The Importance of Automation in Genetic Diagnosis: Lessons from Analyzing an Inherited Retinal Degeneration Cohort with the Mendelian Analysis Toolkit (MATK)

Purpose: In Mendelian disease diagnosis, variant analysis is a repetitive, error-prone, and time-consuming process. To address this, we have developed the Mendelian Analysis Toolkit (MATK), a configurable automated variant ranking program. Methods: MATK aggregates variant information from multiple annotation sources and uses expert-designed rules with parameterized weights to produce a ranked list of potentially causal solutions. MATK performance was measured by a comparison of MATK-aided versus human domain-expert analyses of 1060 inherited retinal degeneration (IRD) families investigated with an IRD-specific gene panel (589 families) and exome sequencing (471 families). Results: When comparing MATK-assisted analysis to expert curation, we found that 97.3% (541/556) of potential solutions found by experts were also identified by the MATK-assisted analysis. Furthermore, MATK-assisted analysis identified 114 additional potential solutions. The software also showed utility in data reanalysis after remapping to the GRCh38 genome build. Conclusion: MATK expedites the process of identifying likely solving variants in Mendelian traits and helps to remove variability coming from human error and researcher bias. MATK facilitates data re-analysis to keep up with the constantly improving annotation sources and NGS processing pipelines. The software is open source and available at https://gitlab.partners.org/meei-ogi-bioinformatics/MendelAnalysis

Mating-Type-Specific Ribosomal Proteins Control Aspects of Sexual Reproduction in Cryptococcus neoformans

The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by an unusually large structure, spanning over 100 kb. MAT genes have been characterized by functional genetics as being involved in sexual reproduction and virulence. However, classical gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, and RPL39), indicating that they are likely essential. In the present study, targeted gene replacement was performed in a diploid strain for both the α and a alleles of the ribosomal genes RPL22 and RPL39. Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential for viability. Ectopic integration of the RPL22 allele of opposite MAT identity in the heterozygous RPL22a/rpl22αΔ or RPL22α/rpl22aΔ mutant strains failed to complement their essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. Furthermore, via CRISPR/Cas9 technology, the RPL22 alleles were exchanged in haploid MATα and MATa strains of C. neoformans. These RPL22 exchange strains displayed morphological and genetic defects during bilateral mating. These results contribute to elucidating functions of C. neoformans essential mating type genes that may constitute a type of imprinting system to promote inheritance of nuclei of both mating types.

Mating-type specific ribosomal proteins control aspects of sexual reproduction in Cryptococcus neoformans

The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by an unusually large structure, spanning over 100 kb. MAT genes have been characterized by functional genetics as being involved in sexual reproduction and virulence. However, classical gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, RPL39), indicating that they are likely essential. In the present study, targeted gene replacement was performed in a diploid strain for both the α and a alleles of the ribosomal genes RPL22 and RPL39. Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential for viability. Ectopic integration of the RPL22 allele of opposite MAT identity in the heterozygous RPL22a/rpl22αΔ or RPL22α/rpl22aΔ mutant strains failed to complement their essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. Furthermore, via CRISPR/Cas9 technology the RPL22 alleles were exchanged in haploid MATα and MATa strains of C. neoformans. These RPL22 exchange strains displayed morphological and genetic defects during bilateral mating. These results contribute to elucidate functions of C. neoformans essential mating type genes that may constitute a type of imprinting system to promote inheritance of nuclei of both mating types.

Using high-resolution variant frequencies to empower clinical genome interpretation

ABSTRACTWhole exome and genome sequencing have transformed the discovery of genetic variants that cause human Mendelian disease, but discriminating pathogenic from benign variants remains a daunting challenge. Rarity is recognised as a necessary, although not sufficient, criterion for pathogenicity, but frequency cutoffs used in Mendelian analysis are often arbitrary and overly lenient. Recent very large reference datasets, such as the Exome Aggregation Consortium (ExAC), provide an unprecedented opportunity to obtain robust frequency estimates even for very rare variants. Here we present a statistical framework for the frequency-based filtering of candidate disease-causing variants, accounting for disease prevalence, genetic and allelic heterogeneity, inheritance mode, penetrance, and sampling variance in reference datasets. Using the example of cardiomyopathy, we show that our approach reduces by two-thirds the number of candidate variants under consideration in the average exome, and identifies 43 variants previously reported as pathogenic that can now be reclassified. We present precomputed allele frequency cutoffs for all variants in the ExAC dataset.

Inheritance of an epigenetic change in the mouse: a new role for RNA

Hereditary epigenetic variation, initially recognized and studied extensively in plants, had not been reported in mammals until recently. We have now identified the Kit locus as the first example of a paramutable gene of the mouse. Kit+/+ homozygotes born from Kittm1Alf/+ heterozygotes maintain and transmit to their progeny the white-spotted phenotype characteristic of the mutant heterozygote. Our observation of unusual amounts of RNA in the sperm of the paramutated (Kit*) males had led us to consider the possibility of RNA-mediated inheritance. A role for RNA was supported further by the efficient establishment of the epigenetic modification following microinjection in one-cell embryos of either sperm RNA of the paramutated males or of the Kit-specific microRNAs miR-221 and -222. In this article, we describe the phenotypes associated with the wild-type genome in the Kit* paramutated animals. Paramutation may be considered to be one possibility of epigenetic modification in the case of familial disease predispositions that are not fully accounted for by Mendelian analysis.

A genetic study on Gracilaria sjoestedtii

In the present study on Gracilaria sjoestedtii, 41 mutants (40 pigmentation and 1 morphological) were isolated. These were characterized in a Mendelian analysis, which showed that 26 of the pigmentation variants are recessive nuclear mutations. The remaining 14 are cytoplasmic mutants having maternal inheritance and no detectable transmission through spermatia. A partial complementation analysis identified seven cistrons among the nuclear pigmentation mutants. Multiple alleles were encountered at five loci and apparent intragenic recombination was observed. No linkage between cistrons was found in recombination tests conducted thus far. Mitotic recombination leading to diploid gametes on tetrasporophytes was observed and used to construct triploid tetrasporophytes. The chromosome number of a wild-type clone of G. sjoestedtii was determined at meiosis as n = 29–30. Trivalent chromosomes were observed during meiosis in tetrasporophytes obtained by crossing female plants with tetrasporophytes. In culture tanks with flowing seawater, female gametophytes grew faster and appeared more suitable for vegetative propagation than male gametophytes and tetrasporophytes.