Bi-allelic genetic variants in the translational GTPases GTPBP1 and GTPBP2 cause a distinct identical neurodevelopmental syndrome.

The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species.

Relevance of genetic testing in the gene-targeted trial era: the Rostock Parkinson's disease study.

Estimates of the spectrum and frequency of pathogenic variants in Parkinson's disease (PD) in different populations are currently limited and biased. Furthermore, although therapeutic modification of several genetic targets has reached the clinical trial stage, a major obstacle in conducting these trials is that PD patients are largely unaware of their genetic status and, therefore, cannot be recruited. Expanding the number of investigated PD-related genes and including genes related to disorders with overlapping clinical features in large, well-phenotyped PD patient groups is a prerequisite for capturing the full variant spectrum underlying PD and for stratifying and prioritizing patients for gene-targeted clinical trials. The Rostock Parkinson's disease (ROPAD) study is an observational clinical study aiming to determine the frequency and spectrum of genetic variants contributing to PD in a large international cohort. We investigated variants in 50 genes with either an established relevance for PD or possible phenotypic overlap in a group of 12 580 PD patients from 16 countries [62.3% male; 92.0% White; 27.0% positive family history (FH+), median age at onset (AAO) 59 years] using a next-generation sequencing panel. Altogether, in 1864 (14.8%) ROPAD participants (58.1% male; 91.0% White, 35.5% FH+, median AAO 55 years), a PD-relevant genetic test (PDGT) was positive based on GBA1 risk variants (10.4%) or pathogenic/likely pathogenic variants in LRRK2 (2.9%), PRKN (0.9%), SNCA (0.2%) or PINK1 (0.1%) or a combination of two genetic findings in two genes (∼0.2%). Of note, the adjusted positive PDGT fraction, i.e. the fraction of positive PDGTs per country weighted by the fraction of the population of the world that they represent, was 14.5%. Positive PDGTs were identified in 19.9% of patients with an AAO ≤ 50 years, in 19.5% of patients with FH+ and in 26.9% with an AAO ≤ 50 years and FH+. In comparison to the idiopathic PD group (6846 patients with benign variants), the positive PDGT group had a significantly lower AAO (4 years, P = 9 × 10-34). The probability of a positive PDGT decreased by 3% with every additional AAO year (P = 1 × 10-35). Female patients were 22% more likely to have a positive PDGT (P = 3 × 10-4), and for individuals with FH+ this likelihood was 55% higher (P = 1 × 10-14). About 0.8% of the ROPAD participants had positive genetic testing findings in parkinsonism-, dystonia/dyskinesia- or dementia-related genes. In the emerging era of gene-targeted PD clinical trials, our finding that ∼15% of patients harbour potentially actionable genetic variants offers an important prospect to affected individuals and their families and underlines the need for genetic testing in PD patients. Thus, the insights from the ROPAD study allow for data-driven, differential genetic counselling across the spectrum of different AAOs and family histories and promote a possible policy change in the application of genetic testing as a routine part of patient evaluation and care in PD.

GBA1-Associated Parkinson's Disease Is a Distinct Entity.

GBA1-associated Parkinson's disease (GBA1-PD) is increasingly recognized as a distinct entity within the spectrum of parkinsonian disorders. This review explores the unique pathophysiological features, clinical progression, and genetic underpinnings that differentiate GBA1-PD from idiopathic Parkinson's disease (iPD). GBA1-PD typically presents with earlier onset and more rapid progression, with a poor response to standard PD medications. It is marked by pronounced cognitive impairment and a higher burden of non-motor symptoms compared to iPD. Additionally, patients with GBA1-PD often exhibit a broader distribution of Lewy bodies within the brain, accentuating neurodegenerative processes. The pathogenesis of GBA1-PD is closely associated with mutations in the GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase). In this review, we discuss two mechanisms by which GBA1 mutations contribute to disease development: 'haploinsufficiency,' where a single functional gene copy fails to produce a sufficient amount of GCase, and 'gain of function,' where the mutated GCase acquires harmful properties that directly impact cellular mechanisms for alpha-synuclein degradation, leading to alpha-synuclein aggregation and neuronal cell damage. Continued research is advancing our understanding of how these mechanisms contribute to the development and progression of GBA1-PD, with the 'gain of function' mechanism appearing to be the most plausible. This review also explores the implications of GBA1 mutations for therapeutic strategies, highlighting the need for early diagnosis and targeted interventions. Currently, small molecular chaperones have shown the most promising clinical results compared to other agents. This synthesis of clinical, pathological, and molecular aspects underscores the assertion that GBA1-PD is a distinct clinical and pathobiological PD phenotype, necessitating specific management and research approaches to better understand and treat this debilitating condition.

Contribution of Glucosylsphingosine (Lyso-Gb1) to Treatment Decisions in Patients with Gaucher Disease.

Glucosylsphingosine (lyso-Gb1), the deacylated form of glucocerebroside, was shown to be the most specific and sensitive biomarker for diagnosing Gaucher disease (GD). The aim of this study is to assess the contribution of lyso-Gb1 at the time of diagnosis for treatment decisions in naïve patients with GD. Newly diagnosed patients from July 2014 to November 2022 were included in this retrospective cohort study. The diagnosis was done by sending a dry blood spot (DBS) sample for GBA1 molecular sequencing and lyso-Gb1 quantification. Treatment decisions were based on symptoms, signs, and routine laboratory tests. We diagnosed 97 patients (41 males), both type 1 (n = 87), and neuronopathic (n = 10). The median (range) age at diagnosis was 22 (1-78), with 36 children. In 65 patients, GD-specific therapy was started with a median (range) lyso-Gb1, 337 (60-1340) ng/mL, significantly higher than in patients who did not go on to treatment, 153.5 (9-442) ng/mL. Using a receiver operating characteristic (ROC) analysis, a cutoff of lyso-Gb1 > 250 ng/mL was associated with treatment with a sensitivity of 71% and specificity of 87.5%. Predictors of treatment were thrombocytopenia, anemia, and elevated lyso-Gb1 (>250 ng/mL). In conclusion, lyso-Gb1 levels contribute to the medical decision related to the initiation of treatment, mainly among mildly affected newly diagnosed patients. For patients with a severe phenotype, as for all patients, the main value of lyso-Gb1 would be to monitor response to therapy. The variable methodology and differences in the units of lyso-Gb1 measurements between laboratories prevent the adaptation of the exact cut-off we found in general practice. However, the concept is that a significant elevation, i.e., a several-fold increase from the diagnostic lyso-Gb1 cutoff, is related to a more severe phenotype and, accordingly, to the decision regarding the initiation of GD-specific therapy.

The Rostock International Parkinson's Disease (ROPAD) Study: Protocol and Initial Findings.

BACKGROUND: Genetic stratification of Parkinson's disease (PD) patients facilitates gene-tailored research studies and clinical trials. The objective of this study was to describe the design of and the initial data from the Rostock International Parkinson's Disease (ROPAD) study, an epidemiological observational study aiming to genetically characterize ~10,000 participants. METHODS: Recruitment criteria included (1) clinical diagnosis of PD, (2) relative of participant with a reportable LRRK2 variant, or (3) North African Berber or Ashkenazi Jew. DNA analysis involved up to 3 successive steps: (1) variant (LRRK2) and gene (GBA) screening, (2) panel sequencing of 68 PD-linked genes, and (3) genome sequencing. RESULTS: Initial data based on the first 1360 participants indicated that the ROPAD enrollment strategy revealed a genetic diagnostic yield of ~14% among a PD cohort from tertiary referral centers. CONCLUSIONS: The ROPAD screening protocol is feasible for high-throughput genetic characterization of PD participants and subsequent prioritization for gene-focused research efforts and clinical trials. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Screening for patients with Gaucher's disease using routine pathology results: PATHFINDER (ferritin, alkaline phosphatase, platelets) study.

AIMS: Lysosomal ß-glucocerebrosidase A (GBA) deficiency causes Gaucher disease (GD), a recessive disorder caused by bi-allelic mutations in GBA. The prevalence of GD is associated with ethnicity but largely unknown and potentially underestimated in many countries. GD may manifest with organomegaly, bone involvement, and neurological symptoms as well as abnormal laboratory biomarkers. This study attempted to screen for GD in patients using abnormal platelet, alkaline phosphatase (ALP), and ferritin results from laboratory databases. METHODS: Electronic laboratory databases were interrogated using a 2- to 4-year time interval to identify from clinical biochemistry records patients with a phenotype of reduced platelets (<150 × 109 /L) and either elevated ALP (>130 iu/L) or ferritin [>150 (female) or >250 µg/L (male)]. The mean value over the screening window was used to reduce variability in results. A dried blood spot sample was collected for the determination of GBA activity in patients meeting these criteria. If low GBA activity was found, then the concentration of the GD-specific biomarker glucosyl-sphingosine (lyso-GB1) was assayed, and the GBA gene sequenced. RESULTS: Samples were obtained from 1058 patients; 232 patients had low GBA activity triggering further analysis. No new cases of GD with homozygosity for pathogenic variants were identified, but 12 patients (1%) were identified to be carriers of a pathogenic variant in GBA. CONCLUSIONS: Pathology databases hold routine information that can be used to screen for patients with inherited errors of metabolism. However, biochemical screening using mean platelets, ALP, and ferritin has a low yield for unidentified cases of GD.

Biphasic zinc compartmentalisation in a human fungal pathogen.

Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH-dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2Δ growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrt1/Zrt2-cellular import, followed by Zrc1-dependent intracellular compartmentalisation.

Hereditary Transthyretin-Related Amyloidosis Ongoing Observational Study: A Baseline Report of the First 3167 Participants.

Background: Hereditary transthyretin-related amyloidosis is a clinically heterogeneous autosomal dominant disease caused by pathogenic variants in the TTR gene (hATTR amyloidosis). Objective: The current study describes the demographic, clinical, and genetic characteristics of patients with suspected hATTR amyloidosis. Methods: This study is part of the "Hereditary transthyretin-related amyloidosis and longitudinal monitoring of TTR-positive patients" (TRAMmoniTTR) study. This study included 3167 participants, along with their clinical details. Principal component (PC) analysis was used to analyze their clinical symptomatology. Next-generation sequencing of the TTR gene was performed and genotype-phenotype relationships were investigated. We compared the demographic and clinical characteristics using the principal components (PCs) and also compared participants with and without the TTR pathogenic variants. Results: We identified five main clinical phenotypes out of 22 single symptoms that explained 49% of the variation. The first two PCs referred to polyneuropathy and cardiomyopathy. We found significant differences between gender and PC-polyneuropathy and PC-cardiomyopathy, with male over-representation in the higher quantiles of PC-polyneuropathy and male under-representation in the lowest quantiles of PC-cardiomyopathy. We identified 92 participants with hATTR (3%), exhibiting 17 unique heterozygous TTR variants. The p.Val50Met variant was the most frequent. Furthermore, 503 participants (20%) were identified with ATTR and no relevant TTR variants (ATTRwt). We detected significant differences between the ATTRwt and hATTR groups, with male gender predominance in only the ATTRwt group and a positive family history of polyneuropathy and/or cardiomyopathy among the hATTR participants. Conclusions: The current clinical and genetic characterization of this cohort serves as a foundation for further longitudinal monitoring and assessment.

Microbial contamination and composition of oral samples subjected to clinical whole genome sequencing.

Biological material from the oral cavity is an excellent source of samples for genetic diagnostics. This is because collection is quick, easy-to-access, and non-invasive. We have set-up clinical whole genome sequence testing for patients with suspected hereditary disease. Beside the excellent quality of human DNA that can be isolated from such samples, we observed the presence of non-human DNA sequences at varying percentages. We investigated the proportion of non-human mapped reads (NHMR) sequenced from buccal swabs and saliva, the type of microbial genomes from which they were derived, and impact on molecular classification. Read sequences that did not map to the human reference genome were aligned to complete reference microbial reference sequences from the National Center for Biotechnology Information's (NCBI) RefSeq database using Kraken2. Out of 765 analyzed samples over 80% demonstrated more than 5% NHMRs. The majority of NHMRs were from bacterial genomes (average 69%, buccal swabs and 54% saliva), while the proportion of viruses was low, averaging 0.32% (buccal swabs) and 0.07% (saliva). We identified more than 30 different bacterial families of which Streptococcus mitis and Rothia mucilaginosa were the most common species. Importantly, the level of contamination did not impact the diagnostic yield.

Secondary findings in a large Pakistani cohort tested with whole genome sequencing.

Studies on genomic secondary findings (SFs) are diverse in participants' characteristics, sequencing methods, and versions of the ACMG SF list. Based on whole genome sequencing and the version 3.1 of the ACMG SF list, we studied SFs in 863 individuals from five different regions in Pakistan. We identified 24 ACMG SFs in 23 (2.7%) of 863 individuals: 18 of 24 were related to cardiovascular disease and four to cancer syndromes. In addition to ACMG SFs, we identified 16 (1.9%) participants with pathogenic and likely pathogenic variants in genes that were not related to the participants' clinical conditions but with clear medical actionability (non-ACMG SFs): 4 of 16 were related to eye diseases, two to metabolic disorders, and two to urinary system disorders. By testing a large Pakistani cohort with whole genome sequencing, we concluded that in countries such as Pakistan, the ACMG SF list could be expanded, and our non-ACMG SF list is one example.

Additional evidence on the phenotype produced by combination of CFTR 1677delTA alleles and their relevance in causing CFTR-related disease.

Cystic fibrosis is the most common, life-threatening, autosomal recessive disease in the Caucasian population. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, which encodes a chloride ion channel expressed on the surface of epithelial cells. There are more than 2000 variants of the cystic fibrosis transmembrane conductance regulator gene reported worldwide. Some of these variants cause classic cystic fibrosis, while others are labeled as variants of unknown significance or variants of varying clinical consequences alleles and associated with atypical disease or cystic fibrosis transmembrane conductance regulator-related disorders. Although these alleles do not directly cause cystic fibrosis, they may predispose compound heterozygous patients to certain clinical phenotypes. Specifically, 1677delTA has been reported as a pathogenic allele in homozygous state or in combination with other cystic fibrosis-causing alleles. However, the L997F allele is considered to be benign or causative of non-classic cystic fibrosis or cystic fibrosis transmembrane conductance regulator-related disorders in combination with other pathogenic alleles. In this case series, we describe three cases with 1677delTA and L997F genotype, and speculate that a co-concurrence of these two alleles in trans does not cause classic cystic fibrosis symptoms; however, because the late-onset of cystic fibrosis is possible in the presence of rare alleles, such as L997F, longer follow-up of these patients and identification of a greater number of adults with 1677delTA/L997F genotype are necessary to make final conclusion about the nature of this genotype.

Insights into the Value of Lyso-Gb1 as a Predictive Biomarker in Treatment-Naïve Patients with Gaucher Disease Type 1 in the LYSO-PROOF Study.

Gaucher disease (GD) is a rare autosomal recessive disorder arising from bi-allelic variants in the GBA1 gene, encoding glucocerebrosidase. Deficiency of this enzyme leads to progressive accumulation of the sphingolipid glucosylsphingosine (lyso-Gb1). The international, multicenter, observational "Lyso-Gb1 as a Long-term Prognostic Biomarker in Gaucher Disease"-LYSO-PROOF study succeeded in enrolling a cohort of 160 treatment-naïve GD patients from diverse geographic regions and evaluated the potential of lyso-Gb1 as a specific biomarker for GD. Using genotypes based on established classifications for clinical presentation, patients were stratified into type 1 GD (n = 114) and further subdivided into mild (n = 66) and severe type 1 GD (n = 48). Due to having previously unreported genotypes, 46 patients could not be classified. Though lyso-Gb1 values at enrollment were widely distributed, they displayed a moderate and statistically highly significant correlation with disease severity measured by the GD-DS3 scoring system in all GD patients (r = 0.602, p < 0.0001). These findings support the utility of lyso-Gb1 as a sensitive biomarker for GD and indicate that it could help to predict the clinical course of patients with undescribed genotypes to improve personalized care in the future.

Genotype-phenotype correlations of cystic fibrosis in siblings compound heterozygotes for rare variant combinations: Review of literature and case report.

Here, we describe a cystic fibrosis (CF) family with affected siblings, two of whom have a combination of I1234V and 1677delTA variants with classic CF features, the third child with a combination of I1234V and L997F variants with atypical CF, and the apparently healthy mother with a combination of 1677delTA and L997F alleles. Interestingly, the sibling with I1234V and L997F variants had normal sweat test results and had a much milder phenotype than the other two siblings with I1234V and 1677delTA variants, suggesting that this combination is causative for atypical CF. The fact that their mother with the combination of 1677delTA and L997F appears to be healthy suggests that the L997F variant causes different phenotypes in different allele combinations. The current cases show that there is a genotype-phenotype correlation in this disease and underline the importance of genotyping individuals with suspected CF to allow prediction of disease severity and effective treatment.

HAE patient self-sampling for biomarker establishment.

BACKGROUND: Hereditary Angioedema (HAE) is a genetic disorder that leads to frequent angioedema attacks in various parts of the body. In most cases it is caused by pathogenic variants in the SERPING1 gene, coding for C1-Inhibitor (C1-INH). The pathogenic variants in the gene result in reduced C1-INH levels and/or activity, which causes aberrant bradykinin production and enhanced vascular permeability. The standard-of-care diagnostic test is performed biochemically via measuring C1-INH level and activity as well as the C4 level. This, however, does not allow for the diagnosis of HAE types with normal C1-INH. There is an urgent need to identify and characterize HAE biomarkers for facilitating diagnostics and personalizing the treatment. The Hereditary Angioedema Kininogen Assay (HAEKA) study aims to measure the dynamics of cleaved High Molecular Weight Kininogen (HKa) and other metabolite levels during the angioedema and non-angioedema state of the disease. The metabolites will be analyzed and verified by liquid chromatography ion mobility high resolution mass spectrometry (LC/IM-QToF MS) of dried blood spot (DBS) cards upon the study completion. The study design is truly innovative: 100 enrolled participants provide blood samples via DBS: (1) every 3 months within 2 years during regular study site visits and (2) by at-home self-sampling during HAE attacks via finger pricking. We are presenting a project design that permits clinical study activities during pandemic contact restrictions and opens the door for other clinical studies during COVID-19. RESULTS: As of October 2020, there are 41 patients from 5 sites in Germany enrolled. 90 blood samples were collected during the regular visits, and 19 of the participants also performed self-sampling during the HAE attacks from which a total of 286 attack blood samples were collected. Participating patients rate the study procedures as easy to implement in their daily lives. The concept of home self-sampling is effective, reproducible, and convenient especially in times of contact restrictions due to the COVID-19 pandemic. CONCLUSIONS: It is the hope that the HAEKA study will complete in 2023, reveal biomarker(s) for monitoring HAE disease activity, and may help to avoid HAE attacks via applying medication prior to the symptom onset.

Hereditary transthyretin-related amyloidosis is frequent in polyneuropathy and cardiomyopathy of no obvious aetiology.

BACKGROUND: Hereditary Transthyretin-Related Amyloidosis, a clinically heterogeneous autosomal dominant disease caused by pathogenic variants in the TTR gene, is characterized by the deposition of insoluble misfolded protein fibrils. The diagnosis, especially in non-endemic areas, is typically delayed by 4-5 years; a misdiagnosis due to clinical heterogeneity is common. The study objective was to define the prevalence of Hereditary Transthyretin-Related Amyloidosis in patients with polyneuropathy and/or cardiomyopathy of no obvious aetiology. METHOD: A multicenter observational "Epidemiological analysis for the hereditary Transthyretin-Related AMyloidosis"-TRAM study was performed in Germany, Austria, and Switzerland. RESULTS: A total of 5141 participants were recruited by 50 neurologic and 27 cardiologic specialized centres. Genetic analysis demonstrated a 1.1% Hereditary Transthyretin-Related Amyloidosis positivity rate among patients with polyneuropathy and/or cardiomyopathy of not obvious aetiology. Twenty-one various TTR variants (TTR-positive) were identified. Body Mass Index was lower in the TTR-positive patients as an indicator for the involvement of the autonomic nervous system; the age of onset of clinical manifestations was higher in TTR-positive patients. There were no other genotype-phenotype correlations or the prevalence of specific clinical manifestations in TTR-positive patients. CONCLUSIONS: Our data support the fact that Hereditary Transthyretin-Related Amyloidosis is underdiagnosed in polyneuropathy and cardiomyopathy patients. Routine implementation of genetic testing is recommended in patients with unexplained polyneuropathy and/or cardiomyopathy to accelerate the earlier diagnosis and the time-sensitive treatment initiation.KEY MESSAGESMore than 5.000 participants with CM and/or PNP of no obvious aetiology were recruited in the observational "Epidemiological analysis for the hereditary Transthyretin-Related AMyloidosis" TRAM study and screened for pathogenic TTR variants.The study demonstrated >1% of patients with CM and/or PNP of unclear aetiology are positive for a pathogenic TTR variant.Routine genetic testing is recommended in patients with unexplained CM and/or PNP to accelerate the initial diagnosis and timely treatment initiation.

LIPAD (LRRK2/Luebeck International Parkinson's Disease) Study Protocol: Deep Phenotyping of an International Genetic Cohort.

Background: Pathogenic variants in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most common known monogenic cause of Parkinson's disease (PD). LRRK2-linked PD is clinically indistinguishable from idiopathic PD and inherited in an autosomal dominant fashion with reduced penetrance and variable expressivity that differ across ethnicities and geographic regions. Objective: To systematically assess clinical signs and symptoms including non-motor features, comorbidities, medication and environmental factors in PD patients, unaffected LRRK2 pathogenic variant carriers, and controls. A further focus is to enable the investigation of modifiers of penetrance and expressivity of LRRK2 pathogenic variants using genetic and environmental data. Methods: Eligible participants are invited for a personal or online examination which comprises completion of a detailed eCRF and collection of blood samples (to obtain DNA, RNA, serum/plasma, immune cells), urine as well as household dust. We plan to enroll 1,000 participants internationally: 300 with LRRK2-linked PD, 200 with LRRK2 pathogenic variants but without PD, 100 PD patients with pathogenic variants in the GBA or PRKN genes, 200 patients with idiopathic PD, and 200 healthy persons without pathogenic variants. Results: The eCRF consists of an investigator-rated (1 h) and a self-rated (1.5 h) part. The first part includes the Movement Disorder Society Unified Parkinson's Disease Rating, Hoehn &Yahr, and Schwab & England Scales, the Brief Smell Identification Test, and Montreal Cognitive Assessment. The self-rating part consists of a PD risk factor, food frequency, autonomic dysfunction, and quality of life questionnaires, the Pittsburgh Sleep Quality Inventory, and the Epworth Sleepiness as well as the Hospital Anxiety and Depression Scales. The first 15 centers have been initiated and the first 150 participants enrolled (as of March 25th, 2021). Conclusions: LIPAD is a large-scale international scientific effort focusing on deep phenotyping of LRRK2-linked PD and healthy pathogenic variant carriers, including the comparison with additional relatively frequent genetic forms of PD, with a future perspective to identify genetic and environmental modifiers of penetrance and expressivity Clinical Trial Registration:ClinicalTrials.gov, NCT04214509.

Rapid Large-Scale COVID-19 Testing During Shortages.

The Coronavirus disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in economic and social lockdowns in most countries all over the globe. Early identification of infected individuals is regarded as one of the most important prerequisites for fighting the pandemic and for returning to a 'New Normal'. Large-scale testing is therefore crucial, but is facing several challenges including shortage of sample collection tools and of molecular biological reagents, and the need for safe electronic communication of medical reports. We present the successful establishment of a holistic SARS-CoV-2 testing platform that covers proband registration, sample collection and shipment, sample testing, and report issuing. The RT-PCR-based virus detection, being central to the platform, was extensively validated: sensitivity and specificity were defined as 96.8% and 100%, respectively; intra-run and inter-run precision were <3%. A novel type of sample swab and an in-house-developed RNA extraction system were shown to perform as good as commercially available products. The resulting flexibility guarantees independence from the current bottlenecks in SARS-CoV-2 testing. Based on our technology, we offered testing at local, national, and global levels. In the present study, we report the results from approx. 18,000 SARS-CoV-2 tests in almost 10,000 individuals from a low-frequency SARS-CoV-2 pandemic area in a homogenous geographical region in north-eastern Germany for a period of 10 weeks (21 March to 31 May 2020). Among the probands, five SARS-CoV-2 positive cases were identified. Comparative analysis of corresponding virus genomes revealed a diverse origin from three of the five currently recognized SARS-CoV-2 phylogenetic clades. Our study exemplifies how preventive SARS-CoV-2 testing can be set up in a rapid and flexible manner. The application of our test has enabled a safe maintenance/resume of critical local infrastructure, e.g., nursing homes where more than 5000 elderlies and caretakers got tested. The strategy outlined by the present study may serve as a blueprint for the implementation of large-scale preventive SARS-CoV-2 testing elsewhere.

Genomic testing in 1019 individuals from 349 Pakistani families results in high diagnostic yield and clinical utility.

We implemented a collaborative diagnostic program in Lahore (Pakistan) aiming to establish the genetic diagnosis, and to asses diagnostic yield and clinical impact in patients with suspected genetic diseases. Local physicians ascertained pediatric patients who had no previous access to genetic testing. More than 1586 genetic tests were performed in 1019 individuals (349 index cases, 670 relatives). Most frequently performed tests were exome/genome sequencing (ES/GS, 284/78 index cases) and specific gene panels (55 index cases). In 61.3% of the patients (n = 214) a genetic diagnosis was established based on pathogenic and likely pathogenic variants. Diagnostic yield was higher in consanguineous families (60.1 vs. 39.5%). In 27 patients, genetic diagnosis relied on additional biochemical testing, allowing rapid assessment of the functional effect of the variants. Remarkably, the genetic diagnosis had a direct impact on clinical management. Most relevant consequences were therapy related such as initiation of the appropriated treatment in a timely manner in 51.9% of the patients (n = 111). Finally, we report 12 candidate genes among 66 cases with no genetic diagnosis. Importantly, three of these genes were validated as 'diagnostic' genes given the strong evidence supporting causality derived from our data repository (CAP2-dilated cardiomyopathy, ITFG2-intellectual disability and USP53-liver cholestasis). The high diagnostic yield, clinical impact, and research findings demonstrate the utility of genomic testing, especially when used as first-line genetic test. For patients with suspected genetic diseases from resource-limited regions, ES can be considered as the test of choice to achieve genetic diagnosis.

Hereditary ATTR Amyloidosis in Austria: Prevalence and Epidemiological Hot Spots.

BACKGROUND: Hereditary transthyretin amyloidosis (hATTR) is an autosomal dominantly inherited disorder caused by an accumulation of amyloid fibrils in tissues due to mutations in the transthyretin (TTR) gene. The prevalence of hATTR is still unclear and likely underestimated in many countries. In order to apply new therapies in a targeted manner, early diagnosis and knowledge of phenotype-genotype correlations are mandatory. This study aimed to assess the prevalence and phenotypic spectrum of hATTR in Austria. METHODS: Within the period of 2014-2019, patients with ATTR-associated cardiomyopathy and/or unexplained progressive polyneuropathies were screened for mutations in the TTR gene. RESULTS: We identified 43 cases from 22 families carrying 10 different TTR missense mutations and confirmed two mutational hot spots at c.323A>G (p.His108Arg) and c.337G>C (p.Val113Leu). Two further patients with late onset ATTR carried TTR variants of unknown significance. The majority of patients initially presented with heart failure symptoms that were subsequently accompanied by progressive polyneuropathy in most cases. A total of 55% had a history of carpal tunnel syndrome before the onset of other organ manifestations. CONCLUSIONS: Our study underlined the relevance of hATTR in the pathogenesis of amyloid-driven cardiomyopathy and axonal polyneuropathy and indicated considerable genetic heterogeneity of this disease in the Austrian population. The estimated prevalence of hATTR in Austria based on this study is 1:200,000 but a potentially higher number of unknown cases must be taken into account. With respect to new therapeutic approaches, we strongly propose genetic testing of the TTR gene in an extended cohort of patients with unexplained heart failure and progressive polyneuropathy.