Pkhd1

Autosomal-recessive polycystic kidney disease (ARPKD; MIM #263200) is a severe, hereditary, hepato-renal fibrocystic disorder that causes early childhood morbidity and mortality. Mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, which encodes the protein fibrocystin/polyductin complex (FPC), cause all typical forms of ARPKD. Several mouse lines carrying diverse, genetically engineered disruptions in the orthologous Pkhd1 gene have been generated, but none expresses the classic ARPKD renal phenotype. In the current study, we characterized a spontaneous mouse Pkhd1 mutation that is transmitted as a recessive trait and causes cysticliver (cyli), similar to the hepato-biliary disease in ARPKD, but which is exacerbated by age, sex, and parity. We mapped the mutation to Chromosome 1 and determined that an insertion/deletion mutation causes a frameshift within Pkhd1 exon 48, which is predicted to result in a premature termination codon (UGA). Pkhd

The Sex Chromosome Trisomy mouse model of XXY and XYY: metabolism and motor performance

BACKGROUND: Klinefelter syndrome (KS), caused by XXY karyotype, is characterized by low testosterone, infertility, cognitive deficits, and increased prevalence of health problems including obesity and diabetes. It has been difficult to separate direct genetic effects from hormonal effects in human studies or in mouse models of KS because low testosterone levels are confounded with sex chromosome complement. METHODS: In this study, we present the Sex Chromosome Trisomy (SCT) mouse model that produces XXY, XYY, XY, and XX mice in the same litters, each genotype with either testes or ovaries. The independence of sex chromosome complement and gonadal type allows for improved recognition of sex chromosome effects that are not dependent on levels of gonadal hormones. All mice were gonadectomized and treated with testosterone for 3 weeks. Body weight, body composition, and motor function were measured. RESULTS: Before hormonal manipulation, XXY mice of both sexes had significantly greater body weight and relative fat mass compared to XY mice. After gonadectomy and testosterone replacement, XXY mice (both sexes) still had significantly greater body weight and relative fat mass, but less relative lean mass compared to XY mice. Liver, gonadal fat pad, and inguinal fat pad weights were also higher in XXY mice, independent of gonadal sex. In several of these measures, XX mice also differed from XY mice, and gonadal males and females differed significantly on almost every metabolic measure. The sex chromosome effects (except for testis size) were also seen in gonadally female mice before and after ovariectomy and testosterone treatment, indicating that they do not reflect group differences in levels of testicular secretions. XYY mice were similar to XY mice on body weight and metabolic variables but performed worse on motor tasks compared to other groups. CONCLUSIONS: We find that the new SCT mouse model for XXY and XYY recapitulates features found in humans with these aneuploidies. We illustrate that this model has significant promise for unveiling the role of genetic effects compared to hormonal effects in these syndromes, because many phenotypes are different in XXY vs. XY gonadal female mice which have never been exposed to testicular secretions

Identification of Genetic Etiology in Disorders of Sex Development

Disorders of Sex Development (DSD) are defined as “congenital conditions in which development of chromosomal, gonadal, or anatomic sex is atypical.” These conditions have an approximate frequency of 0.5-1% of live births and encompass a wide variety of urogenital abnormalities ranging from mild hypospadias to sex reversal. Lack of standardized anatomical/endocrine phenotyping and the limited number of known DSD genes with poor genotype/phenotype correlation have hampered the field of clinical management, leaving many patients without a definitive genetic diagnosis. Thus, the focus of this thesis is to identify the underlying pathogenic genetic mutations that disrupt development of urogenital structures, leading to Disorders of Sex Development in humans.The traditional trend of diagnostic approach for patients with DSD is to select candidate gene testing by searching for additional phenotypic and metabolic information through imaging studies and endocrine tests that could explain the patient’s phenotype. This approach is usually ineffective, costly and time-consuming. To address this issue and identify genetic variants leading to DSD, we utilized exome sequencing, in patients diagnosed with abnormal sex development. We show that exome sequencing has transformed the field of clinical genetic diagnosis by increasing the rate of diagnosis by approximately 30% and has become a method of choice for many clinicians. Although exome sequencing provides much higher diagnostic yields for DSD patients than the conventional techniques, more than half of the patients tested with ES still do not possess a specific genetic diagnosis. Rather, in these patients ES identifies hundreds of variants of unknown clinical significance (VUS). To investigate the role of these variants in the 46,XY subset of DSD patients, we performed gonadal gene expression studies in C57BL/6J-YPOS mice modeling the phenotype of human 46,XY individuals to identify genes important in sex development. We used these genes to filter VUS identified in 46,XY DSD exome negative cases. We identified 15 novel candidate genes with mutations in 46,XY DSD patents that may be associated with disease pathogenesis.Due to innate limitations of exonic short read sequencing, many native variants are not identified by exome sequencing. To this end, we utilize genome sequencing in conjunction with a novel genome mapping technology in order to uncover the full spectrum of variations present in a human genomes. Collectively, these two technologies provide a physical map and a base pair-level DNA resolution allowing for identification of novel pathogenic variants that were previously inaccessible

New technologies to uncover the molecular basis of disorders of sex development.

The elegant developmental biology experiments conducted in the 1940s by French physiologist Alfred Jost demonstrated that the sexual phenotype of a mammalian embryo depended whether the embryonic gonad develops into a testis or not. In humans, anomalies in the processes that regulate development of chromosomal, gonadal or anatomic sex result in a spectrum of conditions termed Disorders/Differences of Sex Development (DSD). Each of these conditions is rare, and understanding of their genetic etiology is still incomplete. Historically, DSD diagnoses have been difficult to establish due to the lack of standardization of anatomical and endocrine phenotyping procedures as well as genetic testing. Yet, a definitive diagnosis is critical for optimal management of the medical and psychosocial challenges associated with DSD conditions. The advent in the clinical realm of next-generation sequencing methods, with constantly decreasing price and turnaround time, has revolutionized the diagnostic process. Here we review the successes and limitations of the genetic methods currently available for DSD diagnosis, including Sanger sequencing, karyotyping, exome sequencing and chromosomal microarrays. While exome sequencing provides higher diagnostic rates, many patients still remain undiagnosed. Newer approaches, such as whole-genome sequencing and whole-genome mapping, along with gene expression studies, have the potential to identify novel DSD-causing genes and significantly increase total diagnostic yield, hopefully shortening the patients journey to an accurate diagnosis and enhancing health-related quality-of-life outcomes for patients and families

nanotatoR: a tool for enhanced annotation of genomic structural variants.

BACKGROUND: Whole genome sequencing is effective at identification of small variants, but because it is based on short reads, assessment of structural variants (SVs) is limited. The advent of Optical Genome Mapping (OGM), which utilizes long fluorescently labeled DNA molecules for de novo genome assembly and SV calling, has allowed for increased sensitivity and specificity in SV detection. However, compared to small variant annotation tools, OGM-based SV annotation software has seen little development, and currently available SV annotation tools do not provide sufficient information for determination of variant pathogenicity. RESULTS: We developed an R-based package, nanotatoR, which provides comprehensive annotation as a tool for SV classification. nanotatoR uses both external (DGV; DECIPHER; Bionano Genomics BNDB) and internal (user-defined) databases to estimate SV frequency. Human genome reference GRCh37/38-based BED files are used to annotate SVs with overlapping, upstream, and downstream genes. Overlap percentages and distances for nearest genes are calculated and can be used for filtration. A primary gene list is extracted from public databases based on the patients phenotype and used to filter genes overlapping SVs, providing the analyst with an easy way to prioritize variants. If available, expression of overlapping or nearby genes of interest is extracted (e.g. from an RNA-Seq dataset, allowing the user to assess the effects of SVs on the transcriptome). Most quality-control filtration parameters are customizable by the user. The output is given in an Excel file format, subdivided into multiple sheets based on SV type and inheritance pattern (INDELs, inversions, translocations, de novo, etc.). nanotatoR passed all quality and run time criteria of Bioconductor, where it was accepted in the April 2019 release. We evaluated nanotatoRs annotation capabilities using publicly available reference datasets: the singleton sample NA12878, mapped with two types of enzyme labeling, and the NA24143 trio. nanotatoR was also able to accurately filter the known pathogenic variants in a cohort of patients with Duchenne Muscular Dystrophy for which we had previously demonstrated the diagnostic ability of OGM. CONCLUSIONS: The extensive annotation enables users to rapidly identify potential pathogenic SVs, a critical step toward use of OGM in the clinical setting

Regulation of sex determination in mice by a non-coding genomic region.

To identify novel genomic regions that regulate sex determination, we utilized the powerful C57BL/6J-Y(POS) (B6-Y(POS)) model of XY sex reversal where mice with autosomes from the B6 strain and a Y chromosome from a wild-derived strain, Mus domesticus poschiavinus (Y(POS)), show complete sex reversal. In B6-Y(POS), the presence of a 55-Mb congenic region on chromosome 11 protects from sex reversal in a dose-dependent manner. Using mouse genetic backcross designs and high-density SNP arrays, we narrowed the congenic region to a 1.62-Mb genomic region on chromosome 11 that confers 80% protection from B6-Y(POS) sex reversal when one copy is present and complete protection when two copies are present. It was previously believed that the protective congenic region originated from the 129S1/SviMJ (129) strain. However, genomic analysis revealed that this region is not derived from 129 and most likely is derived from the semi-inbred strain POSA. We show that the small 1.62-Mb congenic region that protects against B6-Y(POS) sex reversal is located within the Sox9 promoter and promotes the expression of Sox9, thereby driving testis development within the B6-Y(POS) background. Through 30 years of backcrossing, this congenic region was maintained, as it promoted male sex determination and fertility despite the female-promoting B6-Y(POS) genetic background. Our findings demonstrate that long-range enhancer regions are critical to developmental processes and can be used to identify the complex interplay between genome variants, epigenetics, and developmental gene regulation

Comparative Benchmarking of Optical Genome Mapping and Chromosomal Microarray Reveals High Technological Concordance in CNV Identification and Additional Structural Variant Refinement

The recommended practice for individuals suspected of a genetic etiology for disorders including unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), and multiple congenital anomalies (MCA) involves a genetic testing workflow including chromosomal microarray (CMA), Fragile-X testing, karyotype analysis, and/or sequencing-based gene panels. Since genomic imbalances are often found to be causative, CMA is recommended as first tier testing for many indications. Optical genome mapping (OGM) is an emerging next generation cytogenomic technique that can detect not only copy number variants (CNVs), triploidy and absence of heterozygosity (AOH) like CMA, but can also define the location of duplications, and detect other structural variants (SVs), including balanced rearrangements and repeat expansions/contractions. This study compares OGM to CMA for clinically reported genomic variants, some of these samples also have structural characterization by fluorescence in situ hybridization (FISH). OGM was performed on IRB approved, de-identified specimens from 55 individuals with genomic abnormalities previously identified by CMA (61 clinically reported abnormalities). SVs identified by OGM were filtered by a control database to remove polymorphic variants and against an established gene list to prioritize clinically relevant findings before comparing with CMA and FISH results. OGM results showed 100% concordance with CMA findings for pathogenic variants and 98% concordant for all pathogenic/likely pathogenic/variants of uncertain significance (VUS), while also providing additional insight into the genomic structure of abnormalities that CMA was unable to provide. OGM demonstrates equivalent performance to CMA for CNV and AOH detection, enhanced by its ability to determine the structure of the genome. This work adds to an increasing body of evidence on the analytical validity and ability to detect clinically relevant abnormalities identified by CMA. Moreover, OGM identifies translocations, structures of duplications and complex CNVs intractable by CMA, yielding additional clinical utility