Computational biology insights into genotype-clinical phenotype-protein phenotype relationships between novel SLC26A2 variants identified in inherited skeletal dysplasias.

BACKGROUND: Pathogenic variants in the transmembrane sulfate transporter protein SLC26A2 are associated with different phenotypes of inherited chondrodysplasias. As limited data is published from India, in this study we sought to elucidate the molecular basis of inherited chondrodysplasias in an Indian cohort. METHODS: Molecular screening of 32 fetuses with antenatally diagnosed lethal skeletal dysplasia was performed by next generation sequencing and Sanger sequencing. The genotype-protein phenotype characterization was done using computational biology techniques like homology modelling, stability and pathogenicity predictions. RESULTS: We identified five rare autosomal recessive SLC26A2 [NM_000112.4] variants, including three homozygous c.796dupA(p.Thr266Asnfs*12), c.1724delA(p.Lys575Serfs*10), and c.1375_1377dup(p.Val459dup) and two heterozygous variants (c.532C > T(p.Arg178*)) and (c.1382C > T(p.Ala461Val)) in compound heterozygous form in a total of four foetuses. Genotype-protein phenotype annotations highlighted that the clinically severe achondrogenesis 1B causative c.796dupA(p.Thr266Asnfs*12) and c.1724delA(p.Lys575Serfs*10)variants impact SLC26A2 protein structure by deletion of the protein core and transmembrane STAS domains, respectively. In clinically moderate atelosteogenesis type 2 phenotype, the c.1382C > T(p.Ala461Val) variant is predicted to distort alpha helix conformation and alter the bonding properties and free energy dynamics of transmembrane domains and the c.532C > T(p.Arg178*) variant results in loss of both core transmembrane and STAS domains of the SLC26A2 protein. The c.1375_1377dup(p.Val459dup) variant identified in clinically milder atelosteogenesis type II-diastrophic dysplasia spectrum lethal phenotype is predicted to decrease the Qualitative Model Energy Analysis (QMean), which affects major geometrical aspects of the SLC26A2 protein structure. CONCLUSION: We expand the spectrum of SLC26A2 related lethal chondrodysplasia and report three novel variants correlating clinical severity and protein phenotype within the lethal spectrum of this rare dysplasia. We demonstrate the relevance of structural characterization to aid novel variant reclassification to provide better prenatal management and reproductive options to families with lethal antenatal skeletal disorder.

NGS-based expanded carrier screening for genetic disorders in North Indian population reveals unexpected results - a pilot study.

BACKGROUND: To determine the carrier frequency and pathogenic variants of common genetic disorders in the north Indian population by using next generation sequencing (NGS). METHODS: After pre-test counselling, 200 unrelated individuals (including 88 couples) were screened for pathogenic variants in 88 genes by NGS technology. The variants were classified as per American College of Medical Genetics criteria. Pathogenic and likely pathogenic variants were subjected to thorough literature-based curation in addition to the regular filters. Variants of unknown significance were not reported. Individuals were counselled explaining the implications of the results, and cascade screening was advised when necessary. RESULTS: Of the 200 participants, 52 (26%) were found to be carrier of one or more disorders. Twelve individuals were identified to be carriers for congenital deafness, giving a carrier frequency of one in 17 for one of the four genes tested (SLC26A4, GJB2, TMPRSS3 and TMC1 in decreasing order). Nine individuals were observed to be carriers for cystic fibrosis, with a frequency of one in 22. Three individuals were detected to be carriers for Pompe disease (frequency one in 67). None of the 88 couples screened were found to be carriers for the same disorder. The pathogenic variants observed in many disorders (such as deafness, cystic fibrosis, Pompe disease, Canavan disease, primary hyperoxaluria, junctional epidermolysis bullosa, galactosemia, medium chain acyl CoA deficiency etc.) were different from those commonly observed in the West. CONCLUSION: A higher carrier frequency for genetic deafness, cystic fibrosis and Pompe disease was unexpected, and contrary to the generally held view about their prevalence in Asian Indians. In spite of the small sample size, this study would suggest that population-based carrier screening panels for India would differ from those in the West, and need to be selected with due care. Testing should comprise the study of all the coding exons with its boundaries in the genes through NGS, as all the variants are not well characterized. Only study of entire coding regions in the genes will detect carriers with adequate efficiency, in order to reduce the burden of genetic disorders in India and other resource poor countries.

Infantile Systemic Hyalinosis: Novel Founder Mutation in the Initiation Codon among "Malis (Farmers)" in Jodhpur.

Infantile systemic hyalinosis (OMIM 236490) is a progressive autosomal recessive disorder characterized by widespread deposition of hyaline material in many tissues leading to multiple subcutaneous skin nodules, gingival hypertrophy and joint contractures. The authors describe five children from four unrelated families, from the "mali (farmer)" community in Jodhpur, with the disorder. All of them had classical clinical features, and four died from severe infections between age of 7 mo to 3 y. Two affected children had the same, but novel mutation in the initiation codon, in homozygous form c.1 A > G; p. M1? in capillary morphogenesis protein-2 (CMG2), or ANTXR2 gene on chromosome 4q21.21. The other two parents had the same mutation in heterozygous form. It is likely that this is a founder mutation in this community.

Inherited metabolic disorders: prenatal diagnosis of lysosomal storage disorders.

OBJECTIVE: To offer accurate prenatal diagnosis of lysosomal storage disorders in early pregnancy. METHOD: Prenatal enzymatic diagnoses of Gaucher, Fabry, Pompe, Niemann Pick A/B, Tay Sach, Sandoff, GM1, mucoplysaccharidoses, Wolman, Krabbe, Metachromatic leukodystrophy and Batten diseases were made in uncultured chorionic villi samples by fluorometric/spectrophotometric methods. RESULTS: Of 331 prenatal enzymatic diagnosis, 207 fetuses (67%) were normal and 124 (37%) were affected. The interpretation of affected, normal and carrier fetuses was done using their respective reference ranges as well as % enzyme activity of normal mean. The prenatal molecular confirmation was feasible in 43 biochemically diagnosed fetuses. Of the 207 normal reported fetuses, post natal enzymatic confirmation was done in 23 babies, clinical status of another 165 babies was assessed as unaffected via questionnaire on telephone and 19 were lost to follow-up. In affected pregnancies, 123 opted for termination of which 44 were confirmed enzymatically after abortion. A single false positive was determined to be a carrier by prenatal mutation analysis and carried to term. CONCLUSION: We recommend uncultured chorionic villi for reliable prenatal enzymatic diagnosis of various lysosomal storage disorders on account of the low rate of false positive (0.5%) and false negative (2.2%) results.

Identification of a Novel TAZ Gene Mutation in a Family With X-Linked Dilated Cardiomyopathy Barth Syndrome

Abstract Mutations in the tafazzin (TAZ) gene on chromosome Xq28 are responsible for the Barth syndrome (BTHS) phenotype resulting in a loss of function in the protein tafazzin involved in the transacylation of cardiolipin, an essential mitochondrial phospholipid. TAZ gene was investigated in the proband in our study, who died of dilated cardiomyopathy at 8 months of age, and his family by sequencing to identify the genetic cause of BTHS. Molecular analysis revealed a novel mutation in exon 5 (c.520T>G) of the TAZ gene. This novel mutation c.520T>G, pW174G, was also found in female carriers (mother and grandmother of proband) in the family. Bioinformatic analysis was carried out to examine the effect of mutation in the gene and confirmed the deleterious effect of this single mutation to the protein structure. Protein modeling and 3-dimensional structure of TAZ protein demonstrated the significantly visible changes in mutated protein leading to BTHS phenotype. Prenatal diagnosis in a subsequent pregnancy showed a carrier female, and pregnancy was continued. Child is doing well at 1 year of age.

An unforgettable concurrence: Successfully managed gallstone ileus accompanied by diabetic nephropathy.

BACKGROUND: Gallstone ileus is an uncommon complication of gall stones associated with potentially serious morbidity and mortality. CASE REPORT: We reported a 60-year-old male case who presented with renal failure and pain in right hypochondriac region. He also had a history of brain infarcts along with diabetes which is an additional factor for mortality. On Computed Tomography of the abdomen, he was diagnosed to have cholecystocholedochal fistula including air in the gall bladder and obstruction in the distal part of the ileum. Computed tomography plays an important role to make the proper diagnosis and in treatment. CONCLUSIONS: As in our case, diagnosis was challengeable because of renal failure,diabetes, septicaemia and intestinal obstruction (peritonitis). We did surgery on the basis of peritonitis which remains the only choice in such cases. In follow- up of 1 month patient was doing well and asymptomatic.

An unusual presentation of giant extrathoracic tumor in a child--managed successfully.

Lipoma is one of the most common mesenchymal tumors, usually seen over the trunk and proximal portions of the extremities. However, giant lipomas are very rarely seen over the chest wall and axilla in children. In rural areas, patients usually do not bother to be treated, which leads to complications, as seen in our case. Here, we report a rare case of a three-year-old child who presented with a large swelling on the right side of the anterior chest wall and in the axillary region. On histopathology, the diagnosis was lipoma. At the six-month follow-up, the child was normal without any complications. The clinical features, radiological and pathological findings and management of the lesion are discussed. If such a large tumor is not treated in a timely manner, it can cause respiratory discomfort or lead to malignancy.

Mutation analysis of the CFTR gene in 225 children: identification of five novel severe and seven reported severe mutations.

BACKGROUND: Cystic fibrosis (CF) is the most common inherited disorder in Caucasian populations, with more than 1400 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations. The type of mutations and their distributions varies widely between different countries and/or ethnic groups. METHODS: We characterized the mutations in the CFTR gene by single-strand conformation polymorphism followed by sequencing in CF patients. RESULTS: Twelve mutations were found in 79/225 (35.1%) patients. The most frequent mutations were F508 deletion (31.1%), p.R1162× (2.2%), p.M1T (0.8%), and S559N (0.8%). Five novel severe mutations (p.R80N11fs*11, p.R75G, p.Y577×, p.Y808Yfs*10, and p.I331×) and three reported mutations (p.C343×, p.Ile1000×, p.M469V) were detected. CONCLUSION: The protocol for identification of mutations in cases of CF in developing countries would have to include a different set of mutations than those reported from western countries.

Kearns Sayre Syndrome--case report with review of literature.

Kearns-Sayre Syndrome is form of rare mitochondrial cytopathy, first described by Thomas P. Kearns and George Pomeroy Sayre in 1958 and is characterized by progressive external opthalmoplegia, cardiac conduction block, pigmentary retinal degeneration, variable number of red ragged fibers on muscle biopsy. It presents before the child reaches the age of twenty. Kearns-Sayre syndrome may affect many organ systems and additional features may include myopathy, dystonia, bulbar symptoms in the form of dysarthria and nasal regurgitation and bilateral facial weakness. Endocrine abnormalities (e.g., diabetes, growth retardation/short stature, and hypoparathyroidism), bilateral sensorineural deafness, dementia, cataracts, and proximal renal tubular acidosis, skeletal muscle weakness (proximal more than distal) and exercise intolerance are additional features. Kearns Sayre Syndrome occurs as a result of large-scale single deletions (or rearrangements) of mitochondrial DNA (mtDNA), which is usually not inherited but occurs spontaneously, probably at the germ-cell level or very early in embryonic development. No disease-modifying therapy is available for Kearns-Sayre syndrome (KSS). Management is supportive vigilance for detection of associated problems. In the future, potential treatment in patients with Kearns-Sayre syndrome may attempt to inhibit mutant mtDNA replication or encourage replication of wild-type mtDNA.

Past, present & future scenario of thalassaemic care & control in India.

The first case of thalassaemia, described in a non-Mediterranean person, was from India. Subsequently, cases of thalassaemia were documented in all parts of India. Centres for care of thalassaemics were started in the mid-1970s in Mumbai and Delhi, and then in other cities. The parent's associations, with the help of International Thalassemia Federation, greatly helped in improving the care of thalassaemics. Obtaining blood for transfusion was difficult, but the Indian Red Cross Society and the parent's associations played a crucial role in arranging voluntary donations of blood. Chelation with deferoxamine was used sparingly due to the high cost. The Indian physicians conducted trials with deferiprone, and the drug was first approved and marketed in India. Deferasirox is also now being administered. Studies of physical and pubertal growth documented significant retardation, suggesting that generally patients receive inadequate chelation and transfusions. Bone marrow transplantation is available at a number of centres, and cord blood stem cell storage facilities have been established. Information about mutations in different parts of India is available, and ThalInd, an Indian database has been set up. There is a need to set up preimplantation genetic diagnosis and non-invasive prenatal diagnosis. It is argued that too much emphasis should not be placed on premarital screening. The focus should be on screening pregnant women to yield immediate results in reducing the burden of this disorder. Care of thalassaemia has been included in the 12 th 5-year Plan of the Government of India. Many States now provide blood transfusions and chelation free of cost. Although inadequacies in care of thalassaemia remain, but the outlook is bright, and the stage is set for initiating a control programme in the high risk States.

Molecular studies of achondroplasia.

BACKGROUND: Achondroplasia (ACH) is the most frequent form of short-limbed dwarfism, caused by mutations in the FGFR3 gene. It follows an autosomal dominant inheritance, though most cases are sporadic. The molecular techniques are the only available methods to confirm the diagnosis of a skeletal dysplasia. Clinical and radiological features are only suggestive and not confirmatory. The present study was conducted to find out how often the clinical diagnosis of achondroplasia is verified on molecular studies. MATERIALS AND METHODS: From 1998 through 2007, we carried out molecular analysis for the two common mutations in the FGFR3 gene in 130 cases clinically suspected to have ACH. RESULTS: A diagnostic mutation was identified in 53 (40.8%) cases. The common mutation (1138G>A) was present in 50 (94.7%) of the positive cases, while the rare 1138 G>C substitution was found in three (5.3%). CONCLUSION: This study shows that confirmation of clinical diagnosis of ACH by molecular genetic testing is essential to distinguish it from other skeletal dysplasias, to plan therapeutic options, and to offer genetic counseling. Management (medical and surgical) in patients confirmed to have ACH, is briefly discussed.