SwissGenVar: A Platform for Clinical-Grade Interpretation of Genetic Variants to Foster Personalized Healthcare in Switzerland.

Large-scale next-generation sequencing (NGS) germline testing is technically feasible today, but variant interpretation represents a major bottleneck in analysis workflows. This includes extensive variant prioritization, annotation, and time-consuming evidence curation. The scale of the interpretation problem is massive, and variants of uncertain significance (VUSs) are a challenge to personalized medicine. This challenge is further compounded by the complexity and heterogeneity of the standards used to describe genetic variants and the associated phenotypes when searching for relevant information to support clinical decision making. To address this, all five Swiss academic institutions for Medical Genetics joined forces with the Swiss Institute of Bioinformatics (SIB) to create SwissGenVar as a user-friendly nationwide repository and sharing platform for genetic variant data generated during routine diagnostic procedures and research sequencing projects. Its aim is to provide a protected environment for expert evidence sharing about individual variants to harmonize and upscale their significance interpretation at the clinical grade according to international standards. To corroborate the clinical assessment, the variant-related data will be combined with consented high-quality clinical information. Broader visibility will be achieved by interfacing with international databases, thus supporting global initiatives in personalized healthcare.

Recurrent spontaneous pneumothorax in an NF1 patient with a novel causative variant: broadening genotype-phenotype correlations.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance, most commonly known to affect the skin and eyes. Although lung involvement in the form of cysts and bullae occurs in up to 20% of adults, the seemingly intuitive association of NF1 and spontaneous pneumothorax is not widely recognised among clinicians. Here, we report the second case of recurring spontaneous pneumothorax in the context of NF1 with a confirmed molecular diagnosis. In both cases, the NF1 variants featured a premature stop codon in the C-terminal protein domain. Interestingly, our patient had mild skin symptoms, suggesting that spontaneous pneumothorax may not be correlated with cutaneous disease severity. More genotype-phenotype correlation studies are needed for NF1 in general and for its link to spontaneous pneumothorax in particular.

Enhancing fetal outcomes in GCK-MODY pregnancies: a precision medicine approach via non-invasive prenatal GCK mutation detection.

Background: Mutations in the GCK gene cause Maturity Onset Diabetes of the Young (GCK-MODY) by impairing glucose-sensing in pancreatic beta cells. During pregnancy, managing this type of diabetes varies based on fetal genotype. Fetuses carrying a GCK mutation can derive benefit from moderate maternal hyperglycemia, stimulating insulin secretion in fetal islets, whereas this may cause macrosomia in wild-type fetuses. Modulating maternal glycemia can thus be viewed as a form of personalized prenatal therapy, highly beneficial but not justifying the risk of invasive testing. We therefore developed a monogenic non-invasive prenatal diagnostic (NIPD-M) test to reliably detect the transmission of a known maternal GCK mutation to the fetus. Methods: A small amount of fetal circulating cell-free DNA is present in maternal plasma but cannot be distinguished from maternal cell-free DNA. Determining transmission of a maternal mutation to the fetus thus implies sequencing adjacent polymorphisms to determine the balance of maternal haplotypes, the transmitted haplotype being over-represented in maternal plasma. Results: Here we present a series of such tests in which fetal genotype was successfully determined and show that it can be used to guide therapeutic decisions during pregnancy and improve the outcome for the offspring. We discuss several potential hurdles inherent to the technique, and strategies to overcome these. Conclusion: Our NIPD-M test allows reliable determination of the presence of a maternal GCK mutation in the fetus, thereby allowing personalized in utero therapy by modulating maternal glycemia, without incurring the risk of miscarriage inherent to invasive testing.

Homozygous substitution of threonine 191 by proline in polymerase η causes Xeroderma pigmentosum variant.

DNA polymerase eta (Polη) is the only translesion synthesis polymerase capable of error-free bypass of UV-induced cyclobutane pyrimidine dimers. A deficiency in Polη function is associated with the human disease Xeroderma pigmentosum variant (XPV). We hereby report the case of a 60-year-old woman known for XPV and carrying a Polη Thr191Pro variant in homozygosity. We further characterize the variant in vitro and in vivo, providing molecular evidence that the substitution abrogates polymerase activity and results in UV sensitivity through deficient damage bypass. This is the first functional molecular characterization of a missense variant of Polη, whose reported pathogenic variants have thus far been loss of function truncation or frameshift mutations. Our work allows the upgrading of Polη Thr191Pro from 'variant of uncertain significance' to 'likely pathogenic mutant', bearing direct impact on molecular diagnosis and genetic counseling. Furthermore, we have established a robust experimental approach that will allow a precise molecular analysis of further missense mutations possibly linked to XPV. Finally, it provides insight into critical Polη residues that may be targeted to develop small molecule inhibitors for cancer therapeutics.

Substitution of arginine 219 by glycine compromises stability, dimerization, and catalytic activity in a G6PD mutant.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common enzymopathies in humans, present in approximately half a billion people worldwide. More than 230 clinically relevant G6PD mutations of different classes have been reported to date. We hereby describe a patient with chronic hemolysis who presents a substitution of arginine by glycine at position 219 in G6PD protein. The variant was never described in an original publication or characterized on a molecular level. In the present study, we provide structural and biochemical evidence for the molecular basis of its pathogenicity. When compared to the wild-type enzyme, the Arg219Gly mutation markedly reduces the catalytic activity by 50-fold while having a negligible effect on substrate binding affinity. The mutation preserves secondary protein structure, but greatly decreases stability at higher temperatures and to trypsin digestion. Size exclusion chromatography elution profiles show monomeric and dimeric forms for the mutant, but only the latter for the wild-type form, suggesting a critical role of arginine 219 in G6PD dimer formation. Our findings have implications in the development of small molecule activators, with the goal of rescuing the phenotype observed in this and possibly other related mutants.

Noninvasive prenatal diagnosis of Mendelian disorders for consanguineous couples by relative genotype dosage.

Noninvasive prenatal diagnosis relies on the presence in maternal blood of circulating cell-free fetal DNA released by apoptotic trophoblast cells. Widely used for aneuploidy screening, it can also be applied to monogenic diseases (NIPD-M) in case of known parental mutations. Due to the confounding effect of maternal DNA, detection of maternal or biparental mutations requires relative haplotype dosage (RHDO), a method relying on the presence of SNPs that are heterozygous in one parent and homozygous in the other. Unavoidably, there is a risk of test failure by lack of such informative SNPs, an event particularly likely for consanguineous couples who often share common haplotypes in regions of identity-by-descent. Here we present a novel approach, relative genotype dosage (RGDO) that bypasses this predicament by directly assessing fetal genotype with SNPs that are heterozygous in both parents (frequent in regions of identity-by-descent). We show that RGDO is as sensitive as RHDO and that it performs well over a large range of fetal fractions and DNA amounts, thereby opening NIPD-M to most consanguineous couples. We also report examples of couples, consanguineous or not, where combining RGDO and RHDO allowed a diagnosis that would not have been possible with only one approach.

Precision medicine in diabetes: A non-invasive prenatal diagnostic test for the determination of fetal glucokinase mutations.

Hyperglycemia caused by mutations in the glucokinase gene, GCK, is the most common form of monogenic diabetes. Prenatal diagnosis is important, as it impacts on treatment. This study reports a monogenic non-invasive prenatal diagnostic (NIPD-M) test on cell-free DNA in maternal plasma using the relative haplotype dosage. In three pregnancies of two families with known maternal GCK mutations, the fetal genotype was determined unambiguously already at 12 weeks of gestation. In summary, proof is provided of the feasibility for NIPD-M in GCK diabetes.

Molecular characterization of pathogenic OTOA gene conversions in hearing loss patients.

Bi-allelic loss-of-function variants of OTOA are a well-known cause of moderate-to-severe hearing loss. Whereas non-allelic homologous recombination-mediated deletions of the gene are well known, gene conversions to pseudogene OTOAP1 have been reported in the literature but never fully described nor their pathogenicity assessed. Here, we report two unrelated patients with moderate hearing-loss, who were compound heterozygotes for a converted allele and a deletion of OTOA. The conversions were initially detected through sequencing depths anomalies at the OTOA locus after exome sequencing, then confirmed with long range polymerase chain reactions. Both conversions lead to loss-of-function by introducing a premature stop codon in exon 22 (p.Glu787*). Using genomic alignments and long read nanopore sequencing, we found that the two probands carry stretches of converted DNA of widely different lengths (at least 9 kbp and around 900 bp, respectively).

Reliability of liquid biopsy analysis: an inter-laboratory comparison of circulating tumor DNA extraction and sequencing with different platforms.

Liquid biopsy, the analysis of circulating tumor DNA (ctDNA), is a promising tool in oncology, especially in personalized medicine. Although its main applications currently focus on selection and adjustment of therapy, ctDNA may also be used to monitor residual disease, establish prognosis, detect relapses, and possibly screen at-risk individuals. CtDNA represents a small and variable proportion of circulating cell-free DNA (ccfDNA) which is itself present at a low concentration in normal individuals and so analyzing ctDNA is technically challenging. Various commercial systems have recently appeared on the market, but it remains difficult for practitioners to compare their performance and to determine whether they yield comparable results. As a first step toward establishing national guidelines for ctDNA analyses, four laboratories in Switzerland joined a comparative exercise to assess ccfDNA extraction and ctDNA analysis by sequencing. Extraction was performed using six distinct methods and yielded ccfDNA of equally high quality, suitable for sequencing. Sequencing of synthetic samples containing predefined amounts of eight mutations was performed on three different systems, with similar results. In all four laboratories, mutations were easily identified down to 1% allele frequency, whereas detection at 0.1% proved challenging. Linearity was excellent in all cases and while molecular yield was superior with one system this did not impact on sensitivity. This study also led to several additional conclusions: First, national guidelines should concentrate on principles of good laboratory practice rather than recommend a particular system. Second, it is essential that laboratories thoroughly validate every aspect of extraction and sequencing, in particular with respect to initial amount of DNA and average sequencing depth. Finally, as software proved critical for mutation detection, laboratories should validate the performance of variant callers and underlying algorithms with respect to various types of mutations.

Tissue-Plasma TMB Comparison and Plasma TMB Monitoring in Patients With Metastatic Non-small Cell Lung Cancer Receiving Immune Checkpoint Inhibitors.

Immuno-oncology is an ever growing field that has seen important progress across the spectrum of cancers. Responses can be deep and durable. However, as only a minority of patients respond to checkpoint inhibition, predictive biomarkers are needed. Cancer is a genetic disease arising from the accumulation of somatic mutations in the DNA of affected cells. Tumor mutational burden (TMB), represents the number of somatic mutations in a tumor that form neoantigens, responsible for the immunogenicity of tumors. Randomized controlled trials have so far failed to show a survival benefit when stratifying patients by tissue TMB. TMB has also been evaluated in plasma (PTMB). PTMB is anticipated to represent the biology of the entire cancer, whereas obtaining tissue of an amenable primary or a metastatic lesion may be prone to sampling bias because of tumor heterogeneity. For this reason, we are evaluating the correlation between TMB and PTMB, and prospectively evaluating the impact of these biomarkers on clinical outcomes. We also discuss the technical difficulties inherent to performing and comparing these analyses. Furthermore, we evaluate the correlation between the evolution of PTMB during an immunotherapy treatment and response at 3 and 6 months, as we believe PTMB may be a dynamic biomarker. In this paper, we present results from the first 4 patients in this project.

Implementing circulating tumor DNA analysis in a clinical laboratory: A user manual.

Liquid biopsy, the analysis of cell-free circulating tumor DNA (ctDNA), is becoming one of the most promising tools in oncology. It has already shown its usefulness in selecting and modulating therapy via remote analysis of the tumor genome and holds important promises in cancer therapy and management, such as assessing the success of key therapeutic steps, monitoring residual disease, early detection of relapses, and establishing prognosis. Yet, ctDNA analysis is technically challenging and its implementation in the laboratory raises multiple strategic and practical issues. As for oncology clinics, integration of this novel test in well-established therapeutic protocols can also pose numerous questions. The current review is intended as a field guide for (1) diagnostic laboratories wishing to implement, validate and possibly accredit ctDNA testing and (2) clinical oncologists interested in integrating the various applications of liquid biopsies in their daily practice. We provide advice and practical recommendations based on our own experience with the technical validations of these methods and on a review of the current literature, with a focus toward gastro-intestinal, lung and breast cancers.

Circulating tumoral DNA: Preanalytical validation and quality control in a diagnostic laboratory.

We present the results of our technical validation process in establishing the analysis of circulating tumor DNA (ctDNA) as a diagnostic tool. Like most cells in our body, tumor cells shed DNA in the blood flow. Analysis of ctDNA mutational content can provide invaluable information on the genetic makeup of a tumor, and assist oncologists in deciding on therapy, or in following residual disease. However, low absolute amounts of circulating DNA and low tumor fraction constitute formidable analytical challenges. A key step is to avoid contamination with genomic DNA from cell lysis. Several brands of specialized blood collection tubes are available to prevent leukocyte lysis. We show that they are not equally efficient, depending on storage temperature and time before plasma preparation. We report our analysis of preanalytical factors pertaining to ctDNA analysis (tubes, transportation time, temperature) and our conclusions in terms of instructions to prescribing physicians. We also stress the importance of proper DNA quality control and compare several methods, including a differential amplicon length PCR technique which allows determination of multiple QC parameters from minimal amounts of DNA. Altogether, these data provide useful practical information to diagnostic laboratories wishing to implement the assay of ctDNA in clinical practice.

[Hereditary haemorrhagic telangiectasia: importance of a multidisciplinary approach]. / Maladie de Rendu-Osler-Weber: importance d'une prise en charge multidisciplinaire.

The Rendu-Osler-Weber disease, also known as hereditary haemorrhagic telangiectasia, is an autosomal dominant inherited disease. Its main manifestations are nosebleeds and digestive tract bleeding due to angiodysplasia. The presence of arteriovenous malformations in organs such as lung, liver, brain, etc. can cause serious complications (haemorrhage, stroke, brain abscess, hypoxemia, increased cardiac output, pulmonary arterial hypertension). Diagnosis is based on clinical criteria and can be confirmed by genetic analysis. The prevalence of this rare disease is 1/5,000 to 1/10,000 and its expression varies widely, even in the same family. The management must be multidisciplinary and based on prevention and treatment of bleeding complications as well as screening and treatment of arteriovenous malformations.

Genetic instability in human embryonic stem cells: prospects and caveats.

Human embryonic stem cells (hESCs) display a leaky G1/S checkpoint and inefficient nucleotide excision repair activity. Maintenance of genomic stability in these cells mostly relies on the elimination of damaged cells by high rates of apoptosis. However, a subpopulation survives and proliferates actively, bypassing DNA damage by translesion synthesis, a known mutagenic process. Indeed, high levels of damage-induced mutations were observed in hESCs, similar to those in repair-deficient cells. The surviving cells also become more resistant to further damage, leading to a progressive enrichment of cultures in mutant cells. In long-term cultures, hESCs display features characteristic of neoplastic progression, including chromosomal anomalies often similar to those observed in embryo carcinoma. The implication of these facts for stem cell-based therapy and cancer research are discussed.

Deficient DNA damage response and cell cycle checkpoints lead to accumulation of point mutations in human embryonic stem cells.

Human embryonic stem cells (hESCs) tend to lose genomic integrity during long periods of culture in vitro and to acquire a cancer-like phenotype. In this study, we aim at understanding the contribution of point mutations to the adaptation process and at providing a mechanistic explanation for their accumulation. We observed that, due to the absence of p21/Waf1/Cip1, cultured hESCs lack proper cell cycle checkpoints and are vulnerable to the kind of DNA damage usually repaired by the highly versatile nucleotide excision repair (NER) pathway. In response to UV-induced DNA damage, the majority of hESCs succumb to apoptosis; however, a subpopulation continues to proliferate, carrying damaged DNA and accumulating point mutations with a typical UV-induced signature. The UV-resistant cells retain their proliferative capacity and potential for pluripotent differentiation and are markedly less apoptotic to subsequent UV exposure. These findings demonstrate that, due to deficient DNA damage response, the modest NER activity in hESCs is insufficient to prevent increased mutagenesis. This provides for the appearance of genetically aberrant hESCs, paving the way for further major genetic changes.

The Fanconi anemia pathway is downregulated upon macrophage differentiation through two distinct mechanisms.

The Fanconi anaemia (FA) pathway is a DNA-damage inducible pathway critical for genomic stability. FA patients typically display high cancer susceptibility and hypersensitivity to DNA-damaging agents such as cross-linkers and ionizing radiation. A key step in the activation of the FA pathway is monoubiquitination of the FancD2 protein. Here we report that the FA pathway is downregulated by two distinct mechanisms upon differentiation of THP-1 and HL-60 leukaemia cells into macrophages. Firstly, qRT-PCR analysis revealed a transcriptional downregulation of most components of the FA complex, including FancD2. Secondly, DNA damage-induced monoubiquitination of the remaining FancD2 became deficient at various stages of differentiation depending on the type of damage. This was attributed to the differentiation-induced downregulation of Chk1, which phosphorylates FancD2 as a prelude to its ubiquitination. Although Western blotting revealed that levels of FancD2 were greatly reduced in terminally differentiated macrophages and that FancD2 ubiquitination was abolished, double-strand breaks were proficiently repaired, likely through an increase in non-homologous end joining (NHEJ). It has been suggested that the FA pathway promotes repair of double-strand breaks via homologous recombination rather than NHEJ. Its downregulation in macrophages may thus be required to avoid promoting a repair mechanism that is inefficient in post-mitotic cells.

Nucleotide excision repair and B lymphoma: somatic hypermutation is not the only culprit.

Different mechanisms account for the development of B lymphoma. Malignant transformation of B lymphocytes arises from progressive loss of genome integrity combined with uncontrolled cell proliferation, often triggered by foreign or self antigens. It is well established that somatic hypermutation, the pathway responsible for introducing high levels of mutations in immunoglobulin genes, also targets several other genes, contributing mainly to germinal center-derived B-cell lymphoma. We have recently discovered that a major DNA repair pathway, nucleotide excision repair (NER), is downregulated in quiescent B lymphocytes. Upon B-cell stimulation, unrepaired DNA damage results in the accumulation of mutations in a different and likely larger set of genes, including normally silent genes (e.g., oncogenes) as well as cell cycle and activation-induced genes. This mechanism potentially produces a transforming event relevant to a wider palette of B lymphomas. Here we discuss the relative contribution of both mechanisms to lymphomagenesis and possible implications of NER downregulation for other types of malignancies and for B cell-mediated immunity. Given that hematopoietic cancer stem cells remain quiescent for long periods of time, we propose that downregulation of NER during quiescence, in an environment that causes both genotoxic stress and proliferation, could be a general mechanism for carcinogenesis.

Multiple roles of ubiquitination in the control of nucleotide excision repair.

Nucleotide excision repair (NER) is a remarkably versatile DNA repair system, essential for maintenance of genomic stability. Hereditary alterations in NER enzymes can result in increased cancer propensity, but also in developmental, neurodegenerative, and progeroid syndromes. NER can be operationally divided in three subtypes, which share many common steps: global genomic repair (GGR) operates on the whole genome level, transcription domain-associated repair (DAR) is a concentration of NER activity within transcription factories, and transcription-coupled repair (TCR) provides faster repair of the transcribed strand of active genes. Interestingly, ubiquitination plays an important role in all three classes of NER, as well as in associated phenomena, such as damage signalling by histone ubiquitination, and degradation of stalled RNA polymerase II when repair does not occur in a timely manner.

Circulating human B lymphocytes are deficient in nucleotide excision repair and accumulate mutations upon proliferation.

Faithful repair of DNA lesions is a crucial task that dividing cells must actively perform to maintain genome integrity. Strikingly, nucleotide excision repair (NER), the most versatile DNA repair system, is specifically down-regulated in terminally differentiated cells. This prompted us to examine whether NER attenuation might be a common feature of all G0-arrested cells, and in particular of those that retain the capacity to reenter cell cycle and might thus convert unrepaired DNA lesions into mutations, a prerequisite for malignant transformation. Here we report that quiescent primary human B lymphocytes down-regulate NER at the global genome level while maintaining proficient repair of constitutively expressed genes. Quiescent B cells exposed to an environment that causes both DNA damage and proliferation accumulate point mutations in silent and inducible genes crucial for cell replication and differentiation, such as BCL6 and Cyclin D2. Similar to differentiated cells, NER attenuation in quiescent cells is associated with incomplete phosphorylation of the ubiquitin activating enzyme Ube1, which is required for proficient NER. Our data establish a mechanistic link between NER attenuation during quiescence and cell mutagenesis and also support the concept that oncogenic events targeting cell cycle- or activation-induced genes might initiate genomic instability and lymphomagenesis.