Discovering disease causing variants in dogs through whole genome sequencing

Dr. Gary S. Johnson, Dissertation Supervisor.includes vitaThis dissertation focuses on the use of whole genome sequencing (WGS) for the identification of disease causing variants in canine genomes. A brief review on the historical milestones of genetics, the creation and popularization of the fast throughput DNA sequencing technologies and their advantages and potential problems and biases, the importance of the study of canine genetics and the current state of the canine genome assembly is presented. Our lab sequenced [about]100 dogs in the attempt to discover disease-causing variants. So far 20 such variants have been identified. This dissertation contains detailed accounts of the discovery variants likely to be responsible for four canine diseases. Those diseases are: Paroxysmal dyskinesia in Soft Coated Wheaten Terriers that is associated to the missense mutation PIGN:c.398C greater than T; two different forms of neuronal ceroid lipofuscinosis, one in Australian Cattle dogs caused by CLN5:c.619C greater than T, and one in the Cane Corso caused by the splice site mutation PPT1c.124+1 [greater than] A; and a Shiba Inu GM2 gangliosidosis caused by HEXB.c:948_950delCCT. Furthermore, examples of not so successful attempts, possible reasons for failures and suggestions to successfully conclude other ongoing investigations.Dr. Gary S. Johnson, Dissertation Supervisor.|Includes vita.Includes bibliographical references (pages 127-150)

Detection and characterization of genomic regions associated to Dilated Cardiomyopathy in Great Danes

Canine dilated cardiomyopathy (DCM) is a lethal myocardial disease prevalent in large and giant-sized dog breeds, including the Great Dane (GD). The goal of this study was to identify genetic risk factors for DCM and to contribute to a future development of DCM genetic markers in order to facilitate disease diagnosis and risk allele detection. Furthermore, in the long term, the identification of novel genes and pathways that may contribute for development of future therapies. Two different approaches of genome wide association study (GWAS) using data from ~170,000 Single Nucleotide Polymorphism (SNP) markers were conducted in order to identify disease-associated regions of the genome. A previous GWAS to this study used a different cohort of individuals and analytical software. This methodology detected six candidate regions on chromosomes 3, 16, 19, 29 and 30 (two associated regions on chr19). The current GWAS tested nine different models in a dataset containing 127 Great Danes (62 cases and 65 controls). The GenABEL package for R was used for the analysis. The elected model 3 included population stratification and had an inflation factor lambda (λ) equal to 1.04. Two regions on chromosome 19 overlapped with the first GWAS. Region 1: between 23-23.3 Mb containing two rRNAs; Region 2: between 44-46Mb containing three genes: SPOPL, NXPH2 and a LRP1B orthologue. No marker reached Bonferroni statistical significance, and the low power could be due to insufficient number of cases and controls. It is suggested that SNP typing of extra Great Danes and a better use of the available phenotype data would help to improve the statistical power. The correction for complex gene-gene interactions such as epistasis may be an interesting approach to detect novel candidates. The regions detected in the precedent GWAS were re-sequenced using Illumina Next generation sequencing technology in five animals (three controls and two cases), and the data was available for analysis in the current study. Six candidate non-synonymous mutations were detected in the DMXL2, TMC3, ZMAT4, MESDC2 genes and in a paralogue to RPL10A. DMXL2 encodes for a protein important for the Notch pathway in mammals. In humans, a SNP in this gene has been associated with ischemic stroke. The polymorphism in DMXL2 was confirmed through the genotyping of 319 individuals and the non-reference allele is slightly more frequent on verified cases as opposed to verified controls, however, the difference is not statistically significant (t-test p-value= 0.366; 2X3 Fisher test p-value= 0.258). Further analysis on both regions on chr19 should be carried out in order to detect possible novel mutations, copy number variants, genes and regulatory elements that could influence the DCM phenotype

Reliance on Cox10 and oxidative metabolism for antigen-specific NK cell expansion

Natural killer (NK) cell effector functions are dependent on metabolic regulation of cellular function; however, less is known about in vivo metabolic pathways required for NK cell antiviral function. Mice with an inducible NK-specific deletion of Cox10, which encodes a component of electron transport chain complex IV, were generated to investigate the role of oxidative phosphorylation in NK cells during murine cytomegalovirus (MCMV) infection. Ncr1-Cox1

A human STAT3 gain-of-function variant confers T cell dysregulation without predominant Treg dysfunction in mice

Primary immune regulatory disorders (PIRD) represent a group of disorders characterized by immune dysregulation, presenting with a wide range of clinical disease, including autoimmunity, autoinflammation, or lymphoproliferation. Autosomal dominant germline gain-of-function (GOF) variants in STAT3 result in a PIRD with a broad clinical spectrum. Studies in patients have documented a decreased frequency of FOXP3+ Tregs and an increased frequency of Th17 cells in some patients with active disease. However, the mechanisms of disease pathogenesis in STAT3 GOF syndrome remain largely unknown, and treatment is challenging. We developed a knock-in mouse model harboring a de novo pathogenic human STAT3 variant (p.G421R) and found these mice developed T cell dysregulation, lymphoproliferation, and CD4+ Th1 cell skewing. Surprisingly, Treg numbers, phenotype, and function remained largely intact; however, mice had a selective deficiency in the generation of iTregs. In parallel, we performed single-cell RNA-Seq on T cells from STAT3 GOF patients. We demonstrate only minor changes in the Treg transcriptional signature and an expanded, effector CD8+ T cell population. Together, these findings suggest that Tregs are not the primary driver of disease and highlight the importance of preclinical models in the study of disease mechanisms in rare PIRD

Increased susceptibility to Mycobacterium avium complex infection in miniature Schnauzer dogs caused by a codon deletion in CARD9.

Mammals are generally resistant to Mycobacterium avium complex (MAC) infections. We report here on a primary immunodeficiency disorder causing increased susceptibility to MAC infections in a canine breed. Adult Miniature Schnauzers developing progressive systemic MAC infections were related to a common founder, and pedigree analysis was consistent with an autosomal recessive trait. A genome-wide association study and homozygosity mapping using 8 infected, 9 non-infected relatives, and 160 control Miniature Schnauzers detected an associated region on chromosome 9. Whole genome sequencing of 2 MAC-infected dogs identified a codon deletion in the CARD9 gene (c.493_495del; p.Lys165del). Genotyping of Miniature Schnauzers revealed the presence of this mutant CARD9 allele worldwide, and all tested MAC-infected dogs were homozygous mutants. Peripheral blood mononuclear cells from a dog homozygous for the CARD9 variant exhibited a dysfunctional CARD9 protein with impaired TNF-α production upon stimulation with the fungal polysaccharide β-glucan that activates the CARD9-coupled C-type lectin receptor, Dectin-1. While CARD9-deficient knockout mice are susceptible to experimental challenges by fungi and mycobacteria, Miniature Schnauzer dogs with systemic MAC susceptibility represent the first spontaneous animal model of CARD9 deficiency, which will help to further elucidate host defense mechanisms against mycobacteria and fungi and assess potential therapies for animals and humans

Immunological Findings and Clinical Outcomes of Infants With Positive Newborn Screening for Severe Combined Immunodeficiency From a Tertiary Care Center in the U.S.

The implementation of severe combined immunodeficiency (SCID) newborn screening has played a pivotal role in identifying these patients early in life as well as detecting various milder forms of T cell lymphopenia (TCL). In this study we reviewed the diagnostic and clinical outcomes, and interesting immunology findings of term infants referred to a tertiary care center with abnormal newborn SCID screens over a 6-year period. Key findings included a 33% incidence of non-SCID TCL including infants with novel variants in FOXN1, TBX1, MYSM1, POLD1, and CD3E; 57% positivity rate of newborn SCID screening among infants with DiGeorge syndrome; and earlier diagnosis and improved transplant outcomes for SCID in infants diagnosed after compared to before implementation of routine screening. Our study is unique in terms of the extensive laboratory workup of abnormal SCID screens including lymphocyte subsets, measurement of thymic output (TREC and CD4TE), and lymphocyte proliferation to mitogens in nearly all infants. These data allowed us to observe a stronger positive correlation of the absolute CD3 count with CD4RTE than with TREC copies, and a weak positive correlation between CD4RTE and TREC copies. Finally, we did not observe a correlation between risk of TCL and history of prenatal or perinatal complications or low birth weight. Our study demonstrated SCID newborn screening improves disease outcomes, particularly in typical SCID, and allows early detection and discovery of novel variants of certain TCL-associated genetic conditions