Krishna Kumar Kandaswamy

Clinical exome sequencing: results from 2819 samples reflecting 1000 families.

We report our results of 1000 diagnostic WES cases based on 2819 sequenced samples from 54 countries with a wide phenotypic spectrum. Clinical information given by the requesting physicians was translated to HPO terms. WES processes were performed according to standardized settings. We identified the underlying pathogenic or likely pathogenic variants in 307 families (30.7%). In further 253 families (25.3%) a variant of unknown significance, possibly explaining the clinical symptoms of the index patient was identified. WES enabled timely diagnosing of genetic diseases, validation of causality of specific genetic disorders of PTPN23, KCTD3, SCN3A, PPOX, FRMPD4, and SCN1B, and setting dual diagnoses by detecting two causative variants in distinct genes in the same patient. We observed a better diagnostic yield in consanguineous families, in severe and in syndromic phenotypes. Our results suggest that WES has a better yield in patients that present with several symptoms, rather than an isolated abnormality. We also validate the clinical benefit of WES as an effective diagnostic tool, particularly in nonspecific or heterogeneous phenotypes. We recommend WES as a first-line diagnostic in all cases without a clear differential diagnosis, to facilitate personal medical care.

Validation of a semiconductor next‐generation sequencing assay for the clinical genetic screening of CFTR

Genetic testing for cystic fibrosis and CFTR‐related disorders mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the CFTR gene. We have explored a more efficient genetic screening strategy based on next‐generation sequencing (NGS) of the CFTR gene. We validated this approach in a cohort of 177 patients with previously known CFTR mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq™ CFTR panel. The DNA libraries were pooled, barcoded, and sequenced using an Ion Torrent PGM sequencer. The combination of different robust bioinformatics tools allowed us to detect previously known pathogenic mutations and polymorphisms in the 177 samples, without detecting spurious pathogenic calls. In summary, the assay achieves a sensitivity of 94.45% (95% CI: 92% to 96.9%), with a specificity of detecting nonvariant sites from the CFTR reference sequence of 100% (95% CI: 100% to 100%), a positive predictive value of 100% (95% CI: 100% to 100%), and a negative predictive value of 99.99% (95% CI: 99.99% to 100%). In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases. Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly‐thymidine‐guanine and poly‐thymidine (poly‐TG‐T) tracts were also detected. Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene.

Homozygous deletion of exons 2 and 3 of NPC2 associated with Niemann–Pick disease type C

Homozygous Deletion of Exons 2 and 3 of NPC2 Associated with Niemann–Pick Disease Type C Malavika Hebbar, Harsha Prasada L, Aneek Das Bhowmik, Daniel Trujillano, Anju Shukla, Shrijeet Chakraborti, Krishna Kumar Kandaswamy, Arndt Rolfs, Nutan Kamath, Ashwin Dalal, Stephanie Bielas, and Katta Mohan Girisha* Department of Medical Genetics, Kasturba Medical College, Manipal University, Manipal, Karnataka, India Department of Pediatrics, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Andhra Pradesh, India Centogene AG, Rostock, Mecklenburg-Vorpommern, Germany Department of Pathology, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India Albrecht-Kossel-Institute for Neuroregeneration, Medical University Rostock, Rostock, Mecklenburg-Vorpommern, Germany Department of Human Genetics, University of Michigan, Ann Arbor, Michigan

Development of an evidence-based algorithm that optimizes sensitivity and specificity in ES-based diagnostics of a clinically heterogeneous patient population

PurposeNext-generation sequencing (NGS) is rapidly replacing Sanger sequencing in genetic diagnostics. Sensitivity and specificity of NGS approaches are not well-defined, but can be estimated from applying NGS and Sanger sequencing in parallel. Utilizing this strategy, we aimed at optimizing exome sequencing (ES)–based diagnostics of a clinically diverse patient population.MethodsConsecutive DNA samples from unrelated patients with suspected genetic disease were exome-sequenced; comparatively nonstringent criteria were applied in variant calling. One thousand forty-eight variants in genes compatible with the clinical diagnosis were followed up by Sanger sequencing. Based on a set of variant-specific features, predictors for true positives and true negatives were developed.ResultsSanger sequencing confirmed 81.9% of ES-derived variants. Calls from the lower end of stringency accounted for the majority of the false positives, but also contained ~5% of the true positives. A predictor incorporating three variant-specific features classified 91.7% of variants with 100% specificity and 99.75% sensitivity. Confirmation status of the remaining variants (8.3%) was not predictable.ConclusionsCriteria for variant calling in ES-based diagnostics impact on specificity and sensitivity. Confirmatory sequencing for a proportion of variants, therefore, remains a necessity. Our study exemplifies how these variants can be defined on an empirical basis.

A founder nonsense variant in NUDT2 causes a recessive neurodevelopmental disorder in Saudi Arab children

We identified the homozygous p.Arg12* variant in 5 patients with neurodevelopmental delay, but variation databases list many truncating heterozygous variants for this small 2-exon gene. As most of these affect the proteins C-terminus, loss-of-function mediated pathogenicity may be confined to bi-allelic truncating variants in exon 1 (nonsense-mediated decay!) or in the catalytically active Nudix box.

Expanding the clinical and genetic spectra of NKX6-2-related disorder

Hypomyelinating leukodystrophies (HLDs) affect the white matter of the central nervous system and manifest as neurological disorders. They are genetically heterogeneous. Very recently, biallelic variants in NKX6‐2 have been suggested to cause a novel form of autosomal recessive HLD. Using whole‐exome or whole‐genome sequencing, we identified the previously reported c.196delC and c.487C>G variants in NKX6‐2 in 3 and 2 unrelated index cases, respectively; the novel c.608G>A variant was identified in a sixth patient. All variants were homozygous in affected family members only. Our patients share a primary diagnosis of psychomotor delay, and they show spastic quadriparesis, nystagmus and hypotonia. Seizures and dysmorphic features (observed in 2 families each) represent an addition to the phenotype, while developmental regression (observed in 3 families) appears to be a notable and previously underestimated clinical feature. Our findings extend the clinical and mutational spectra associated with this novel form of HLD. Comparative analysis of our 10 patients and the 15 reported previously did, however, not reveal clear evidence for a genotype‐phenotype correlation.