Background About half of malignant hyperthermia (MH) cases are associated with skeletal muscle ryanodine receptor 1 (RYR1) and calcium channel, voltage-dependent, L type, α1S subunit (CACNA1S) gene mutations, leaving many with an unknown cause. We chose to apply a sequencing approach to uncover causal variants in unknown cases. Sequencing the exome, the protein-coding region of the genome, has power at low sample sizes and identified the cause of over a dozen Mendelian disorders. Methods We considered four families with multiple MH cases but in whom no mutations in RYR1 and CACNA1S had been identified by Sanger sequencing of complementary DNA. Exome sequencing of two affecteds per family, chosen for maximum genetic distance, were compared. Variants were ranked by allele frequency, protein change, and measures of conservation among mammals to assess likelihood of causation. Finally, putative pathogenic mutations were genotyped in other family members to verify cosegregation with MH. Results Exome sequencing revealed 1 rare RYR1 nonsynonymous variant in each of 3 families (Asp1056His, Val2627Met, Val4234Leu), and 1 CACNA1S variant (Thr1009Lys) in a 4th family. These were not seen in variant databases or in our control population sample of 5379 exomes. Follow-up sequencing in other family members verified cosegregation of alleles with MH. Conclusions Using both exome sequencing and allele frequency data from large sequencing efforts may aid genetic diagnosis of MH. In our sample, it was more sensitive for variant detection in known genes than Sanger sequencing of complementary DNA, and allows for the possibility of novel gene discovery.

Nasal polyposis are common presentations in patients of chronic rhinosinusitis and are considered to be associated with more severe forms of disease with poor treatment outcome. The presentation and treatment outcome after endoscopic sinus surgery in patients of chronic rhinosinusitis and nasal polyposis have been analysed in this study. A prospective analysis of 90 patients of chronic rhinosinusitis who were classified into two groups depending on presence and absence of nasal polyps was performed in the study. The two groups were evaluated using subjective (patient complaints) and objective (computed tomography scan and endoscopy scores) criteria. Preoperative data were compared with data obtained 12 months post endoscopic sinus surgery. The study included 38 patients of chronic rhinosinusitis and 52 patients of nasal polyps. The patients of nasal polyp group presented with increased severity of symptoms of nasal blockage, nasal discharge and reduced sense of smell as compared to the chronic rhinosinusitis group who had significantly higher presentation of headache and facial pain. The preoperative CT scan revealed significantly higher bilateral disease with increased involvement of multiple sinuses in nasal polyp group. Post endoscopic sinus surgery both the groups showed significant improvement in their symptoms with the nasal polyp group demonstrating reduction in improvement on 1 year follow up. In our study we have found the patients with chronic rhinosinusitis and nasal polyp have varied severity of symptoms with the nasal polyp group having higher nasal symptoms and increased severity as compared to chronic rhinosinusitis group. Though the universal rationale of management by adequate drainage and ventilation of sinus is similar in both groups, there is a reduction in both objective and subjective scores during 1 year follow up in the nasal polyp group.

Purpose: Detection of all major classes of genomic variants in a single test would decrease cost and increase the efficiency of genomic diagnostics. Genome sequencing (GS) has the potential to provide this level of comprehensive detection. We sought to demonstrate the utility of GS in the molecular diagnosis of 18 patients with clinically defined Alagille syndrome (ALGS), who had a negative or inconclusive result by standard-of-care testing. Methods: We performed GS on 16 pathogenic variant-negative probands and two probands with inconclusive results (of 406 ALGS probands) and analyzed the data for sequence, copy-number, and structural variants in JAG1 and NOTCH2. Results: GS identified four novel pathogenic alterations including a copy-neutral inversion, a partial deletion, and a promoter variant in JAG1, and a partial NOTCH2 deletion, for an additional diagnostic yield of 0.9%. Furthermore, GS resolved two complex rearrangements, resulting in identification of a pathogenic variant in 97.5% (n = 396/406) of patients after GS. Conclusion: GS provided an increased diagnostic yield for individuals with clinically defined ALGS who had prior negative or incomplete genetic testing by other methods. Our results show that GS can detect all major classes of variants and has potential to become a single first-tier diagnostic test for Mendelian disorders.

Gastrointestinal motility disorders include a spectrum of mild to severe clinical phenotypes that are caused by smooth muscle dysfunction. We investigated the genetic etiology of severe esophageal, gastric, and colonic dysmotility in two unrelated families with autosomal dominant disease presentation. Using exome sequencing, we identified a 2 base pair insertion at the end of the myosin heavy chain 11 (MYH11) gene in all affected members of Family 1 [NM_001040113:c. 5819_5820insCA(p.Gln1941Asnfs*91)] and a 1 base pair deletion at the same genetic locus in Proband 2 [NM_001040113:c.5819del(p.Pro1940Hisfs*91)]. Both variants are predicted to result in a similarly elongated protein product. Heterozygous dominant negative MYH11 pathogenic variants have been associated with thoracic aortic aneurysm and dissection while biallelic null alleles have been associated with megacystis microcolon intestinal hypoperistalsis syndrome. This report highlights heterozygous protein-elongating MYH11 variants affecting the SM2 isoforms of MYH11 as a cause for severe gastrointestinal dysmotility, and we hypothesize that the mechanistic pathogenesis of this disease, dominant hypercontractile loss-of-function, is distinct from those implicated in other diseases involving MYH11 dysfunction. K E Y W O R D S dysmotility, gastroparesis, hiatal hernia, MYH11, pseudo-obstruction

Mutations in CDH23 are known to cause autosomal-recessive nonsyndromic hearing loss (DFNB12). Until now, there was only one study describing its frequency in Indian population. We screened for CDH23 mutations to identify prevalent and recurring mutations among South Indian assortative mating hearing-impaired individuals who were identified as non-DFNB1 (GJB2 and GJB6). Whole-exome sequencing was performed in individuals found to be heterozygous for CDH23 to determine whether there was a second pathogenic allele. In our study, 19 variants including 6 pathogenic missense mutations were identified. The allelic frequency of pathogenic mutations accounts to 4.7% in our cohort, which is higher than that reported previously; three mutations (c.429+4G>A, c.2968G>A, and c.5660C>T) reported in the previous Indian study were found to recur. DFNB12 was found to be the etiology in 3.4% of our cohort, with missense mutation c.2968G>A (p.Asp990Asn) being the most prevalent (2.6%). These results suggest a need to investigate the possibility for higher proportion of CDH23 mutations in the South Indian hearing-impaired population.

BackgroundDFNB1, the first locus to have been associated with deafness, has two major genes GJB2 & GJB6, whose mutations have played vital role in hearing impairment across many ethnicities in the world. In our present study we have focused on the role of these mutations in assortative mating hearing impaired families from south India.MethodsOne hundred and six assortatively mating hearing impaired (HI) families of south Indian origin comprising of two subsets: 60 deaf marrying deaf (DXD) families and 46 deaf marrying normal hearing (DXN) families were recruited for this study. In the 60 DXD families, 335 members comprising of 118 HI mates, 63 other HI members and 154 normal hearing members and in the 46 DXN families, 281 members comprising of 46 HI and their 43 normal hearing partners, 50 other HI members and 142 normal hearing family members, participated in the molecular study. One hundred and sixty five (165) healthy normal hearing volunteers were recruited as controls for this study. All the participating members were screened for variants in GJB2 and GJB6 genes and the outcome of gene mutations were compared in the subsequent generation in begetting deaf offspring.ResultsThe DFNB1 allele frequencies for DXD mates and their offspring were 36.98 and 38.67%, respectively and for the DXN mates and their offspring were 22.84 and 24.38%, respectively. There was a 4.6% increase in the subsequent generation in the DXD families, while a 6.75% increase in the DXN families, which demonstrates the role of assortative mating along with consanguinity in the increase of DFNB1 mutations in consecutive generations. Four novel variants, p.E42D (in GJB2 gene), p.Q57R, p.E101Q, p.R104H (in GJB6 gene) were also identified in this study.ConclusionThis is the first study from an Indian subcontinent reporting novel variants in the coding region of GJB6 gene. This is perhaps the first study in the world to test real-time, the hypothesis proposed by Nance et al. in 2000 (intense phenotypic assortative mating mechanism can double the frequency of the commonest forms of recessive deafness [DFNB1]) in assortative mating HI parental generation and their offspring.

SummaryHearing loss is the most common sensory disorder and is genetically heterogeneous. Apart from nuclear gene mutations, a number of inherited mitochondrial mutations have also been implicated. The m.1555A>G mutation in the mitochondrial MT-RNR1 gene is reported as the most common mutation causing nonsyndromic hearing loss in various ethnic populations. We report here for the first time the clinical, genetic and molecular characterisation of a single large five-generational Tamil-speaking South Indian family with maternally inherited nonsyndromic postlingual hearing loss. Molecular analysis led to identification of m.1555A>G in 28 maternal relatives with variable degree of phenotypic expression. The penetrance of hearing loss among the maternal relatives in this family was 55%. Sequence analysis of the complete mitochondrial genome in 36 members of this pedigree identified 25 known variants and one novel variant co-transmitted along with m.1555A>G mutation. The mtDNA haplotype analysis revealed that the maternal relatives carry the R * T 2 haplotype similar to Europeans and South Asians. Sequencing of the coding exon of GJB2 nuclear gene did not show any pathogenic mutations. The results suggest that other nuclear or environmental modifying factors could have played a role in the differential expression of mutation m.1555A>G in postlingual hearing loss in this family.

SummaryMutations in the GJB2 gene encoding the gap junction protein Connexin 26 have been associated with autosomal recessive as well as dominant nonsyndromic hearing loss. Owing to the involvement of connexins in skin homeostasis, GJB2 mutations have also been associated with syndromic forms of hearing loss showing various skin manifestations. We report an assortatively mating hearing impaired family of south Indian origin with three affected members spread over two generations, having p.R75Q mutation in the GJB2 gene in the heterozygous condition. The inheritance pattern was autosomal dominant with mother and son being affected. Dermatological and histopathologic examinations showed absence of palmoplantar keratoderma. To the best of our knowledge, this is the first report from India on p.R75Q mutation in the GJB2 gene with nonsyndromic hearing loss.