SCN1A Gene Mutations in Indian Children With Epilepsy: Single Center Experience.

OBJECTIVE: To study prevalence of SCN1A gene mutations in complex seizure disorders. METHODS: Retrospective laboratory based study on samples sent for molecular diagnosis in complex seizure disorders. Exome sequencing was performed. Phenotype- genotype correlation was done for patients showing variants in SCN1A gene. RESULTS: 364 samples were evaluated; of which, 54% were of children below 5 years of age. SCN1A mutations were seen in 50 samples of patients with complex seizure disorders; 44 variants were identified. Types of seizure disorders commonly associated were Dravet syndrome and genetic epilepsy with febrile seizures. CONCLUSIONS: SCN1A mutations are common in complex seizure disorders, especially Dravet syndrome. Early identification of SCN1A gene in etiology is important for selection of correct antiepileptic and counselling.

Neonatal Screening for Congenital Adrenal Hyperplasia in Indian Newborns with Reflex Genetic Analysis of 21-Hydroxylase Deficiency.

Congenital adrenal hyperplasia (CAH), screened for in neonates, is the second most common endocrinopathy after congenital hypothyroidism.Newborn screening for CAH due to CYP21A2 deficiency is performed by immunologic assay for 17-hydroxyprogesterone (17-OHP). The second-tier test for confirmation of diagnosis is carried out on recall venous blood sample from screen positives measuring 17-OHP, or other metabolites of steroid metabolism by liquid chromatography-tandem mass spectroscopy. However, as steroid metabolism is dynamic, it can affect these parameters even in the recall sample of a stressed neonate. Moreover, there is some time delay in recalling the neonate for repeat testing. Reflex genetic analysis of blood spot from the initial Guthrie cards of screen positive neonates, if used for confirmatory testing, can avoid this time delay as well as the effect of stress on steroid metabolism. In this study, we used Sanger sequencing and MLPA in a reflex manner for molecular genetic analysis to confirm CYP21A2-mediated CAH. Out of 220,000 newborns screened, 97 were positive on the initial biochemical screen, of which 54 were confirmed true positives with genetic reflex testing, giving incidence of CAH as 1:4074. Point mutations were more common than deletions, indicating that Sanger sequencing should be used ahead of MLPA for molecular diagnosis in India. Amongst the variants detected, the most common was I2G-Splice variant (44.5%), followed by c.955C>T (p.Gln319Ter) (21.2%); Del 8 bp and c.-113G>A were detected with frequencies of 20.3% and 20%, respectively. In conclusion, reflex genetic testing is an effective strategy for identifying true positives in CAH screening in neonates. This will obviate need for recall samples and also aid effective counselling and timely prenatal diagnosis in the future. In Indian newborns, as point mutations are more common than large deletions, Sanger sequencing should be the initial method of choice for genotyping, ahead of MLPA.

Characterization of 40 novel HLA class I and class II alleles identified in umbilical cord blood units.

Forty novel HLA class I and class II alleles were identified in umbilical cord blood (UCB) samples and categorized based on various types of mutations: non-synonymous, synonymous, frameshift, and premature termination codon. This study described 14 novel HLA-A alleles, 9 novel HLA-B alleles, 4 novel HLA-C alleles, 3 novel HLA-DRB1 alleles and 10 novel HLA-DQB1 alleles. Comparing the new allele sequence with the most homologous sequence, 60% of the novel alleles exhibited non-synonymous substitution, 32.5% observed with synonymous substitution, 5% displayed premature stop codon, and 2.5% presented with frameshift mutation. The majority of the new alleles contained a single nucleotide variation when compared with the most similar sequence.

Correlation of first-trimester serum levels of pregnancy-associated plasma protein A with small-for-gestational-age neonates and preterm births.

OBJECTIVE: To analyze the relationship between first-trimester levels of pregnancy-associated plasma protein A (PAPP-A) and small-for-gestational-age (SGA) neonates and preterm births, and to assess predictive utility for these events. METHODS: A prospective study was conducted among women undergoing first-trimester screening between January 1, 2012, and December 31, 2013, at two centers in Pune, India. Serum PAPP-A levels, pregnancy course, and outcome were assessed. RESULTS: Overall, 1474 women were included. An association was found between the lowest quintile of PAPP-A levels (<0.4 multiples of median) for both SGA (<10th centile; 20.9% of cases in this PAPP-A quintile) and preterm birth (<37weeks; 15.8%). Women in the lowest quintile of PAPP-A concentration had a significantly increased risk of SGA (<10th centile) than did those with higher concentrations (adjusted odds ratio 2.92, 95% confidence interval 2.00-4.27). Their risk of preterm birth (<37weeks) was also increased (adjusted odds ratio 1.84, 95% confidence interval 1.25-2.72). The predictive sensitivities of the lowest quintile of PAPP-A were 35.85% for SGA (<10th centile) and 27.92% for preterm birth (<37weeks). CONCLUSION: Low levels of PAPP-A were associated with SGA and preterm births; however, poor predictive sensitivity could restrict clinical utility of this marker when used alone.

Nucleotide sequence analysis of NIPBL gene in Indian Cornelia de Lange syndrome cases.

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a multisystem developmental disorder in children. The disorder is caused mainly due to mutations in Nipped-B-like protein. The molecular data for CdLS is available from developed countries, but not available in developing countries like India. In the present study, the hotspot region of NIPBL gene was screened by Polymerase Chain Reaction which includes exon 2, 22, 42, and a biggest exon 10, in six CdLS patients and ten controls. MATERIALS AND METHODS: The method adopted in present study was amplification of the target exon by using polymerase chain reaction, qualitative confirmation of amplicons by Agarose Gel Electrophoresis and use of amplicons for Conformation Sensitive Gel Electrophoresis to find heteroduplex formation followed by sequencing. RESULTS: We report two polymorphisms in the studied region of gene NIPBL. The polymorphisms are in the region of intron 1 and in exon 10. The polymorphism C/A is present in intron 1 region and polymorphism T/G in exon 10. CONCLUSION: The intronic region polymorphism may have a role in intron splicing whereas the polymorphism in exon 10 results in amino acid change (Val to Gly). These polymorphisms are disease associated as these are found in CdLS patients only and not in controls.

Is chromium from stainless steel utensils responsible for epidemic of type 2 diabetes?

Type 2 diabetes prevalence is rising rapidly in developing world especially in India in last few decades. 'Thrifty phenotype' and 'westernization of lifestyle' is used to explain this epidemic. Chromium is an important modulator in insulin and glucose metabolism. Preconceptional chromium exposure has been demonstrated to raise the corticosterone and glucose levels in offspring of rodent model. Chromium is chelated when acidic food is stored in the stainless steel utensils. Chromium levels are shown to be high in Asian Indians. The hyperinsulinemia and insulin resistance is demonstrated in Indians from the newborn stage. We hypothesize that increased exposure to chromium in preconceptional and/or fetal stage leads to altered epigenetic control and altered endocrine and metabolic functioning. Increasing urbanization has led to increasing use of stainless steel and resultant exposure to chromium is at the least partly responsible for rising prevalence of type 2 diabetes. If proven avoiding or modifying the use of stainless steel utensils will halt the present epidemic of type 2 diabetes.

Craniomicromelic syndrome: first report in a male.

We report on craniomicromelic syndrome in a male fetus. This case had the previously reported features of prenatal onset growth retardation, underossified cranial bones, wide sutures and fontanels, small face as compared to head, small palpebral fissures, pinched nose, microstomia, micrognathia, and narrow thorax. The consistent combination of these features with short appearing limbs as observed in this case establishes this syndrome as a distinct entity.

Chronic umbilical cord entanglements causing intrauterine fetal demise in the second trimester.

Entanglement of the umbilical cord with fetal body parts is known to occur in early pregnancy. This can potentially compromise the cord blood flow and cause fetal demise. We report 3 instances of intrauterine fetal deaths in the 2nd trimester of pregnancy with longstanding cord entanglement. The cord had left impressions of entanglement on the entrapped growing fetal part. Restricted movements of the fetus due to cord entanglement led to reduced spiraling of the umbilical cord. Our case series demonstrates that tight entanglement of fetal body parts by the umbilical cord can cause fetal demise in the 2nd trimester. This event is associated with a straight umbilical cord. Thus, the presence of reduced spiraling in intrauterine fetal demise warrants a search for possible cord entanglement along with established causes, such as chromosomal and congenital anomalies.

Plasma chitotriosidase activity in children with lysosomal storage disorders.

Chitotriosidase (ChT) is an enzyme that is selectively activated in tissue macrophage. This property of ChT makes it a potential marker for many disease process and prognostication. Present study has been carried out to know the significance of ChT as a screening marker in lysosomal storage disorders (LSDs) where tissue macrophage activation is commonly observed due to accumulation of substrate in various organs of the body. Study comprises of 20 healthy children in the age range of 10 days to 5 yrs and 56 children in the age range of 2.5 months to 13 yrs with regression of milestones, skeletal dysplasia, neuroregression and hepatosplenomegaly were selected for plasma ChT who had confirmed LSDs as carried out by specific lysosomal enzyme study from the leukocytes or fibroblasts. Plasma ChT was 55.21 +/- 20.81 nmol/ml/hr in twenty healthy age matched controls. Plasma ChT level was 42.88 to 79.78 nmol/ml/hr in thirteen of 56 (23.21%) children with LSDs like Morquio-B, Pompe, Metachromatic leucodystrophy (MLD), Sandhoff and Niemann-Pick disease type C (NPD-C). While in 43 (76.78%) children it was in the range of 213.74 to 23,511.40 nmol/ml/hr. who had LSDs like Morquio-B, Sly syndrome, MLD, GM2 Gangliosidosis, NPD-A/B and Gaucher disease (GD). Marked elevated ChT (4,000 to 23,511 nmol/ml/hr) was observed in all cases of GD (n=7) and NDP-A/B. It can be concluded from the present study that moderately raised activity of ChT can be utilized as a positive predictive test for certain LSD's. Those with marked elevated ChT have confirmed GD or NPD-A/B making it a strong screening marker for this group of diseases.

Genomewide scan for nonsyndromic cleft lip and palate in multigenerational Indian families reveals significant evidence of linkage at 13q33.1-34.

Nonsyndromic cleft lip with or without cleft palate (CL-P) is a common congenital anomaly with incidence ranging from 1 in 300 to 1 in 2,500 live births. We analyzed two Indian pedigrees (UR017 and UR019) with isolated, nonsyndromic CL-P, in which the anomaly segregates as an autosomal dominant trait. The phenotype was variable, ranging from unilateral to bilateral CL-P. A genomewide linkage scan that used approximately 10,000 SNPs was performed. Nonparametric linkage (NPL) analysis identified 11 genomic regions (NPL>3.5; P<.005) that could potentially harbor CL-P susceptibility variations. Among those, the most significant evidence was for chromosome 13q33.1-34 at marker rs1830756 (NPL=5.57; P=.00024). This was also supported by parametric linkage; MOD score (LOD scores maximized over genetic model parameters) analysis favored an autosomal dominant model. The maximum LOD score was 4.45, and heterogeneity LOD was 4.45 (alpha =100%). Haplotype analysis with informative crossovers enabled the mapping of the CL-P locus to a region of approximately 20.17 cM (7.42 Mb) between SNPs rs951095 and rs726455. Thus, we have identified a novel genomic region on 13q33.1-34 that harbors a high-risk variant for CL-P in these Indian families.