Integration of genomics and transcriptomics allows the identification of rare DNA damage defects in PID patients with a cancer predisposition
Lynn BackersBram PartonMattias Van HeetveldeMarieke De BruyneKim De LeeneerSimon J. TavernierAnne VralFilomeen HaerynckKathleen Claes
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Abstract:
Introduction
Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders caused by genetically determined defects of the immune system, predisposing to life-threatening complications such as severe and recurrent infections, auto-immunity and malignancies. A subset of PIDs is caused by pathogenic germline variants in DNA repair genes. As a consequence these patients are radiosensitive and present with increased cancer risk. Since PID patients are exposed to radiation for several reasons (bone marrow transplantation, radiotherapy, diagnostic imaging), identification of the genetic defect is important for risk stratification and improved therapeutic management. As in only 5-20% of the patients pathogenic variants are found in currently known PID genes, we hypothesize that defects in additional DNA damage response genes may be involved in PID patients prone to malignancies. We applied an integrated approach of whole exome sequencing (WES) and transcriptomics to identify the underlying genetic defect in a patient with primary dwarfism, facial dysmorphia, skeletal abnormalities, severe microcephaly, mental retardation and immunodeficiency.
Materials and Methods
WES data is analyzed by means of a PID gene panel (460 genes) and a DNA damage response panel consisting of >1230 genes. Extensive filtering results in a long list of variants of unknown clinical significance (VUS). To facilitate variant prioritization we are complementing WES with transcriptomics on RNA extracted from short term lymphocyte cultures. We search for expression and splicing outliers in the transcriptome.
Results
WES did not reveal a pathogenic variant in the PID gene panel, but a homozygous variant at position +5 in intron 5 of the DNA repair gene ATRIP was found in the patient. RNA-Seq showed skipping of exon 5, leading to a premature stop codon. This was confirmed using Sanger sequencing at the cDNA level. No full length ATRIP transcripts were detected. Additionally western blot analysis shows complete absence of ATRIP protein in our patient.
Our patient only is the second ATRIP-deficient patient. The first patient ((Ogi et al. 2012)) had a clinical diagnosis of Seckel syndrome. Clinically our patient resembles SS, but additionally presents with an immunodeficiency. Previously a link between this gene and PID has not been established. Further functional validations are ongoing to provide insights into the link with the immune phenotype.
Conclusions
In summary, these first findings encourage the complementation of a PID gene panel with a panel of DNA damage genes for the molecular work-up of PID patients with a (presumed) cancer predisposition. Furthermore, transcriptomics allows prioritization of variants and improves diagnosis and patient management. We expanded the molecular and clinical spectrum of Seckel syndrome by describing the second ATRIP-deficient patient. Further functional work-up will contribute to the disease mechanism.Keywords:
Primary Immunodeficiency
PALB2
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Genetic disorders of the skeleton comprise a large group of more than 450 clinically distinct and genetically heterogeneous diseases associated with mutations in more than 300 genes. Achieving a definitive diagnosis is complicated due to the genetic heterogeneity of these disorders, their individual rarity and their diverse radiographic presentations. We used targeted exome sequencing and designed a 1.4Mb panel for simultaneous testing of more than 4,800 exons in 309 genes involved in skeletal disorders. DNA from 69 individuals from 66 families with a known or suspected clinical diagnosis of a skeletal disorder was analyzed. Of 36 cases with a specific clinical hypothesis with a known genetic basis, mutations were identified for eight cases (22%). Of 20 cases with a suspected skeletal disorder but without a specific diagnosis, four causative mutations were identified. Also included were 11 cases with a specific skeletal disorder but for which there was at the time no known associated gene. For these cases, one mutation was identified in a known skeletal disease genes, and re-evaluation of the clinical phenotype in this case changed the diagnoses from osteodysplasia syndrome to Apert syndrome. These results suggest that the NGS panel provides a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in a highly genetically heterogeneous set of disorders such as genetic skeletal disorders. The data also stress the importance of a thorough clinical evaluation before DNA sequencing. The strategy should be applicable to other groups of disorders in which the molecular basis is largely known.
Exome
Medical genetics
Molecular Genetics
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Bone marrow failure
Genetic predisposition
Aplastic anemia
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Bloom syndrome
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Primary Immunodeficiency
Penetrance
Genome-wide Association Study
Immune Dysregulation
Genetic Association
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Exome
Genetic predisposition
Candidate gene
Genetic Association
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Exome
Candidate gene
Compound heterozygosity
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With the advent of high-throughput genomic sequencing techniques, novel genetic etiologies are being uncovered for previously unexplained Mendelian phenotypes, and the underlying genetic architecture of disease is being unraveled. Although most of these "mendelizing" disease traits represent phenotypes caused by single-gene defects, a percentage of patients have blended phenotypes caused by pathogenic variants in multiple genes. We describe an adult patient with susceptibility to bacterial, herpesviral, and fungal infections. Immunologic defects included CD8+ T cell lymphopenia, decreased T cell proliferative responses to mitogens, hypogammaglobulinemia, and radiation sensitivity. Whole-exome sequencing revealed compound heterozygous variants in ZAP70. Biallelic mutations in ZAP70 are known to produce a spectrum of immune deficiency that includes the T cell abnormalities observed in this patient. Analyses for variants in genes associated with radiation sensitivity identified the presence of a homozygous RNF168 variant of unknown significance. RNF168 deficiency causes radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome and may account for the radiation sensitivity. Thus, the patient was found to have a novel blended phenotype associated with multilocus genomic variation: i.e., separate and distinct genetic defects. These findings further illustrate the clinical utility of applying genomic testing in patients with primary immunodeficiency diseases.
Immune Dysregulation
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Radiation sensitivity
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Background
Multiple genes have been implicated by association studies in altering inflammatory bowel disease (IBD) predisposition. Paediatric patients often manifest more extensive disease and a particularly severe disease course. It is likely that genetic predisposition plays a more substantial role in this group.Objective
To identify the spectrum of rare and novel variation in known IBD susceptibility genes using exome sequencing analysis in eight individual cases of childhood onset severe disease.Design
DNA samples from the eight patients underwent targeted exome capture and sequencing. Data were processed through an analytical pipeline to align sequence reads, conduct quality checks, and identify and annotate variants where patient sequence differed from the reference sequence. For each patient, the entire complement of rare variation within strongly associated candidate genes was catalogued.Results
Across the panel of 169 known IBD susceptibility genes, approximately 300 variants in 104 genes were found. Excluding splicing and HLA-class variants, 58 variants across 39 of these genes were classified as rare, with an alternative allele frequency of <5%, of which 17 were novel. Only two patients with early onset Crohn9s disease exhibited rare deleterious variations within NOD2: the previously described R702W variant was the sole NOD2 variant in one patient, while the second patient also carried the L1007 frameshift insertion. Both patients harboured other potentially damaging mutations in the GSDMB, ERAP2 and SEC16A genes. The two patients severely affected with ulcerative colitis exhibited a distinct profile: both carried potentially detrimental variation in the BACH2 and IL10 genes not seen in other patients.Conclusion
For each of the eight individuals studied, all non-synonymous, truncating and frameshift mutations across all known IBD genes were identified. A unique profile of rare and potentially damaging variants was evident for each patient with this complex disease.Exome
Candidate gene
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Prioritization
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Etiology
Exome
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