Clinical application of a novel next generation sequencing assay for CYP21A2 gene in 310 cases of 21- hydroxylase congenital adrenal hyperplasia from India.

PURPOSE: Accurate diagnosis is required for management of Congenital adrenal hyperplasia (CAH). The conventional method for detection of mutations in the CYP21A2 gene is targeted capillary sequencing which is labor intensive and has limited multiplexing capability. Next generation sequencing (NGS) provides data with high sequence coverage and depth. Our objective was to develop an accurate NGS-based assay to characterize the mutation spectrum in CYP21A2 gene in Indian patients suspected to have 21-OH CAH. METHODS: Cases with 21-OH CAH from 12 endocrine units across India were studied. DNA was extracted from proband's and parent's(subset) blood. Locus-specific long-range PCR and gel electrophoresis of amplicons was followed by NGS where no visible 30 kb homozygous/whole gene deletion was observed. Orthogonal confirmation was performed by capillary sequencing (ABI 3500) and Multiplex Ligation-dependent Probe Amplification (MLPA, MRC-Holland). PCR products were purified and individual libraries were pooled and sequenced (Illumina). RESULTS: Of the 310 CAH cases, biallelic mutations (pathogenic/ likely pathogenic variants involving both CYP21A2 gene copies) were detected in 256 (82.6%), heterozygous mutations in 13 (4.2 %), and none in 41 (13.2%). Most common mutation was c.293-13A/C>G (29.03%), followed by 30 kb deletion (18.24%). Thirty samples tested orthogonally (by capillary sequencing or MLPA) showed 100% concordance with NGS assay. Nine novel variants were identified. CONCLUSIONS: We have developed and validated a comprehensive NGS-based assay for detection of variants in CYP21A2 gene in patients with 21-OH CAH. We describe CYP21A2 mutation spectrum and novel variants in a large cohort of Indian patients with CAH.

Association of Fat Mass and Obesity-associated Gene Variant with Lifestyle Factors and Body Fat in Indian Children.

CONTEXT: Common intronic variants of the fat mass and obesity-associated (FTO) gene have been associated with obesity-related traits in humans. AIMS: (1) The aim of this study is to study the distribution of FTO gene variants across different body mass index (BMI) categories and (2) to explore the association between FTO gene variants and lifestyle factors in obese and normal weight Indian children. SUBJECTS AND METHODS: Fifty-six children (26 boys, mean age 10.3 ± 2.2 years) were studied. Height, weight, and waist and hip circumference were measured. Physical activity (questionnaire) and food intake (food frequency questionnaire) were assessed. Body fat percentage (%BF) was measured by dual-energy X-ray absorptiometry. FTO allelic variants at rs9939609 site were detected by SYBR Green Amplification Refractory Mutation System real-time polymerase chain reaction using allele-specific primers. Generalized linear model was used to investigate the simultaneous influence of genetic and lifestyle factors on %BF. RESULTS: Mean height, weight, and BMI of normal and obese children were 130.6 ± 7.1 versus 143.2 ± 15.6, 24.0 ± 5.2 versus 53.1 ± 15.8, and 13.9 ± 2.1 versus 25.3 ± 3.2, respectively. The frequency of AA allele was 57% among obese children and 35% in normal weight children. Children with the AA allele who were obese had least physical activity, whereas children with AT allele and obesity had the highest intake of calories when compared to children who had AT allele and were normal. %BF was positively associated with AA alleles and junk food intake and negatively with healthy food intake and moderate physical activity. CONCLUSIONS: Healthy lifestyle with high physical activity and diet low in calories and fat may help in modifying the risk imposed by FTO variants in children.

Molecular genetics of growth hormone deficient children: correlation with auxology and response to first year of growth hormone therapy.

BACKGROUND: With the paucity of available literature correlating genetic mutation and response to treatment, we aimed to study the genetic makeup of children with growth hormone (GH) deficiency in Western India and correlate the mutation with auxology and response to GH treatment at end of 1 year. METHODS: Fifty-three (31 boys and 22 girls) children with severe short stature (height for age z-score <-3) and failed GH stimulation test were studied. Those having concomitant thyroid hormone or cortisol deficiencies were appropriately replaced prior to starting GH treatment. A magnetic resonance imaging (MRI) brain scan was done in all. Genetic mutations were tested for in GH1, GHRH, LHX3, LHX4 and PROP1, POU1F1 and HESX1 genes. RESULTS: Mean age at presentation was 9.7±5.1 years. Thirty-seven children (Group A) had no genetic mutation detected. Six children (Group B) had mutations in the GH releasing hormone receptor (GHRHR) gene, while eight children (Group C) had mutation in the GH1 gene. In two children, one each had a mutation in PROP1 and LHX3. There was no statistically significant difference in baseline height, weight and BMI for age z-score and height velocity for age z-score (HVZ). HVZ was significantly lower, post 1 year GH treatment in the group with homozygous GH1 deletion than in children with no genetic defect. CONCLUSIONS: Response to GH at the end of 1 year was poor in children with the homozygous GH1 deletion as compared to those with GHRHR mutation or without a known mutation.

Influence of Vitamin D Receptor Gene Fok1 Polymorphism on Bone Mass Accrual Post Calcium and Vitamin D Supplementation.

OBJECTIVE: To examine the association of vitamin D receptor (VDR) gene polymorphisms of the Fok1 locus on bone mass accrual in Indian girls used to a low calcium intake. METHODS: An intervention trial was undertaken in 102 girls aged 8-16 y, attending a state run school in Pune city, India. All girls received 500 mg calcium daily and 30,000 IU of vitamin D3 quarterly for one year. Dietary calcium intake was evaluated. Bone mineral content (BMC), bone area (BA) and bone mineral density (BMD) were measured at total body using Dual Energy X-ray Absorptiometry (Lunar DPX-PRO). Polymorphisms of the Fok1 locus of the vitamin D Receptor (VDR) gene were detected using SYBR Green quantitative polymerase chain reaction. RESULTS: The prevalence of Fok1 polymorphism was 43.1% (Ff), 9.8% (ff) and 47.1% (FF). At baseline, FF genotype had significantly lower BMD as compared to ff and Ff genotype (p < 0.05). At baseline, majority of girls (82.4%) were hypocalcemic with low calcium intake. Post-supplementation, FF genotype had significantly lower bone mass as compared to ff and Ff genotype. Significant increase in BMC [Ff (17.9%); ff (18.1%); FF (17.4%)], and BMD [Ff (5.4 %); ff (6.3%); FF (4.8%)] was observed post supplementation (p value < 0.05), though percentage increase in BMC and BMD was similar for three Fok1 polymorphisms (p > 0.1). CONCLUSIONS: VDR gene polymorphism, as defined by Fok1 genotype had no positive influence on bone mass accrual in response to calcium supplementation.

Clinical applicability of rapid detection of SRY and DYS14 genes in patients with disorders of sex development using an indigenously developed 5' exonuclease based assay.

BACKGROUND: Life threatening conditions are associated with atypical genitalia in newborns. Analysis of genetic sex provides a clue to the underlying etiology in newborns with disorders of sex development (DSD) and can guide further endocrine investigations. Rapid diagnosis of genetic sex would be immensely useful in this situation. Traditionally used methods such as karyotype and fluorescence in situ hybridisation are time-consuming. OBJECTIVES: To study the clinical applicability of an indigenously developed rapid real-time polymerase chain reaction (RT-PCR) assay for the sex determining region on the Y chromosome (SRY gene) and the DYS14 locus in newborns with DSD. METHODS: Clinical examination, endocrinological tests, RT-PCR analysis of SRY and DYS14 and karyotype was performed in 15 newborns with DSD. RESULTS: RESULTS of PCR were available within 4 h. Based on this report, in SRY/DYS14 positive cases, further tests for assessment of testicular function were done. In SRY negative cases, tests for congenital adrenal hyperplasia were done. On comparing PCR results with other tests, the Y chromosome was present on karyotype and testicular tissue was detected by endocrinological and/or histological methods in all (8/15) SRY positive cases. The SRY and DYS14 negative cases (7/15) did not have Y chromosome in the karyotype. Congenital adrenal hyperplasia (CAH) was the most common diagnosis in this group. CONCLUSIONS: The indigenously developed PCR for dual Y chromosome markers is rapid and sensitive. Further endocrine evaluation of newborns with DSD can be based on these results. Information of genetic sex partly allays the psychosocial distress associated with the condition.

Entire DAX1 gene deletion in an Indian boy with adrenal hypoplasia congenita.

OBJECTIVE: To report a case of Adrenal hypoplasia congenita (AHC) in an Indian boy presenting with adrenal failure in the neonatal period. Molecular diagnosis demonstrated absence of the entire DAX1 gene sequence region. METHODS: Real-time SYBR Green Polymerase Chain Reaction (PCR) amplification followed by melt curve analysis was the molecular analytical method used. Analysis of the PCR products by Agarose gel electrophoresis was also performed. RESULTS: Real-time SYBR Green PCR amplification carried out on a 240 bp region of Exon 1 and 320 bp region of Exon 2 of DAX1 gene did not result in any amplification for two independent DNA extractions of the patient sample. The melt curve analysis also failed to show the characteristic melt peaks. Additional analysis of the PCR products performed by Agarose gel electrophoresis of the patient samples did not reveal any DNA bands. CONCLUSIONS: Inability to amplify two distinct regions located on two distinct exons of the DAX1 gene of the patient sample point to the possible absence of the entire DAX1 gene sequence region in the index patient. Such molecular diagnostic techniques may prove very useful in making a diagnosis as well as for genetic counseling.

Vitamin D receptor gene polymorphisms and bone mass indices in post-menarchal Indian adolescent girls.

To study the association between vitamin D receptor (VDR) gene polymorphisms and bone mass indices in adolescent girls, a cross-sectional study was conducted in 120 post-menarchal girls aged 15-18 years in Pune city, India. Serum levels of ionised calcium, inorganic phosphorous, parathyroid hormone and 25-hydroxy vitamin-D were measured. Bone mineral content (BMC), bone area (BA) and bone mineral density (BMD) were measured at total body (TB), lumbar spine (LS) and left femoral neck (FN) using dual energy X-ray absorptiometry. Polymorphisms of the VDR gene at the Fok1 and Bsm1 loci were detected using SYBR Green quantitative polymerase chain reaction. The overall distribution of genotypes at the Bsm1 locus in this study was 33.3 % Bb, 29.2 % bb and 37.5 % BB while that for the Fok1 locus was 44.2 % Ff, 7.5 % ff and 48.3 % FF. There were no significant differences in the blood parameters when classified according to Bsm1 or Fok1 genotypes. Subjects with BB genotype have significantly higher mean TBBMC, TBBA, TBBMD and LSBMD than Bb and bb (p < 0.05) and showed a tendency for association with LSBMC and LSBA (p < 0.1). Subjects with Ff genotype showed a tendency for association with left FNBMC and FNBA (p < 0.1). Bsm1 genotype did not show an association with FN bone indices whereas Fok1 genotype did not show association with TB or LS bone indices. In conclusion, the present study demonstrates VDR gene polymorphism, defined by Bsm1 genotype, has an influence on total body and lumbar spine bone mass indices in post-menarchal Indian girls.

Molecular characterization in a case of isolated growth hormone deficiency and further prenatal diagnosis of an unborn sibling.

Familial isolated growth hormone deficiency (GHD) type 1 is characterized by an autosomal recessive pattern of inheritance with varying degrees of phenotypic severity. We report a proband, with isolated GHD (IGHD) with very early growth arrest and undetectable levels of GH. Homozygous complete deletion of the GH1 gene was identified by real-time/quantitative polymerase chain reaction (RT/q-PCR) and confirmed by an independent molecular genetic method; the multiplex ligation-dependent probe amplification (MLPA) technique. Prenatal diagnosis was offered for the subsequent pregnancy in the mother of our proband. Identical heterozygous deletion of the GH1 gene was detected in both parents. The fetus had a similar homozygous deletion of the GH1 gene. We thus report a unique case with a confirmed mutation in GH1 gene in the proband followed by prenatal detection of the same mutation in the amniotic fluid which to our knowledge hitherto has not been documented from India.

Allele-specific binding of CTCF to the multipartite imprinting control region KvDMR1.

Paternal deletion of the imprinting control region (ICR) KvDMR1 results in loss of expression of the Kcnq1ot1 noncoding RNA and derepression of flanking paternally silenced genes. Truncation of Kcnq1ot1 also results in the loss of imprinted expression of these genes in most cases, demonstrating a role for the RNA or its transcription in gene silencing. However, enhancer-blocking studies indicate that KvDMR1 also contains chromatin insulator or silencer activity. In this report we demonstrate by electrophoretic mobility shift assays and chromatin immunoprecipitation the existence of two CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated paternally derived allele. Methylation interference and mutagenesis allowed the precise mapping of protein-DNA contact sites for CTCF within KvDMR1. Using a luciferase reporter assay, we mapped the putative transcriptional promoter for Kcnq1ot1 upstream and to a site functionally separable from enhancer-blocking activity and CTCF binding sites. Luciferase reporter assays also suggest the presence of an additional cis-acting element in KvDMR1 upstream of the putative promoter that can function as an enhancer. These results suggest that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Dissection of KvDMR1 into its functional components should help elucidate the mechanism of its function in vivo.