFR) gene variation with T2DM and diabetic nephropathy (DN). This case-control study assessed the association of MTHFR C677T mutation in T2DM and DN cases. Genotyping of MTHFR was carried out for 236 T2DM cases with diabetes diagnosed for >8 years, having either normoalbuminuria (n=100) or established DN (n=136). One hundred age- and sex-matched healthy individuals with normal blood sugars and no family history of T2DM were selected as controls. This first report from India gives a highly significant odds ratio of 4.0423 (95% confidence interval=1.8753-8.7133), indicating that the MTHFR 677T allele confers a fourfold risk of developing DM in our population. The frequency of the T allele in both the DM and DN groups was similar (i.e., 0.16 and 0.11, respectively), showing no association with the initiation or progression of DN. Individuals with a family history of diabetes or with risk factors such as obesity, hypertension, and impaired glucose tolerance should be screened for MTHFR C677T mutation and may be prescribed folic acid, vitamin B6, and vitamin B12 to assess if this helps in delaying the onset of diabetes.

Relationship of angiotensin-converting enzyme gene polymorphism with nephropathy associated with Type 2 diabetes mellitus in Asian Indians.

OBJECTIVE: Diabetic nephropathy (DN) has become the leading cause of end-stage renal disease, but the pathogenesis of this condition is not exactly understood. Several studies from different parts of the world have examined angiotensin-converting enzyme (ACE) gene polymorphism as a candidate for DN. Two studies yielding controversial results have been reported from India. To rule out this discrepancy, we carried out a hospital-based study on a cohort from our population to determine whether ACE gene polymorphism is associated with DN. RESEARCH DESIGN AND METHODS: ACE gene polymorphism was analyzed by polymerase chain reaction in 460 individuals consisting of 174 cases of DN, 175 cases of Type 2 diabetes mellitus (DM), and 111 controls. The DN cases included in the study were Type 2 DM cases with serum creatinine >1.5 mg/dl and serum albumin >30 mg/dl in a 24-h urine sample. RESULTS: ACE insertion/deletion genotyping analysis showed DD genotype in 22.75% of DN cases, 15.42% of Type 2 DM cases, and 21.62% of controls. Chi-square test between the DN group and the control group did not show a significant difference in D allele. However, the difference was significant at P<.05 between the DN group and the DM group. The odds ratio was 2.0953 (95% confidence interval=1.35-3.2522), indicating a significant association of DD genotype and D allele with DN. CONCLUSION: Our data enable us to conclude that Asian Indians with D allele and Type 2 DM are at greater risk for developing DN.