Lessons learned from additional research analyses of unsolved clinical exome cases

Abstract

Background: Given the rarity of most single-gene Mendelian disorders, concerted efforts of data exchange between clinical and scientific communities are critical to optimize molecular diagnosis and novel disease gene discovery.

Methods: We designed and implemented protocols for the study of cases for which a plausible molecular diagnosis was not achieved in a clinical genomics diagnostic laboratory (i.e. unsolved clinical exomes). Such cases were recruited to a research laboratory for further analyses, in order to potentially: (1) accelerate novel disease gene discovery; (2) increase the molecular diagnostic yield of whole exome sequencing (WES); and (3) gain insight into the genetic mechanisms of disease. Pilot project data included 74 families, consisting mostly of parent-offspring trios. Analyses performed on a research basis employed both WES from additional family members and complementary bioinformatics approaches and protocols.

Results: Analysis of all possible modes of Mendelian inheritance, focusing on both single nucleotide variants (SNV) and copy number variant (CNV) alleles, yielded a likely contributory variant in 36% (27/74) of cases. If one includes candidate genes with variants identified within a single family, a potential contributory variant was identified in a total of ~51% (38/74) of cases enrolled in this pilot study. The molecular diagnosis was achieved in 30/63 trios (47.6%). Besides this, the analysis workflow yielded evidence for pathogenic variants in disease-associated genes in 4/6 singleton cases (66.6%), 1/1 multiplex family involving three affected siblings, and 3/4 (75%) quartet families. Both the analytical pipeline and the collaborative efforts between the diagnostic and research laboratories provided insights that allowed recent disease gene discoveries (PURA, TANGO2, EMC1, GNB5, ATAD3A, and MIPEP) and increased the number of novel genes, defined in this study as genes identified in more than one family (DHX30 and EBF3).

Conclusion: An efficient genomics pipeline in which clinical sequencing in a diagnostic laboratory is followed by the detailed reanalysis of unsolved cases in a research environment, supplemented with WES data from additional family members, and subject to adjuvant bioinformatics analyses including relaxed variant filtering parameters in informatics pipelines, can enhance the molecular diagnostic yield and provide mechanistic insights into Mendelian disorders. Implementing these approaches requires collaborative clinical molecular diagnostic and research efforts.

Fig. 1

Analysis of WES data. a SNVs were filtered and prioritized according to specific criteria, including mode of inheritance, mutation type, variant frequency, conservation, and predictions of pathogenicity. b Candidate genes were further prioritized by data mining, taking into consideration gene function, expression, and networks. In addition, other cohorts were interrogated for additional families with variants in the same candidate gene. MutationMapper (http://www.cbioportal.org/mutation_mapper.jsp), ARIC Atherosclerosis Risk in Communities Study, AR-Hom autosomal recessive-homozygous, BHCMG Baylor-Hopkins Center for Mendelian Genomics, BG Baylor Genetics laboratories, CNV copy number variation, Comp compound, db database, ExAC Exome Aggregation Consortium, Het heterozygous, HGMD Human Gene Mutation Database, MAF minor allele frequency; SNV single nucleotide variant, XLR-Hem X-linked recessive-hemizygous

Fig. 2

Overview of the study design and results. a Clinical WES cases that lacked a definitive molecular diagnosis (left) were eligible for recruitment into a research environment. In a pilot study of 74 families (right), we identified strong candidate genes for 51% (38/74) of cases. Identified variants were categorized into six major classes based on mode of inheritance and known or novel gene. b According to stringent criteria, a potential contributory variant was achieved in 27 of 74 (36%) cases. Of these, 12 were independently solved by the clinical exome laboratory upon reanalysis of WES data and updated literature review. When taking into account strong candidate genes identified in only a single family to date, a potential molecular diagnostic rate of 51% was achieved

Fig. 3

Location of DHX30 and GNB5 variants, dinucleotide variants culled from WES data and UPD. a Variants identified in DHX30 and GNB5 are located in specific protein domains. b Sanger confirmation of a de novo dinucleotide variant in SYN3. c The B-allele frequency extracted from WES data in the patient with the homozygous SLC1A4 variant showed a single region of AOH in the genome (chromosome 2), suggestive of uniparental disomy (UPD) of chromosome 2. d Segregation analysis of the SLC1A4 homozygous variant did not conform to Mendelian expectations