Heterozygous mutations in the C-terminal domain of COPA underlie a complex autoinflammatory syndrome.

Mutations in the N-terminal WD40 domain of coatomer protein complex subunit α (COPA) cause a type I interferonopathy, typically characterized by alveolar hemorrhage, arthritis, and nephritis. We described 3 heterozygous mutations in the C-terminal domain (CTD) of COPA (p.C1013S, p.R1058C, and p.R1142X) in 6 children from 3 unrelated families with a similar syndrome of autoinflammation and autoimmunity. We showed that these CTD COPA mutations disrupt the integrity and the function of coat protein complex I (COPI). In COPAR1142X and COPAR1058C fibroblasts, we demonstrated that COPI dysfunction causes both an anterograde ER-to-Golgi and a retrograde Golgi-to-ER trafficking defect. The disturbed intracellular trafficking resulted in a cGAS/STING-dependent upregulation of the type I IFN signaling in patients and patient-derived cell lines, albeit through a distinct molecular mechanism in comparison with mutations in the WD40 domain of COPA. We showed that CTD COPA mutations induce an activation of ER stress and NF-κB signaling in patient-derived primary cell lines. These results demonstrate the importance of the integrity of the CTD of COPA for COPI function and homeostatic intracellular trafficking, essential to ER homeostasis. CTD COPA mutations result in disease by increased ER stress, disturbed intracellular transport, and increased proinflammatory signaling.

Tools to differentiate between Filamin C and Titin truncating variant carriers: value of MRI.

Whereas truncating variants of the giant protein Titin (TTNtv) are the main cause of familial dilated cardiomyopathy (DCM), recently Filamin C truncating variants (FLNCtv) were identified as a cause of arrhythmogenic cardiomyopathy (ACM). Our aim was to characterize and compare clinical and MRI features of TTNtv and FLNCtv in the Belgian population. In index patients referred for genetic testing of ACM/DCM, FLNCtv and TTNtv were found in 17 (3.6%) and 33 (12.3%) subjects, respectively. Further family cascade screening yielded 24 and 19 additional truncating variant carriers in FLNC and TTN, respectively. The main phenotype was ACM in FLNCtv carriers whereas TTNtv carriers showed either an ACM or DCM phenotype. Non-sustained Ventricular Tachycardia was frequent in both populations. MRI data, available in 28/40 FLNCtv and 32/52 TTNtv patients, showed lower Left Ventricular (LV) ejection fraction and lower LV strain in TTNtv patients (p < 0.01). Conversely, both the frequency (68% vs 22%) and extent of non-ischemic myocardial late gadolinium enhancement (LGE) was significantly higher in FLNCtv patients (p < 0.01). Hereby, ring-like LGE was found in 16/19 (84%) FLNCtv versus 1/7 (14%) of TTNtv patients (p < 0.01). In conclusion, a large number of FLNCtv and TTNtv patients present with an ACM phenotype but can be separated by cardiac MRI. Whereas FLNCtv patients often have extensive myocardial fibrosis, typically following a ring-like pattern, LV dysfunction without or limited replacement fibrosis is the common TTNtv phenotype.

Clinical application of multigene panel testing for bleeding, thrombotic, and platelet disorders: a 3-year Belgian experience.

BACKGROUND: The international study ThromboGenomics has evaluated the diagnostic rate using a targeted multigene panel test for the screening of inherited bleeding, thrombotic and platelet disorders. OBJECTIVES: We retrospectively analyzed the results of the implementation of genetic testing for inherited bleeding, thrombotic and platelet disorders in Belgian clinical practice and evaluated possible reclassification of reported variants. PATIENTS/METHODS: We implemented a Thrombosis-Hemostasis multigene panel test using whole exome sequencing to diagnose 487 patients recruited by 27 different Belgian hospitals with the implementation of stringent laboratory accreditation standards and by studying up to 100 diagnostic-grade genes. RESULTS: This Thrombosis-Hemostasis multigene panel test was able to detect at least one genetic variant in 58% of the 487 patients of which 50% were (likely) pathogenic variants and the others were variants of unknown significance. Polygenic variants were detected in 65 patients (13%). A multi-step workflow for results discussion by multidisciplinary team meetings and patients' recalls for segregation studies and additional laboratory testing was set up. Variants were also submitted to the GoldVariants database from the International Society on Thrombosis and Haemostasis (ISTH). The aim of these approaches is to optimize variant interpretation and to (re)classify variants of unknown significance as (likely) pathogenic or (likely) benign. CONCLUSIONS: The growing implementation of multigene panel tests in clinical diagnostics comes with difficulties in interpreting genetic results. Additional efforts are needed to continuously optimize the diagnostic outcome.

Common variable immunodeficiency in two kindreds with heterogeneous phenotypes caused by novel heterozygous NFKB1 mutations.

NFKB1 haploinsufficiengcy was first described in 2015 in three families with common variable immunodeficiency (CVID), presenting heterogeneously with symptoms of increased infectious susceptibility, skin lesions, malignant lymphoproliferation and autoimmunity. The described mutations all led to a rapid degradation of the mutant protein, resulting in a p50 haploinsufficient state. Since then, more than 50 other mutations have been reported, located throughout different domains of NFKB1 with the majority situated in the N-terminal Rel homology domain (RHD). The clinical spectrum has also expanded with possible disease manifestations in almost any organ system. In silico prediction tools are often used to estimate the pathogenicity of NFKB1 variants but to prove causality between disease and genetic findings, further downstream functional validation is required. In this report, we studied 2 families with CVID and two novel variants in NFKB1 (c.1638-2A>G and c.787G>C). Both mutations affected mRNA and/or protein expression of NFKB1 and resulted in excessive NLRP3 inflammasome activation in patient macrophages and upregulated interferon stimulated gene expression. Protein-protein interaction analysis demonstrated a loss of interaction with NFKB1 interaction partners for the p.V263L mutation. In conclusion, we proved pathogenicity of two novel variants in NFKB1 in two families with CVID characterized by variable and incomplete penetrance.

Child Neurology: Familial Hemophagocytic Lymphohistiocytosis Underlying Isolated CNS Inflammation.

Encephalitis and encephalopathy in children represent a diagnostic challenge. We describe a patient with relapsing encephalitis in whom the differential diagnosis included acute disseminated encephalomyelitis, human herpesvirus 6 encephalitis, and hemophagocytic lymphohistiocytosis (HLH). Because of its rarity, HLH is often overlooked as a differential diagnosis in encephalitis, especially in the isolated CNS forms. As this case illustrates, inborn errors of immunity can underlie isolated encephalitis and should be included in the differential diagnosis of these presentations.

Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel NaV1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on NaV1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings.

Guidelines for Genetic Testing and Management of Alport Syndrome.

Genetic testing for pathogenic COL4A3-5 variants is usually undertaken to investigate the cause of persistent hematuria, especially with a family history of hematuria or kidney function impairment. Alport syndrome experts now advocate genetic testing for persistent hematuria, even when a heterozygous pathogenic COL4A3 or COL4A4 is suspected, and cascade testing of their first-degree family members because of their risk of impaired kidney function. The experts recommend too that COL4A3 or COL4A4 heterozygotes do not act as kidney donors. Testing for variants in the COL4A3-COL4A5 genes should also be performed for persistent proteinuria and steroid-resistant nephrotic syndrome due to suspected inherited FSGS and for familial IgA glomerulonephritis and kidney failure of unknown cause.

Frequency of Participation in External Quality Assessment Programs Focused on Rare Diseases: Belgian Guidelines for Human Genetics Centers.

BACKGROUND: Participation in quality controls, also called external quality assessment (EQA) schemes, is required for the ISO15189 accreditation of the Medical Centers of Human Genetics. However, directives on the minimal frequency of participation in genetic quality control schemes are lacking or too heterogeneous, with a possible impact on health care quality. OBJECTIVE: The aim of this project is to develop Belgian guidelines on the frequency of participation in quality controls for genetic testing in the context of rare diseases. METHODS: A group of experts analyzed 90 EQA schemes offered by accredited providers and focused on analyses used for the diagnosis of rare diseases. On that basis, the experts developed practical recommendations about the minimal frequencies of participation of the Medical Centers of Human Genetics in quality controls and how to deal with poor performances and change management. These guidelines were submitted to the Belgian Accreditation Body and then reviewed and approved by the Belgian College of Human Genetics and Rare Diseases and by the National Institute for Health and Disability Insurance. RESULTS: The guidelines offer a decisional algorithm for the minimal frequency of participation in human genetics EQA schemes. This algorithm has been developed taking into account the scopes of the EQA schemes, the levels of experience, and the annual volumes of the Centers of Human Genetics in the performance of the tests considered. They include three key principles: (1) the recommended annual assessment of all genetic techniques and technological platforms, if possible through EQAs covering the technique, genotyping, and clinical interpretation; (2) the triennial assessment of the genotyping and interpretation of specific germline mutations and pharmacogenomics analyses; and (3) the documentation of actions undertaken in the case of poor performances and the participation to quality control the following year. The use of a Bayesian statistical model has been proposed to help the Centers of Human Genetics to determine the theoretical number of tests that should be annually performed to achieve a certain threshold of performance (eg, a maximal error rate of 1%). Besides, the guidelines insist on the role and responsibility of the national public health authorities in the follow-up of the quality of analyses performed by the Medical Centers of Human Genetics and in demonstrating the cost-effectiveness and rationalization of participation frequency in these quality controls. CONCLUSIONS: These guidelines have been developed based on the analysis of a large panel of EQA schemes and data collected from the Belgian Medical Centers of Human Genetics. They are applicable to other countries and will facilitate and improve the quality management and financing systems of the Medical Centers of Human Genetics.

Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome: refining the ACMG criteria.

The recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3 and COL4A4 genes beyond the classical Alport phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3-5). It identified 'mutational hotspots' (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that 'well-established' functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3 and COL4A4 variants were common 'incidental' findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3-COL4A5 genes remains a challenge.

Clinical characterization of the first Belgian SCN5A founder mutation cohort.

AIMS: We identified the first Belgian SCN5A founder mutation, c.4813 + 3_4813 + 6dupGGGT. To describe the clinical spectrum and disease severity associated with this mutation, clinical data of 101 SCN5A founder mutation carriers and 46 non-mutation carrying family members from 25 Belgian families were collected. METHODS AND RESULTS: The SCN5A founder mutation was confirmed by haplotype analysis. The clinical history and electrocardiographic parameters of the mutation carriers and their family members were gathered and compared. A cardiac electrical abnormality was observed in the majority (82%) of the mutation carriers. Cardiac conduction defects, defined as PR or QRS prolongation on electrocardiogram (ECG), were most frequent, occurring in 65% of the mutation carriers. Brugada syndrome (BrS) was the second most prevalent phenotype identified in 52%, followed by atrial dysrythmia in 11%. Overall, 33% of tested mutation carriers had a normal sodium channel blocker test. Negative tests were more common in family members distantly related to the proband. Overall, 23% of the mutation carriers were symptomatic, with 8% displaying major adverse events. As many as 13% of the patients tested with a sodium blocker developed ventricular arrhythmia. One family member who did not carry the founder mutation was diagnosed with BrS. CONCLUSION: The high prevalence of symptoms and sensitivity to sodium channel blockers in our founder population highlights the adverse effect of the founder mutation on cardiac conduction. The large phenotypical heterogeneity, variable penetrance, and even non-segregation suggest that other genetic (and environmental) factors modify the disease expression, severity, and outcome in these families.

Enhancing rare variant interpretation in inherited arrhythmias through quantitative analysis of consortium disease cohorts and population controls.

PURPOSE: Stringent variant interpretation guidelines can lead to high rates of variants of uncertain significance (VUS) for genetically heterogeneous disease like long QT syndrome (LQTS) and Brugada syndrome (BrS). Quantitative and disease-specific customization of American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines can address this false negative rate. METHODS: We compared rare variant frequencies from 1847 LQTS (KCNQ1/KCNH2/SCN5A) and 3335 BrS (SCN5A) cases from the International LQTS/BrS Genetics Consortia to population-specific gnomAD data and developed disease-specific criteria for ACMG/AMP evidence classes-rarity (PM2/BS1 rules) and case enrichment of individual (PS4) and domain-specific (PM1) variants. RESULTS: Rare SCN5A variant prevalence differed between European (20.8%) and Japanese (8.9%) BrS patients (p = 5.7 × 10-18) and diagnosis with spontaneous (28.7%) versus induced (15.8%) Brugada type 1 electrocardiogram (ECG) (p = 1.3 × 10-13). Ion channel transmembrane regions and specific N-terminus (KCNH2) and C-terminus (KCNQ1/KCNH2) domains were characterized by high enrichment of case variants and >95% probability of pathogenicity. Applying the customized rules, 17.4% of European BrS and 74.8% of European LQTS cases had (likely) pathogenic variants, compared with estimated diagnostic yields (case excess over gnomAD) of 19.2%/82.1%, reducing VUS prevalence to close to background rare variant frequency. CONCLUSION: Large case-control data sets enable quantitative implementation of ACMG/AMP guidelines and increased sensitivity for inherited arrhythmia genetic testing.

Adult-Onset ANCA-Associated Vasculitis in SAVI: Extension of the Phenotypic Spectrum, Case Report and Review of the Literature.

STING-associated vasculopathy with onset in infancy (SAVI) is an autosomal dominant disorder due to gain-of-function mutations in STING1, also known as TMEM173, encoding for STING. It was reported as a vasculopathy of infancy. However, since its description a wider spectrum of associated manifestations and disease-onset has been observed. We report a kindred with a heterozygous STING mutation (p.V155M) in which the 19-year-old proband suffered from isolated adult-onset ANCA-associated vasculitis. His father suffered from childhood-onset pulmonary fibrosis and renal failure attributed to ANCA-associated vasculitis, and died at the age of 30 years due to respiratory failure. In addition, an overview of the phenotypic spectrum of SAVI is provided highlighting (a) a high phenotypic variability with in some cases isolated manifestations, (b) the potential of adult-onset disease, and (c) a novel manifestation with ANCA-associated vasculitis.

Next-generation sequencing in prenatal setting: Some examples of unexpected variant association.

The application of next-generation sequencing to fetal pathology has proved to increase the diagnostic yield in fetuses with abnormal ultrasounds. We retrospectively reviewed genetic data of 30 selected cases studied through targeted resequencing of OMIM genes. In our experience, clinical data proved to be essential to support diagnostic reasoning and enhance variants' assessment. The molecular diagnosis was reached in 19/30 (63%) cases. Only in 7/19 cases the molecular diagnosis confirmed the initial diagnostic hypothesis, showing the relevance of the genotype-first approach. According to the genotype-phenotype correlation, we were able to divide the solved cases into three groups: i) the correlation is well established but it was missed due to lack of specificity, unusual presentation or recent description; ii) the clinical presentation is much more severe than currently known for the underlying condition; iii) the correlation does not recapitulate the entire phenotype, possibly due to the fetal presentation or multiple coexisting conditions. Moreover, we found a higher proportion of recessive diagnosis in abnormal fetuses compared to cohorts of individuals with developmental delay. Our findings suggest that fetal pathology may be enriched in rare alleles and/or in unusual combinations, counter-selected in postnatal genomes and thus contributing to both phenotypic extremeness and atypical presentation.

A double-edged sword.

In medicine, not everything is what it seems and sometimes you need all the pieces of the puzzle in order to complete the diagnosis. This is a case of a disease with two different faces and a review of its respiratory impact. https://bit.ly/2SDKwE5.