Segmental and total uniparental isodisomy (UPiD) as a disease mechanism in autosomal recessive lysosomal disorders : evidence from SNP arrays

Analyses in our diagnostic DNA laboratory include genes involved in autosomal recessive (AR) lysosomal storage disorders such as glycogenosis type II (Pompe disease) and mucopolysaccharidosis type I (MPSI, Hurler disease). We encountered 4 cases with apparent homozygosity for a disease-causing sequence variant that could be traced to one parent only. In addition, in a young child with cardiomyopathy, in the absence of other symptoms, a diagnosis of Pompe disease was considered. Remarkably, he presented with different enzymatic and genotypic features between leukocytes and skin fibroblasts. All cases were examined with microsatellite markers and SNP genotyping arrays. We identified one case of total uniparental disomy (UPD) of chromosome 17 leading to Pompe disease and three cases of segmental uniparental isodisomy (UPiD) causing Hurler-(4p) or Pompe disease (17q). One Pompe patient with unusual combinations of features was shown to have a mosaic segmental UPiD of chromosome 17q. The chromosome 17 UPD cases amount to 11% of our diagnostic cohort of homozygous Pompe patients (plus one case of pseudoheterozygosity) where segregation analysis was possible. We conclude that inclusion of parental DNA is mandatory for reliable DNA diagnostics. Mild or unusual phenotypes of AR diseases should alert physicians to the possibility of mosaic segmental UPiD. SNP genotyping arrays are used in diagnostic workup of patients with developmental delay. Our results show that even small Regions of Homozygosity that include telomeric areas are worth reporting, regardless of the imprinting status of the chromosome, as they might indicate segmental UPiD.Peer reviewe

A unified data infrastructure to support large-scale rare disease research

The Solve-RD project brings together clinicians, scientists, and patient representatives from 51 institutes spanning 15 countries to collaborate on genetically diagnosing ("solving") rare diseases (RDs). The project aims to significantly increase the diagnostic success rate by co-analysing data from thousands of RD cases, including phenotypes, pedigrees, exome/genome sequencing and multi-omics data. Here we report on the data infrastructure devised and created to support this co-analysis. This infrastructure enables users to store, find, connect, and analyse data and metadata in a collaborative manner. Pseudonymised phenotypic and raw experimental data are submitted to the RD-Connect Genome-Phenome Analysis Platform and processed through standardised pipelines. Resulting files and novel produced omics data are sent to the European Genome-phenome Archive, which adds unique file identifiers and provides long-term storage and controlled access services. MOLGENIS "RD3" and Cafe Variome "Discovery Nexus" connect data and metadata and offer discovery services, and secure cloud-based "Sandboxes" support multi-party data analysis. This proven infrastructure design provides a blueprint for other projects that need to analyse large amounts of heterogeneous data.3. Good health and well-bein

The vascular response in chronic periodontitis

This thesis describes work done at the Institute of Dental Research in Sydney between February of 1986 and January 1990. The broad subject of the work is the role of vascular endothelial cells (ECs) in chronic inflammation. Periodontitis has been used as an example of chronic inflammatory disease, and provides the focus for this study of endothelial biology. In Chapter 1, aspects of the endothelial literature which provide relevant background information for work described in later chapters are reviewed. In Chapter 2, literature relating to aetiology and pathogenesis of chronic inflammatory periodontal disease is discussed. To maintain relevance of literature reviews to experimental work, each subsequent chapter contains a small literature review of material relating to the subject of the specific chapter. Early laboratory work is described in Chapter 3, and consisted of a morphological survey of the vascular changes occurring in gingival tissues with development of chronic periodontitis. Expansion of the vasculature and appearance of phenotypically specialised high endothelial cells (HECs), were associated with progression of the disease. Vessels with HECs and had a similar appearance to those known to be responsible for lymphocyte recirculation described in lymphoid tissues and chronic inflammatory sites. In the course of performing this survey, a perivascular hyaline material was noted surrounding capillaries close to the bacterial plaque irritant. The incidence, distribution, extent, ultrastructre and immuo-histochemistry of this material was more closely investigated, and the possible pathogenesis and significance of the material discussed in Chapter 4. In Chapter 5, the ultrastructural, histochemical and functional properties of gingival HECs are described, and compared with the well characterised HECs of rat lymph nodes. It was found that periodontal vessels were very similar to those in rat lymph nodes, with the exception however, that the gingival vessels appeared to exchange polymorphonuclear leukocytes almost exclusively, while vessels with HECs in lymph nodes and other locations are known as sites of lymphocyte recirculation. This observation indicated that the function of HECs requires further investigation, with particular regard to the synthetic activity of the cells. HECs were consistently alkaline phosphatise (AP) negative. The negative association between leukocyte emigration and AP activity (APA), as well as evidence in the literature illustrating both the wide substrate specificity of this enzyme and the importance of phosphorylation in the control of protein activation, suggested that AP could play a role in regulating leukocyte emigration. A pre-requisite for the investigation of this possibility, is the identification of a rich source of the identical iso-enzyme of AP to what is present in ECs. In Chapter 6, the sensitivity of endothelial AP to a panel of inhibitors is compared with that of a number of tissues for which isoenzyme has been identified. Endothelial AP was identified as the liver/bone/kidney isoenzyme. This allows the use of kidney tissue as a relevant source of AP for use in further study of the role of this enzyme in EC biology. It was clear that in order to study both the synthetic activity of HECs, as well as the role of AP in the control of leukocyte emigration, a method for obtaining high density primary cultures of HECs had to be established. Chapter 7 describes work done towards the development of such a culture system. The availability in the latter phase of the work of suitable probe for the technique of the in-situ hybridization allowed the possibility of testing the hypothesis that HECs are important cytokine producers. It was felt that this would provide some basis for the further study of those cells in-virtro. This work is described in the appendix. The general discussion in Chapter 8, summarises the work, and develops potential areas of study arising from the finding of this thesis

Copy Number Variant Analysis of Spinocerebellar Ataxia Genes in a Cohort of Dutch Patients with Cerebellar Ataxia

Background and ObjectivesThe spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of neurodegenerative disorders generally caused by single nucleotide variants (SNVs) or indels in coding regions or by repeat expansions in coding and noncoding regions of SCA genes. Copy number variants (CNVs) have now also been reported for 3 genes - ITPR1, FGF14, and SPTBN2 - but not all SCA genes have been screened for CNVs as the underlying cause of the disease in patients. In this study, we aim to assess the prevalence of CNVs encompassing 36 known SCA genes.MethodsA cohort of patients with cerebellar ataxia who were referred to the University Medical Center Groningen for SCA genetic diagnostics was selected for this study. Genome-wide single nucleotide polymorphism (SNP) genotyping was performed using the Infinium Global Screening Array. Following data processing, genotyping data were uploaded into NxClinical software to perform CNV analysis per patient and to visualize identified CNVs in 36 genes with allocated SCA symbols. The clinical relevance of detected CNVs was determined using evidence from studies based on PubMed literature searches for similar CNVs and phenotypic features.ResultsOf the 338 patients with cerebellar ataxia, we identified putative clinically relevant CNV deletions in 3 patients: an identical deletion encompassing ITPR1 in 2 patients, who turned out to be related, and a deletion involving PPP2R2B in another patient. Although the CNV deletion in ITPR1 was clearly the underlying cause of SCA15 in the 2 related patients, the clinical significance of the deletion in PPP2R2B remained unknown.DiscussionWe showed that CNVs detectable with the limited resolution of SNP array are a very rare cause of SCA. Nevertheless, we suggest adding CNV analysis alongside SNV analysis to SCA gene diagnostics using next-generation sequencing approaches, at least for ITPR1, to improve the genetic diagnostics for patients

Segmental and total uniparental isodisomy (UPiD) as a disease mechanism in autosomal recessive lysosomal disorders: evidence from SNP arrays

Analyses in our diagnostic DNA laboratory include genes involved in autosomal recessive (AR) lysosomal storage disorders such as glycogenosis type II (Pompe disease) and mucopolysaccharidosis type I (MPSI, Hurler disease). We encountered 4 cases with apparent homozygosity for a disease-causing sequence variant that could be traced to one parent only. In addition, in a young child with cardiomyopathy, in the absence of other symptoms, a diagnosis of Pompe disease was considered. Remarkably, he presented with different enzymatic and genotypic features between leukocytes and skin fibroblasts. All cases were examined with microsatellite markers and SNP genotyping arrays. We identified one case of total uniparental disomy (UPD) of chromosome 17 leading to Pompe disease and three cases of segmental uniparental isodisomy (UPiD) causing Hurler-(4p) or Pompe disease (17q). One Pompe patient with unusual combinations of features was shown to have a mosaic segmental UPiD of chromosome 17q. The chromosome 17 UPD cases amount to 11% of our diagnostic cohort of homozygous Pompe patients (plus one case of pseudoheterozygosity) where segregation analysis was possible. We conclude that inclusion of parental DNA is mandatory for reliable DNA diagnostics. Mild or unusual phenotypes of AR diseases should alert physicians to the possibility of mosaic segmental UPiD. SNP genotyping arrays are used in diagnostic workup of patients with developmental delay. Our results show that even small Regions of Homozygosity that include telomeric areas are worth reporting, regardless of the imprinting status of the chromosome, as they might indicate segmental UPiD

Toward an effective exome-based genetic testing strategy in pediatric dilated cardiomyopathy

Purpose: We evaluated the diagnostic yield in pediatric dilated cardiomyopathy (DCM) of combining exome sequencing (ES)based targeted analysis and genome-wide copy-number variation (CNV) analysis. Based on our findings, we retrospectively designed an effective approach for genetic testing in pediatric DCM. Methods: We identified 95 patients (in 85 families) with pediatric onset of DCM. We initially excluded 13 of these families because they already had a genetic diagnosis, leaving a total of 31 probands for singlenucleotide polymorphism (SNP) array and trio-ES. We used Human Phenotype Ontology (HPO)-based filtering for our data analysis. Results: We reached a genetic diagnosis in 15/31 (48.4%) families. ES yielded a diagnosis in 13 probands (13/15; 86.7%), with most variants being found in genes encoding structural cardiomyocyte components. Two large deletions were identified using SNP array. If we had included the 13 excluded families, our estimated yield would have been 54%. Conclusion: We propose a standardized, stepwise analysis of (i) wellknown cardiomyopathy genes, (ii) CNVs, (iii) all genes assigned to HPO cardiomyopathy, and (iv) if appropriate, genes assigned to other HPO terms. This diagnostic approach yields the highest increase at each subsequent step and reduces analytic effort, cost, the number of variants of unknown clinical significance, and the chance of incidental findings