Next-generation sequencing improves precision medicine in hearing loss.

Background: An early etiological diagnosis of hearing loss positively impacts children's quality of life including language and cognitive development. Even though hearing loss associates with extremely high genetic and allelic heterogeneity, several studies have proven that Next-Generation Sequencing (NGS)-based gene panel testing significantly reduces the time between onset and diagnosis. Methods: In order to assess the clinical utility of our custom NGS GHELP panel, the prevalence of pathogenic single nucleotide variants, indels or copy number variants was assessed by sequencing 171 nuclear and 8 mitochondrial genes in 155 Spanish individuals with hearing loss. Results: A genetic diagnosis of hearing loss was achieved in 34% (52/155) of the individuals (5 out of 52 were syndromic). Among the diagnosed cases, 87% (45/52) and 12% (6/52) associated with autosomal recessive and dominant inheritance patterns respectively; remarkably, 2% (1/52) associated with mitochondrial inheritance pattern. Although the most frequently mutated genes in this cohort were consistent with those described in the literature (GJB2, OTOF or MYO7A), causative variants in less frequent genes such as TMC1, FGF3 or mitCOX1 were also identified. Moreover, 5% of the diagnosed cases (3/52) were associated with pathogenic copy number variants. Conclusion: The clinical utility of NGS panels that allows identification of different types of pathogenic variants-not only single nucleotide variants/indels in both nuclear and mitochondrial genes but also copy number variants-has been demonstrated to reduce the clinical diagnostic odyssey in hearing loss. Thus, clinical implementation of genomic strategies within the regular clinical practice, and, more significantly, within the newborn screening protocols, is warranted.

Continued lessons from the INS gene: an intronic mutation causing diabetes through a novel mechanism.

BACKGROUND: Diabetes in neonates usually has a monogenic aetiology; however, the cause remains unknown in 20-30%. Heterozygous INS mutations represent one of the most common gene causes of neonatal diabetes mellitus. METHODS: Clinical and functional characterisation of a novel homozygous intronic mutation (c.187+241G>A) in the insulin gene in a child identified through the Monogenic Diabetes Registry (http://monogenicdiabetes.uchicago.edu). RESULTS: The proband had insulin-requiring diabetes from birth. Ultrasonography revealed a structurally normal pancreas and C-peptide was undetectable despite readily detectable amylin, suggesting the presence of dysfunctional ß cells. Whole-exome sequencing revealed the novel mutation. In silico analysis predicted a mutant mRNA product resulting from preferential recognition of a newly created splice site. Wild-type and mutant human insulin gene constructs were derived and transiently expressed in INS-1 cells. We confirmed the predicted transcript and found an additional transcript created via an ectopic splice acceptor site. CONCLUSIONS: Dominant INS mutations cause diabetes via a mutated translational product causing endoplasmic reticulum stress. We describe a novel mechanism of diabetes, without ß cell death, due to creation of two unstable mutant transcripts predicted to undergo nonsense and non-stop-mediated decay, respectively. Our discovery may have broader implications for those with insulin deficiency later in life.

Phenotypic heterogeneity in monogenic diabetes: the clinical and diagnostic utility of a gene panel-based next-generation sequencing approach.

Single gene mutations that primarily affect pancreatic ß-cell function account for approximately 1-2% of all cases of diabetes. Overlapping clinical features with common forms of diabetes makes diagnosis of monogenic diabetes challenging. A genetic diagnosis often leads to significant alterations in treatment, allows better prediction of disease prognosis and progression, and has implications for family members. Currently, genetic testing for monogenic diabetes relies on selection of appropriate individual genes for analysis based on the availability of often-limited phenotypic information, decreasing the likelihood of making a genetic diagnosis. We thus developed a targeted next-generation sequencing (NGS) assay for the detection of mutations in 36 genes known to cause monogenic forms of diabetes, including transient or permanent neonatal diabetes mellitus (TNDM or PNDM), maturity-onset diabetes of the young (MODY) and rare syndromic forms of diabetes. A total of 95 patient samples were analyzed: 19 with known causal mutations and 76 with a clinically suggestive phenotype but lacking a genetic diagnosis. All previously identified mutations were detected, validating our assay. Pathogenic sequence changes were identified in 19 out of 76 (25%) patients: 7 of 32 (22%) NDM cases, and 12 of 44 (27%) MODY cases. In 2 NDM patients the causal mutation was not expected as consanguinity was not reported and there were no clinical features aside from diabetes. A 3 year old patient with NDM diagnosed at 3 months of age, who previously tested negative for INS, KCNJ11 and ABCC8 mutations, was found to carry a novel homozygous mutation in EIF2AK3 (associated with Wolcott-Rallison syndrome), a gene not previously suspected because consanguinity, delayed growth, abnormal bone development and hepatic complications had not been reported. Similarly, another infant without a history of consanguinity was found to have a homozygous GCK mutation causing PNDM at birth. This study demonstrates the effectiveness of multi-gene panel analysis in uncovering molecular diagnoses in patients with monogenic forms of diabetes.

Replication of twelve association studies for Huntington's disease residual age of onset in large Venezuelan kindreds.

BACKGROUND: The major determinant of age of onset in Huntington's disease is the length of the causative triplet CAG repeat. Significant variance remains, however, in residual age of onset even after repeat length is factored out. Many genetic polymorphisms have previously shown evidence of association with age of onset of Huntington's disease in several different populations. OBJECTIVE: To replicate these genetic association tests in 443 affected people from a large set of kindreds from Venezuela. METHODS: Previously tested polymorphisms were analysed in the HD gene itself (HD), the GluR6 kainate glutamate receptor (GRIK2), apolipoprotein E (APOE), the transcriptional coactivator CA150 (TCERG1), the ubiquitin carboxy-terminal hydrolase L1 (UCHL1), p53 (TP53), caspase-activated DNase (DFFB), and the NR2A and NR2B glutamate receptor subunits (GRIN2A, GRIN2B). RESULTS: The GRIN2A single-nucleotide polymorphism explains a small but considerable amount of additional variance in residual age of onset in our sample. The TCERG1 microsatellite shows a trend towards association but does not reach statistical significance, perhaps because of the uninformative nature of the polymorphism caused by extreme allele frequencies. We did not replicate the genetic association of any of the other genes. CONCLUSIONS: GRIN2A and TCERG1 may show true association with residual age of onset for Huntington's disease. The most surprising negative result is for the GRIK2 (TAA)(n) polymorphism, which has previously shown association with age of onset in four independent populations with Huntington's disease. The lack of association in the Venezuelan kindreds may be due to the extremely low frequency of the key (TAA)(16) allele in this population.