Strategies to Uplift Novel Mendelian Gene Discovery for Improved Clinical Outcomes

Rare genetic disorders, while individually rare, are collectively common. They represent some of the most severe disorders affecting patients worldwide with significant morbidity and mortality. Over the last decade, advances in genomic methods have significantly uplifted diagnostic rates for patients and facilitated novel and targeted therapies. However, many patients with rare genetic disorders still remain undiagnosed as the genetic etiology of only a proportion of Mendelian conditions has been discovered to date. This article explores existing strategies to identify novel Mendelian genes and how these discoveries impact clinical care and therapeutics. We discuss the importance of data sharing, phenotype-driven approaches, patient-led approaches, utilization of large-scale genomic sequencing projects, constraint-based methods, integration of multi-omics data, and gene-to-patient methods. We further consider the health economic advantages of novel gene discovery and speculate on potential future methods for improved clinical outcomes.

Co-located quantitative trait loci mediate resistance to Agrobacterium tumefaciens, Phytophthora cinnamomi, and P. pini in Juglans microcarpa × J. regia hybrids

Soil-borne plant pathogens represent a serious threat that undermines commercial walnut (Juglans regia) production worldwide. Crown gall, caused by Agrobacterium tumefaciens, and Phytophthora root and crown rots, caused by various Phytophthora spp., are among the most devastating walnut soil-borne diseases. A recognized strategy to combat soil-borne diseases is adoption of resistant rootstocks. Here, resistance to A. tumefaciens, P. cinnamomi, and P. pini is mapped in the genome of Juglans microcarpa, a North American wild relative of cultivated walnut. Half-sib J. microcarpa mother trees DJUG 31.01 and DJUG 31.09 were crossed with J. regia cv. Serr, producing 353 and 400 hybrids, respectively. Clonally propagated hybrids were genotyped by sequencing to construct genetic maps for the two populations and challenged with the three pathogens. Resistance to each of the three pathogens was mapped as a major QTL on the long arm of J. microcarpa chromosome 4D and was associated with the same haplotype, designated as haplotype b, raising the possibility that the two mother trees were heterozygous for a single Mendelian gene conferring resistance to all three pathogens. The deployment of this haplotype in rootstock breeding will facilitate breeding of a walnut rootstock resistant to both crown gall and Phytophthora root and crown rots.

AMELIE 3: Fully Automated Mendelian Patient Reanalysis at Under 1 Alert per Patient per Year

BackgroundMany thousands of patients with a suspected Mendelian disease have their exomes/genomes sequenced every year, but only about 30% receive a definitive diagnosis. Since a novel Mendelian gene-disease association is published on average every business day, thousands of undiagnosed patient cases could receive a diagnosis each year if their genomes were regularly compared to the latest literature. With millions of genomes expected to be sequenced for rare disease analysis by 2025, and considering the current publication rate of 1.1 million new articles per annum in PubMed, manually reanalyzing the growing cases of undiagnosed patients is not sustainable.MethodsWe describe a fully automated reanalysis framework for patients with suspected, but undiagnosed, Mendelian disorders. The presented framework was tested by automatically parsing all ∼100,000 newly published peer reviewed papers every month and matching them on genotype and phenotype with all stored undiagnosed patients. If a new article contains a possible diagnosis for an undiagnosed patient, the system provides notification. We test the accuracy of the automatic reanalysis system on 110 patients, including 61 with available trio data.ResultsEven when trained only on older data, our system identifies 80% of reanalysis diagnoses, while sending only 0.5-1 alerts per patient per year, a 100-1,000-fold efficiency gain over manual literature surveillance of equivalent yield.ConclusionWe show that automatic reanalysis of patients with suspected Mendelian disease is feasible and has the potential to greatly streamline diagnosis. Our system is not intended to replace clinical judgment. Rather, clinical diagnostic services could greatly benefit from a modest re-allocation of time from manual literature exploration to review of automated reanalysis alerts. Our system additionally supports a new paradigm for medical IT systems: proactive, continuously learning and consequently able to autonomously identify valuable insights as they emerge in digital health records. We have launched automated patient reanalysis, trained on the latest data, with user accounts and daily literature updates at https://AMELIE.stanford.edu.

Exploiting the Autozygome to Support Previously Published Mendelian Gene-Disease Associations: An Update

There is a growing interest in standardizing gene-disease associations for the purpose of facilitating the proper classification of variants in the context of Mendelian diseases. One key line of evidence is the independent observation of pathogenic variants in unrelated individuals with similar phenotypes. Here, we expand on our previous effort to exploit the power of autozygosity to produce homozygous pathogenic variants that are otherwise very difficult to encounter in the homozygous state due to their rarity. The identification of such variants in genes with only tentative associations to Mendelian diseases can add to the existing evidence when observed in the context of compatible phenotypes. In this study, we report 20 homozygous variants in 18 genes (ADAMTS18, ARNT2, ASTN1, C3, DMBX1, DUT, GABRB3, GM2A, KIF12, LOXL3, NUP160, PTRHD1, RAP1GDS1, RHOBTB2, SIGMAR1, SPAST, TENM3, and WASHC5) that satisfy the ACMG classification for pathogenic/likely pathogenic if the involved genes had confirmed rather than tentative links to diseases. These variants were selected because they were truncating, founder with compelling segregation or supported by robust functional assays as with the DUT variant that we present its validation using yeast model. Our findings support the previously reported disease associations for these genes and represent a step toward their confirmation.

Inheritance of resistance and response of Provisia™ rice to quizalofop-p-ethyl under U.S. field conditions

Provisia™ rice was developed recently by the BASF Corporation for control of grass weeds and is complementary to existing Clearfield® technology. Our previous research showed that resistance of Provisia™ rice to the acetyl coenzyme-A carboxylase herbicide quizalofop-p-ethyl (QPE) in laboratory and greenhouse environments is governed by a single dominant Mendelian gene. However, these results may not be consistent in different populations or field environments. Therefore, the first objective of the current research is to determine the inheritance of resistance to QPE in rice using different segregating populations evaluated under U.S. field environments. The second objective is to evaluate the response of QPE-resistant breeding lines to various herbicide concentrations at two U.S. locations. Chi-square tests of 12 F2 populations evaluated in Louisiana during 2014 and 2015 indicated that QPE seedling resistance at 240 g ai ha−1 was governed by a single dominant Mendelian gene with no observable maternal effects. Similar results were obtained in five F3 populations derived from the aforementioned F2 populations. Allele-specific SNP markers for QPE resistance also followed Mendelian segregation in the five F2 populations. For the second objective, six QPE-resistant inbred lines showed transient leaf injury at 1× (120 g ai ha−1) or 2× (240 g ai ha−1) field rates 7 and 21 d after treatment (DAT). However, a trend of reduced injury (recovery) from 7 through 33 DAT was observed for all breeding material. No differences in grain yield were found between untreated QPE-resistant lines and those treated with 1× or 2× QPE field rate. Single gene inheritance and good levels of QPE herbicide field resistance in different genetic populations suggest feasibility for rapid and effective development of new QPE-resistant varieties and effective stewardship of the Provisia™ technology.

Diagnosing Cornelia de Lange syndrome and related neurodevelopmental disorders using RNA-sequencing

ABSTRACTPurposeNeurodevelopmental phenotypes represent major indications for children undergoing clinical exome sequencing. However, 50% of cases remain undiagnosed even upon exome reanalysis. Here we show RNA sequencing (RNA-seq) on human B lymphoblastoid cell lines (LCL) is highly suitable for neurodevelopmental Mendelian gene testing and demonstrate the utility of this approach in suspected cases of Cornelia de Lange syndrome (CdLS).MethodsGenotype-Tissue Expression project transcriptome data for LCL, blood, and brain was assessed for neurodevelopmental Mendelian gene expression. Detection of abnormal splicing and pathogenic variants in these genes was performed with a novel RNA-seq diagnostic pipeline and using a validation CdLS-LCL cohort (n=10) and test cohort of patients who carry a clinical diagnosis of CdLS but negative genetic testing (n=5).ResultsLCLs share isoform diversity of brain tissue for a large subset of neurodevelopmental genes and express 1.8-fold more of these genes compared to blood (LCL, n=1706; whole blood, n=917). This enables testing of over 1000 genetic syndromes. The RNA-seq pipeline had 90% sensitivity for detecting pathogenic events and revealed novel diagnoses such as abnormal splice products in NIPBL and pathogenic coding variants in BRD4 and ANKRD11.ConclusionThe LCL transcriptome enables robust frontline and/or reflexive diagnostic testing for neurodevelopmental disorders.

Inheritance of Resistance to Gummy Stem Blight in Watermelon

Gummy stem blight (GSB), caused by three related species of Stagonosporopsis [Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), Stagonosporopsis citrulli, and Stagonosporopsis caricae], is a major disease of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] in most production areas of the United States. We studied the inheritance of resistance to GSB using three PI accessions of watermelon. Four families of six progenies (Pr, Ps, F1, F2, BC1Pr, and BC1Ps) were developed from four crosses of resistant PI accessions by susceptible cultivars. Each family was tested in 2002 and 2003 in North Carolina under field and greenhouse conditions for resistance to GSB. Artificial inoculation was used to induce uniform and strong epidemics. The effect of the Mendelian gene for resistance, db, was tested. Partial failure of the data to fit the single-gene inheritance suggested that resistance to GSB of PI 482283 and PI 526233 may be under the control of a more complex genetic system.