To evaluate the molecular aberrations at 11p15.5 locus in thirty-two patients with isolated lateralized overgrowth (ILO). Among selected 32 cases of ILO, methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) was performed initially followed by short tandem repeats (STR) marker analysis to confirm uniparental disomy (UPD). In those patients with normal MLPA reports, cyclin dependent kinase inhibitor 1C (CDKN1C) gene and whole exome sequencing was performed. Molecular analysis by MS-MLPA showed methylation aberrations in 28

Primary microcephaly and Seckel syndrome are rare genetically and clinically heterogenous brain development disorders. Several exonic/splicing mutations are reported for these disorders to date, but ∼40% of all cases remain unexplained. We aimed to uncover the genetic correlate(s) in a family of multiple siblings with microcephaly. A novel homozygous intronic variant (NC_000013.10:g.25459823T>C) in CENPJ (13q12) segregating with all four affected male siblings was identified by exome sequencing and validated by targeted linkage approach (logarithm of the odds score 1.8 at θ 0.0). RT-PCR of CENPJ in affected siblings using their EBV derived cell lines showed aberrant transcripts suggestive of exon skipping confirmed by Sanger sequencing. Significantly reduced wild type transcript/protein in the affected siblings having the splice variant indicates a leaky gene expression of pathological relevance. Based on known CENPJ function, assessing for mitotic alterations revealed defect in centrosome duplication causing mono/multicentrosome(s) at prophase, delayed metaphase, and unequal chromosomal segregation in patient cells. Clinical features witnessed in this study expand the spectrum of CENPJ-associated primary microcephaly and Seckel syndrome. Furthermore, besides the importance of regulatory variants in classical monogenic disorders these findings provide new insights into splice site biology with possible implications for ASO-based therapies.

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. (C) 2020 National Lipid Association. All rights reserved.

Purpose: To evaluate the frequency and the association of Thrombospondin 1 (THBS1) gene single nucleotide polymorphisms (SNPs) in Asian Indian patients with optical full thickness corneal grafting surgery. Methods: Prospective case–control analysis of optical penetrating keratoplasty patients with and without immune rejection and controls for genotyping of 3 THBS1 gene SNPs (rs1478604 A>G; rs2228261 C>T; rs2228262 A>G) by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS PCR). Results: Among 58 patients [45 with immune allograft rejection (DNA isolation was possible in 38 samples) and 13 without immune corneal allograft rejection] and 65 controls, allele frequencies observed for rs1478604 (A>G) are A: 69.7% and 72.6%, G: 30.2% and 27.3%; for rs2228261 (C>T) are T: 70.2% and 62.3%, C: 29.7% and 37.6%; and for rs2228262 (A>G) A: 97.4% and 98.4%; G 2.5% and 1.5% respectively. Genotype frequencies were rs1478604 (A>G) AA: 57.8% and 59.3%, AG 23.6% and 26.5%; GG 18.4% and 14%; for rs2228261 (C>T) TT: 40.5% and 33.8%, TC: 59% and 56.9%, CC: 0% and 9.2%; for rs2228262 (A>G) AA: 94.8% and 96.8%, AG: 5.1% and 3.1% in rejection and controls respectively. The allele and genotype frequency for the 3 described THSB1 SNPs did not show any difference between the corneal graft immune rejection patients and controls. Conclusion: Asian Indian population evaluated for THBS1 gene SNPs by ARMS PCR genotyping in Asian Indian population did not show any genetic association to immune rejection occurrence in our study.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) accounts for 2.6% of the patients with chronic kidney disease in India. ADPKD is caused by pathogenic variants in either PKD1 or PKD2 gene. There is no comprehensive genetic data from Indian subcontinent. We aimed to identify the pathogenic variants in the heterogeneous Indian population. PKD1 and PKD2 variants were identified by direct gene sequencing and/or multiplex ligation-dependent probe amplification (MLPA) in 125 unrelated patients of ADPKD. The pathogenic potential of the variants was evaluated computationally and were classified according to ACMG guidelines. Overall 300 variants were observed in PKD1 and PKD2 genes, of which 141 (47%) have been reported previously as benign. The remaining 159 variants were categorized into different classes based on their pathogenicity. Pathogenic variants were observed in 105 (84%) of 125 patients, of which 99 (94.3%) were linked to PKD1 gene and 6 (6.1%) to PKD2 gene. Of 159 variants, 97 were novel variants, of which 43 (44.33%) were pathogenic, and 10 (10.31%) were of uncertain significance. Our data demonstrate the diverse genotypic makeup of single gene disorders in India as compared to the West. These data would be valuable in counseling and further identification of probable donors among the relatives of patients with ADPKD.

BackgroundLong QT syndromes (LQTS) are characterized by prolonged QTc interval on electrocardiogram (ECG) and manifest with syncope, seizures or sudden cardiac death. Long QT 1–3 constitute about 75% of all inherited LQTS. We classified a cohort of Indian patients for the common LQTS based on T wave morphology and triggering factors to prioritize the gene to be tested. We sought to identify the causative mutations and mutation spectrum, perform genotype-phenotype correlation and screen family members.MethodsThirty patients who fulfilled the criteria were enrolled. The most probable candidate gene among KCNQ1, KCNH2 and SCN5A were sequenced.ResultsOf the 30 patients, 22 were classified at LQT1, two as LQT2 and six as LQT3. Mutations in KCNQ1 were identified in 17 (77%) of 22 LQT1 patients, KCNH2 mutation in one of two LQT2 and SCN5A mutations in two of six LQT3 patients. We correlated the presence of the specific ECG morphology in all mutation positive cases. Eight mutations in KCNQ1 and one in SCN5A were novel and predicted to be pathogenic by in-silico analysis. Of all parents with heterozygous mutations, 24 (92%) of 26 were asymptomatic. Ten available siblings of nine probands were screened and three were homozygous and symptomatic, five heterozygous and asymptomatic.ConclusionsThis study in a cohort of Asian Indian patients highlights the mutation spectrum of common Long QT syndromes. The clinical utility for prevention of unexplained sudden cardiac deaths is an important sequel to identification of the mutation in at-risk family members.

Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in the ATP7B resulting in copper overload in the liver and brain. Direct sequencing is routinely used to confirm WD diagnosis; however, partial and whole gene deletions in the heterozygous state cannot be detected by exon amplification since the normal allele will mask its presence. The aim of the present work was to search for unusual mutational events in the unexplained WD cases and to provide insight into the mechanisms. Out of 1420 clinically and biochemically confirmed WD samples received between 2000 and 2014 for routine mutation analysis, we were unable to detect mutant alleles in 142 samples, after extensive sequencing analysis. We used selective amplification and MLPA to identify the partial gene deletions and identified three different partial gene deletions in seven different families. All three deletions were fully characterized at the DNA sequence level. We report the first hemizygous case with WD due to intragenic deletion in the ATP7B (c.3134_3556+689del). This novel deletion resulted from an excision event mediated by consensus sequences in an AluSq2 repeat element and could be traced to micro homologous end joining (MMEJ). Finally, we determined the prevalence of the three deletions in DNA samples from a multinational group of WD patients. Our results emphasize the need for searching mutant alleles beyond routine methods and highlight that large ATP7B deletions are rare, but account for a detectable proportion in some WD patients. Screening for gene aberrations will further improve mutation detection in patients with unidentified ATP7B mutations presenting with clinical manifestations of WD.

Temporomandibular joint (TMJ) ankylosis is a significant problem in Asian countries and the most common etiology is trauma to the mandibular condyle. Other less common etiologies are infection, forceps delivery trauma and rheumatoid arthritis. Many hypotheses are given to explain the pathogenesis. All the proposed hypotheses revolve around trauma to the joint and subsequent healing causing ankylosis. This is however true for only few patients, most of the others do not progress to ankylosis after trauma to TMJ irrespective of seeking treatment or not. In this paper, we try to answer the question that why only a minor subset of condylar injuries progress to ankylosis and why others do not? The hypothesis follows a report of 4 cases that had bilateral TMJ ankylosis with extrahepatic portal venous obstruction (EHPVO) secondary to protein C deficiency. It is postulated that hypercoagulability/reduced fibrinolytic activity in these as well as non-EHPVO patients with TMJ ankylosis cases may have predisposed them to the development of joint ankylosis. The possible mechanism is explained and correlated with other causes of TMJ ankylosis and known facts of protein C deficiency/activated protein C resistance.

POSTERSmitochondrial isobutyryl-CoA dehydrogenase was located.Genomic DNA sequencing revealed a single nucleotide mutation at -17 of the first intron of Acad8 in affected mice.cDNA sequencing revealed an intronic 28-base-pair insertion at the site of the mutation, which caused a frame shift with a premature stop codon.In vitro splicing assay confirmed that the mutation was sufficient to activate an upstream, aberrant 3 splice site.There was a reduction in the expression of Acad8 at both the mRNA and protein levels.The mutant mice grew normally but demonstrated cold intolerance at young age with a progressive hepatic steatosis.Homozygous mutant mice hepatocytes had abnormal mitochondria with crystalline inclusions, suggestive of mitochondriopathy.This is the first mouse model of isobutyryl-CoA dehydrogenase deficiency; an inborn error of valine metabolism provides us a better understanding of the pathophysiology and the anticipated results of untreated severe cases during metabolic stress.

BACKGROUND:Global developmental delay is a common reason for referral to a paediatrician. We examined the aetiological yield of an extensive diagnostic work-up in young children with developmental delay in a tertiary referral centre.METHODS:To assess the diagnostic possibilities, we systematically examined 100 consecutive children with global developmental delay (< 5 years of age) who visited the paediatric outpatient department over a period of 18 months. An association between the presence of features at initial contact and aetiology was analysed by the 2-tailed Fisher exact test and chi-square test.RESULTS:Of the 100 children, 65 were < 2 years of age (mean age 23.6 months) at presentation. The presence of birth asphyxia, sepsis, seizures, abnormal neurological findings, and dysmorphism were significant predictors of aetiology. Four diagnostic categories--chromosomal disorders including Down syndrome, hypoxic-ischaemic encephalopathy, multiple malformation syndromes and cerebral dysgenesis--were the most common causes of global development delay in 20%, 15%, 14% and 11%, respectively. Moderate delay was seen in 42%, severe in 33% and mild in 25% of the patients. The aetiological yield did not differ with the severity of global developmental delay. Additional investigations such as neuroimaging, cytogenetic analysis, metabolic tests and specific molecular tests contributed to a diagnosis in 73% of the children, while in 23% these were the sole means of arriving at a diagnosis. Neuroimaging for a specific indication was almost twice more likely to yield an aetiology when compared with neuroimaging performed as a screening tool (65% v. 35%; p = 0.003).CONCLUSION:The aetiological yield in this selected cohort with global developmental delay was 73%. A step-wise investigational approach is justified in all children with developmental delay, regardless of the severity of delay or the absence of findings on history and physical examination. This study is an attempt to formulate an investigative approach in a child with global developmental delay, especially in developing countries where advanced molecular and cytogenetic studies are not routinely available.

Shukla, Pallavi; Gupta, Neerja; Balakrishnan, Prahlad; Ghosh, Manju; Gupta, Arun K.; Sharma, Raju; Kabra, Madhulika Author Information

Objective: The purposes of this study were to identify the occurrence of fibrillin-1 gene polymorphisms or mutations in exons 24 to 28 and to identify the relationship between "DNA sequence variants" and aortic dilatation in the presence of abnormal aortic histopathology and other variables in patients undergoing intracardiac repair of tetralogy of Fallot. Methods: Operatively excised full-thickness aortic wall tissue and 5 to 10-mL venous blood samples from 74 consecutive patients undergoing intracardiac repair of tetralogy of Fallot were studied. Histopathologic evaluation was done by light microscopy. Polymerase chain reaction amplification of fibrillin-1 gene was carried out for 5 exons (24-28), and amplified products were subjected to single-strand conformation polymorphism analysis to identify sequence alterations, if any. Logistic regression analysis was done to identify the relationship between patients with and without "exonic DNA variants" with other risk factors causing aortic dilatation. Results: Sixteen aortic tissue specimens (21.6%) were indicated as histologically normal and used as controls. Of 51 patients with dilated aorta, 48 (94.1%) exhibited histologic abnormalities. The incidences of significant lamellar loss, abnormal histopathology, and fibrillin-1 "DNA sequence variants" in tetralogy of Fallot with dilated aorta were 78.4%, 96.1%, and 50.9%, respectively. The risk of aortic dilatation was 8.83 (1.94-13.99) times greater in patients with histologically abnormal aorta and 8.11 (1.93-34.04) times greater in patients with fibrillin-1 "exonic DNA variants." Conclusion: Our findings indicate the existence of "exonic DNA variants" involving the fibrillin-1 gene in 1 or more exons (exon 24-28). The "DNA sequence variants" are more pronounced in patients with tetralogy of Fallot and dilated aorta in the presence of abnormal aortic histopathology.

Prenatal DiagnosisVolume 28, Issue 4 p. 357-359 Research Letter Prenatal diagnosis of megalencephalic leukodystrophy Pallavi Shukla, Pallavi Shukla Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorPrahlad Balakrishnan, Prahlad Balakrishnan Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorNeerja Agarwal, Neerja Agarwal Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorManju Ghosh, Manju Ghosh Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorMadhulika Kabra, Corresponding Author Madhulika Kabra [email protected] Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaAdditional Professor, Genetics Division, Department of Pediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, India, 110029.Search for more papers by this authorRaju Sharma, Raju Sharma Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorDeepika Deka, Deepika Deka Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorVeena Kalra, Veena Kalra Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this author Pallavi Shukla, Pallavi Shukla Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorPrahlad Balakrishnan, Prahlad Balakrishnan Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorNeerja Agarwal, Neerja Agarwal Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorManju Ghosh, Manju Ghosh Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorMadhulika Kabra, Corresponding Author Madhulika Kabra [email protected] Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaAdditional Professor, Genetics Division, Department of Pediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, India, 110029.Search for more papers by this authorRaju Sharma, Raju Sharma Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorDeepika Deka, Deepika Deka Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this authorVeena Kalra, Veena Kalra Genetics Division, Department of Paediatrics, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, IndiaSearch for more papers by this author First published: 10 March 2008 https://doi.org/10.1002/pd.1931Citations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES Ben-Zeev B, Levy-Nissenbaum E, Lahat H, et al. 2002. Megalencephalic with subcortical cysts; a founder effect in Israeli patients and a higher than expected carrier rate among Libean Jews. Hum Genet 111: 214– 218. Budowle B, Chakraborty R, Giusti AM, Eisenberg AJ, Allen RC. 1991. Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE. Am J Hum Genet 48: 137– 144. Decorte R, Cuppens H, Marynen P, Cassiman JJ. 1990. Rapid detection of hypervariable regions by the polymerase chain reaction technique. DNA Cell Biol 9: 461– 469. Goutieres F, Boulloche J, Bourgeois M, Aicardi J. 1996. Leukoencephalopathy, megalencephaly, and mild clinical course. A recently individualized familial leukodystrophy. Report on five new cases. J Child Neurol 11: 439– 444. Gorospe JR, Singhal BS, Kainu T, et al. 2004. Indian Agarwal megalencephalic leukodystrophy with cysts is caused by a common MLC1 mutation. 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Citing Literature Volume28, Issue4April 2008Pages 357-359 ReferencesRelatedInformation

Background: Cystic fibrosis (CF) is considered to be very rare in Indian subcontinent. Based on reports of CF in migrants from Indian subcontinent to United Kingdom and United States of America, the prevalence of CF is estimated to be between 1/10,000 and 1/40,000 in this ethnic group. The present study was done to estimate the carrier frequency of F508del mutation among neonates using cord blood samples to reflect the prevalence of CF in the study population. Methods: 955 mothers delivering at our hospital between December 1999 and November 2000 were enrolled. Cord blood samples were analyzed for F508del mutation using polymerase chain reaction and gel electrophoresis. The frequency of patients homozygous for F508del mutation in the population was estimated using Hardy–Weinberg principle. The prevalence of CF was estimated by using the proportion of F508del homozygous cases out of all CF patients, as reported in various studies (19–44%) from Indian subcontinent. Results: Out of 955 cord blood samples, 4 were positive for F508del mutation. The carrier frequency and gene frequency of F508del mutation in the Indian population was calculated to be 1/238 (0.42%) and 1/477 (0.21%), respectively. Frequency of CF patients homozygous for F508del mutation is 1/228,006. The estimated prevalence of CF is 1/43,321 to 1/100,323 in Indian population. Conclusion: CF does occur in Indian subcontinent though the prevalence is lesser than the Caucasian population.

BACKGROUND: First trimester pregnancy loss is a very common complication and a matter of concern for couples planning pregnancy. Balanced chromosomal rearrangements in either parent is an important cause of recurrent pregnancy loss particularly in the first trimester. AIMS: In this study an evaluation of the contribution of chromosomal anomalies in causing repeated spontaneous abortions was made. METHODS AND MATERIALS: A review of the cytogenetic data in 742 couples (1484 individuals) with recurrent spontaneous abortions who were examined for chromosomal aberrations in the period 1990-2003 is presented. Women who had at least two abortions, or spontaneous abortions preceded or followed by fetal deaths or birth of a malformed child, and patients who had recurrent spontaneous abortions (> 3) with normal live issue/s were studied. RESULTS: Chromosomal rearrangements were found in 31 individuals (2%). These abnormalities included 22 (2.9%) structural aberrations, 9 (1.2%) numerical anomalies. In addition to these abnormalities, 21 (3.2%) chromosomal variants were also found. CONCLUSION: Chromosomal analysis is an important etiological investigation in couples with repeated spontaneous abortions as it helps in genetic counseling and deciding about further reproductive options.