Mono- and biallelic variant effects on disease at biobank scale.

Identifying causal factors for Mendelian and common diseases is an ongoing challenge in medical genetics1. Population bottleneck events, such as those that occurred in the history of the Finnish population, enrich some homozygous variants to higher frequencies, which facilitates the identification of variants that cause diseases with recessive inheritance2,3. Here we examine the homozygous and heterozygous effects of 44,370 coding variants on 2,444 disease phenotypes using data from the nationwide electronic health records of 176,899 Finnish individuals. We find associations for homozygous genotypes across a broad spectrum of phenotypes, including known associations with retinal dystrophy and novel associations with adult-onset cataract and female infertility. Of the recessive disease associations that we identify, 13 out of 20 would have been missed by the additive model that is typically used in genome-wide association studies. We use these results to find many known Mendelian variants whose inheritance cannot be adequately described by a conventional definition of dominant or recessive. In particular, we find variants that are known to cause diseases with recessive inheritance with significant heterozygous phenotypic effects. Similarly, we find presumed benign variants with disease effects. Our results show how biobanks, particularly in founder populations, can broaden our understanding of complex dosage effects of Mendelian variants on disease.

A framework for automated gene selection in genomic applications.

PURPOSE: An efficient framework to identify disease-associated genes is needed to evaluate genomic data for both individuals with an unknown disease etiology and those undergoing genomic screening. Here, we propose a framework for gene selection used in genomic analyses, including applications limited to genes with strong or established evidence levels and applications including genes with less or emerging evidence of disease association. METHODS: We extracted genes with evidence for gene-disease association from the Human Gene Mutation Database, OMIM, and ClinVar to build a comprehensive gene list of 6,145 genes. Next, we applied stringent filters in conjunction with computationally curated evidence (DisGeNET) to create a restrictive list limited to 3,929 genes with stronger disease associations. RESULTS: When compared to manual gene curation efforts, including the Clinical Genome Resource, genes with strong or definitive disease associations are included in both gene lists at high percentages, while genes with limited evidence are largely removed. We further confirmed the utility of this approach in identifying pathogenic and likely pathogenic variants in 45 genomes. CONCLUSION: Our approach efficiently creates highly sensitive gene lists for genomic applications, while remaining dynamic and updatable, enabling time savings in genomic applications.

Guidelines for investigating causality of sequence variants in human disease.

The discovery of rare genetic variants is accelerating, and clear guidelines for distinguishing disease-causing sequence variants from the many potentially functional variants present in any human genome are urgently needed. Without rigorous standards we risk an acceleration of false-positive reports of causality, which would impede the translation of genomic research findings into the clinical diagnostic setting and hinder biological understanding of disease. Here we discuss the key challenges of assessing sequence variants in human disease, integrating both gene-level and variant-level support for causality. We propose guidelines for summarizing confidence in variant pathogenicity and highlight several areas that require further resource development.

A systematic approach to assessing the clinical significance of genetic variants.

Molecular genetic testing informs diagnosis, prognosis, and risk assessment for patients and their family members. Recent advances in low-cost, high-throughput DNA sequencing and computing technologies have enabled the rapid expansion of genetic test content, resulting in dramatically increased numbers of DNA variants identified per test. To address this challenge, our laboratory has developed a systematic approach to thorough and efficient assessments of variants for pathogenicity determination. We first search for existing data in publications and databases including internal, collaborative and public resources. We then perform full evidence-based assessments through statistical analyses of observations in the general population and disease cohorts, evaluation of experimental data from in vivo or in vitro studies, and computational predictions of potential impacts of each variant. Finally, we weigh all evidence to reach an overall conclusion on the potential for each variant to be disease causing. In this report, we highlight the principles of variant assessment, address the caveats and pitfalls, and provide examples to illustrate the process. By sharing our experience and providing a framework for variant assessment, including access to a freely available customizable tool, we hope to help move towards standardized and consistent approaches to variant assessment.

Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa.

Usher syndrome type IIa (OMIM 276901), an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa, maps to the long arm of human chromosome 1q41 between markers AFM268ZD1 and AFM144XF2. Three biologically important mutations in Usher syndrome type IIa patients were identified in a gene (USH2A) isolated from this critical region. The USH2A gene encodes a protein with a predicted size of 171.5 kilodaltons that has laminin epidermal growth factor and fibronectin type III motifs; these motifs are most commonly observed in proteins comprising components of the basal lamina and extracellular matrixes and in cell adhesion molecules.

Connexin 26 studies in patients with sensorineural hearing loss.

OBJECTIVE: To determine the spectrum of connexin 26 (Cx26) mutations and their phenotypes in children with sensorineural hearing loss (SNHL) or mixed hearing loss (MHL). DESIGN: Children with SNHL or MHL were prospectively tested for mutations in the entire coding region of the Cx26 gene. PATIENTS: Children with SNHL or MHL with no obvious etiology for the hearing loss. RESULTS: Between December 1, 1998, and July 1, 2000, 107 patients with SNHL or MHL from 99 families underwent Cx26 testing. Most patients were aged 1 week to 16 years (61 boys and 46 girls). Thirty (30%) of 99 probands had Cx26 mutations: biallelic mutations were detected in 18 (9 homozygous and 9 compound heterozygous) and single mutations were detected in 12. Twelve previously reported mutations (35delG, 167delT, E47X, L90P, M34T, G12V, V37I, R143W, V84L, V153I, V27I, and 310del14) and 3 novel mutations (E129K, T8M, and N206S) were found. Hearing loss in patients with biallelic Cx26 mutations ranged from unilateral high frequency to bilateral profound. Four children, 2 with biallelic mutations, had temporal bone abnormalities. CONCLUSIONS: Connexin 26 mutations are common in children with SNHL, and it is likely that the homozygous and compound heterozygous mutations cause the SNHL. However, pathogenicity is less certain when only a single Cx26 mutation is present. Patients with biallelic Cx26 mutations had a slightly higher incidence of milder hearing loss than in previous studies. Children with SNHL or MHL should be tested for Cx26 mutations early in their evaluation.

A new age in the genetics of deafness.

Recent advancements have been made in understanding, diagnosing, and treating deafness. In particular, much has been learned from the discovery of a small fraction of the genes responsible for deafness. This understanding will doubtless increase as additional genes are cloned and their functions elucidated. Trailing close behind these achievements will be more clinical advancements facilitating diagnosis of the etiologies of deafness. Integrating these genetic and clinical perspectives is critical to the development of better treatments and interventional strategies for deafness and its associated difficulties. Although opinions toward these advancements are likely to vary between the hearing population and the Deaf community, a growing understanding of the hearing process and how genetic variations result in deafness is ultimately likely to offer benefits to both groups.

Coordinate synthesis and turnover of heat shock proteins in Borrelia burgdorferi: degradation of DnaK during recovery from heat shock.

The synthesis and turnover of heat shock proteins (Hsps) by Borrelia burgdorferi, the Lyme disease spirochete, was investigated by radiolabeling of whole spirochetes and spheroplasts, comparison of one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and use of immunochemistry. The approximately 72-kDa DnaK homolog and three additional Hsps of 39, 27, and 21 kDa increased in amount by 3- to 15-fold between 2 and 6 h following temperature upshift from 28 to 39 degrees C. Temperature downshift experiments following the transfer of spirochetes from 40 to 28 degrees C showed that within 15 to 30 min, synthesis of most of the major Hsps returned to levels seen in spirochetes statically maintained at the lower temperature. Spheroplasts of B. burgdorferi produced by treatment with EDTA and lysozyme were radiolabeled, and specific Hsps were localized to either the cytoplasm or membrane fraction. Further analysis by two-dimensional electrophoresis demonstrated three constitutively expressed DnaK isoforms with pIs near 5.5. A pattern suggestive of DnaK degradation was observed following recovery from heat shock but not in spirochetes maintained entirely at a low temperature. Some of these putative degradation products were recognized by monoclonal antibodies directed against the B. burgdorferi DnaK protein. These data suggest that following a period of peak synthesis, DnaK is actively degraded as the spirochete reestablishes its metabolic thermometer. These findings provide a new interpretation of previous work suggesting that 10 to 15 B. burgdorferi polypeptides, including DnaK have a common epitope.

Norrie disease gene mutation in a large Costa Rican kindred with a novel phenotype including venous insufficiency.

A large Costa Rican kindred has been identified with 15 males affected with congenital blindness, progressive bearing loss, and venous insufficiency. Due to ophthalmological and audio-otological findings, including bilateral retinal dysplasia and detachment, progressive bilateral sensorineural hearing loss, and an X-linked pattern of inheritance, a tentative diagnosis of Norrie disease was considered. However, venous insufficiency is a clinical finding not reportedly associated with Norrie disease. Genetic linkage analysis using microsatellite repeat markers demonstrated linkage to Xp11.23-11.4 (z = 2.723 at theta = 0.0). A candidate gene approach using the Norrie disease gene (NDP), which maps to Xp11.3, revealed a point mutation in the third exon resulting in substitution of phenylalanine for leucine at position 61. The precise function of the gene product, norrin, has yet to be elucidated; however, it has been postulated to be involved in the regulation of neural cell differentiation and proliferation, although hypotheses have been considered for its role in vascular development in the eye. The finding of a mutation in NDP in association with peripheral vascular disease may provide valuable insight into the potential role of this gene in cellular processes.