Streamlined two-step fragment analysis PCR and exome sequencing of RFC1 for diagnostic testing of suspected CANVAS patients.

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is caused by biallelic pathogenic expansions, or compound heterozygosity with other pathogenic variants in the RFC1 gene. CANVAS is estimated to be underdiagnosed, both because of the lack of formal diagnostic criteria and molecular challenges that translate to lesser access and high cost of routine testing. Our aim was to address the need for making CANVAS genetic testing routine, by designing a streamlined two-step PCR consisting of a short-allele screening PCR and a confirmatory PCR with fragment capillary electrophoresis detection. Exome sequencing of RFC1 was additionally foreseen to resolve potential compound heterozygosity cases. Specificity of our approach was evaluated using ataxia patients with known non-CANVAS diagnoses, and optimized using Southern blot confirmed CANVAS patients. We evaluated our approach by testing patients consecutively referred for clinically suspected CANVAS using first the two-step PCR, followed by exome sequencing. Our approach was able to accurately identify negative and confirm positive cases in prospectively collected suspected CANVAS patients presenting with at least three typical clinical signs. The proposed testing approach provides an alternative method able to clearly distinguish between CANVAS negative and positive cases and can be easily incorporated into the genetic diagnostic laboratory workflow.

Consensus-based expert recommendations on the management of MPS IVa and VI in Saudi Arabia.

BACKGROUND: Mucopolysaccharidosis type IVa (Morquio A syndrome) and mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome) are rare inherited lysosomal storage diseases associated with significant functional impairment and a wide spectrum of debilitating clinical manifestations. These conditions are thought to have higher-than-average prevalence rates in Saudi Arabia due to high rates of consanguineous marriage in the country. There are several unmet needs associated with the management of these diseases in Saudi Arabia. MAIN BODY: The aim of this manuscript is to contextualize unmet management needs and provide recommendations to optimize diagnosis, multidisciplinary care delivery, and local data generation in this disease area. An expert panel was assembled comprising seven consultant geneticists from across Saudi Arabia. The Delphi methodology was used to obtain a consensus on statements relating to several aspects of mucopolysaccharidosis types IVa and VI. A consensus was reached for all statements by means of an online, anonymized voting system. The consensus statements pertain to screening and diagnosis, management approaches, including recommendations pertaining to enzyme replacement therapy, and local data generation. CONCLUSION: The consensus statements presented provide specific recommendations to improve diagnostic and treatment approaches, promote multidisciplinary care and data sharing, and optimize the overall management of these rare inherited diseases in Saudi Arabia.

Circulating biomarkers of kidney angiomyolipoma and cysts in tuberous sclerosis complex patients.

Patients with tuberous sclerosis complex (TSC) develop multi-organ disease manifestations, with kidney angiomyolipomas (AML) and cysts being one of the most common and deadly. Early and regular AML/cyst detection and monitoring are vital to lower TSC patient morbidity and mortality. However, the current standard of care involves imaging-based methods that are not designed for rapid screening, posing challenges for early detection. To identify potential diagnostic screening biomarkers of AML/cysts, we performed global untargeted metabolomics in blood samples from 283 kidney AML/cyst-positive or -negative TSC patients using mass spectrometry. We identified 7 highly sensitive chemical features, including octanoic acid, that predict kidney AML/cysts in TSC patients. Patients with elevated octanoic acid have lower levels of very long-chain fatty acids (VLCFAs), suggesting that dysregulated peroxisome activity leads to overproduction of octanoic acid via VLCFA oxidation. These data highlight AML/cysts blood biomarkers for TSC patients and offers valuable metabolic insights into the disease.

Identification of mitochondria-related gene biomarkers associated with immune infiltration in acute myocardial infarction.

Mitochondrial dysfunction has been known to contribute to the worsening of acute myocardial infarction (AMI). We screened differentially expressed genes (DEGs) between AMI and healthy individuals based on the GSE66360 dataset. We took the intersection of the obtained DEGs with 1,136 mitochondria-related genes. Finally, we screened out mitochondria-related DEGs (MitoDEGs). Eight MitoDEGs were identified as hub genes based on the random forest algorithm. Two mitochondria-related robust molecular clusters were identified by consensus clustering. Immune infiltration analysis showed that immune cell infiltration was significantly increased in the high-expression group of MitoDEGs. We obtained the potential drugs targeted at ALDH2, PMAIP1, and BCL2A1, such as disulfiram, obatoclax mesylate, and bortezomib. Quantitative reverse-transcription polymerase chain reaction further validated the expression of the MitoDEGs in the cell model of AMI. These findings reveal the potential role of MitoDEGs in AMI and provide new insights into risk stratification and individualized treatment of AMI patients.

Re-analysis of whole genome sequencing ends a diagnostic odyssey: Case report of an RNU4-2 related neurodevelopmental disorder.

Despite increasing knowledge of disease-causing genes in human genetics, approximately half of the individuals affected by neurodevelopmental disorders remain genetically undiagnosed. Part of this missing heritability might be caused by genetic variants outside of protein-coding genes, which are not routinely diagnostically investigated. A recent preprint identified de novo variants in the non-coding spliceosomal snRNA gene RNU4-2 as a cause of a frequent novel syndromic neurodevelopmental disorder. Here we mined 164 whole genome sequencing (WGS) trios from individuals with neurodevelopmental or multiple congenital anomaly disorders that received diagnostic genomic investigations at our clinic. We identify a recurrent de novo RNU4-2 variant (NR_003137.2(RNU4-2):n.64_65insT) in a 5-year-old girl with severe global developmental delay, hypotonia, microcephaly, and seizures that likely explains her phenotype, given that extensive previous genetic investigations failed to identify an alternative cause. We present detailed phenotyping of the individual obtained during a 5-year follow-up. This includes photographs showing recognizable facial features for this novel disorder, which might allow prioritizing other currently unexplained affected individuals sharing similar facial features for targeted investigations of RNU4-2. This case illustrates the power of re-analysis to solve previously unexplained cases even when a diagnostic genome remains negative.

Satisfaction with mode of delivery of genomic sequencing results in a diverse national sample of research participants through the Clinical Sequencing Evidence-Generating Research Consortium.

PURPOSE: Research that includes diverse patient populations is necessary to optimize implementation of telehealth. METHODS: As part of a Clinical Sequencing Evidence-Generating Research Consortium cross-site study, we assessed satisfaction with mode of return of results (RoR) delivery across a diverse sample of participants receiving genetic testing results in person vs telemedicine (TM). RESULTS: Ninety-eight percent of participants were satisfied with their mode of results delivery. Participants receiving results by TM were more likely to report a preference for receiving results in a different way and challenges with providers noticing difficulties with understanding. More than 90% reported satisfaction across all items measuring support and interaction during sessions. Participants self-reporting Hispanic/Latino or Black/African American race and ethnicity compared with White/European American, fewer years of education, and having lower health literacy were more likely to report challenges with understanding the information or asking questions. Participants who were White/European American, had more years of education, and higher health literacy reported higher communication scores, reflecting more positive evaluations of the communication experience. CONCLUSION: TM is an acceptable mode of return of results delivery across diverse settings and populations. Research optimizing approaches for underrepresented populations, populations with lower levels of education and health literacy, and multilingual populations is necessary.

Delivering Telegenetics Services: Review and Synthesis of Best Practices.

Introduction: Before the COVID-19 public health emergency, few genetics providers used telehealth. As a response to this, many genetics providers began conducting telehealth care, referred to as telegenetics, usually with guidance from their institutions but without specific guidance related to the uniqueness of genetic services. Objectives: The Telegenetics Workgroup of the National Coordinating Center for Regional Genetics Networks convened a panel of experts in the fields of telemedicine, genetics, and genomics to review the existing literature on telegenetics and synthesize best operating practices for medical geneticists, genetic counselors, and metabolic dietitians providing telegenetics services. Methods: The group searched PubMed using the terms "telegenetics," "telemedicine + genetics," and "telehealth + genetics." The group also reviewed the Northeast Telehealth Resource Center's telegenetics webliography. Websites were searched, including the American Telemedicine Association's website, Center for Connected Health Policy, and National Telehealth Resource Center for position statements, standards documents, and guidelines. The group met frequently by videoconference and discussed the literature, and using expert consensus, the group determined best practices in providing telegenetics services. Results: These telegenetics best practices cover important aspects of telegenetics services, including, but not limited to, ongoing delivery of telegenetics services, use of special technology, legal and regulatory requirements, and considerations regarding special settings and circumstances in which telegenetics may be conducted. Conclusions: Recognizing the growing use of telegenetics and a future in which telegenetics continues to be part of the regular practice of genetics, this guide informs genetics providers of best practices for delivering telegenetics services to patients.

Limitations of the Commercially Available Gene Expression Test in Predicting Cutaneous Squamous Cell Carcinoma Metastasis and Clinical Outcomes.

BACKGROUND: Clinical genetic tests are integral to healthcare decision-making. However, the unclear regulatory framework, especially regarding products that evade stringent FDA oversight, may compromise test validity and transparency. OBJECTIVE: To critically evaluate the DecisionDx® cutaneous squamous cell carcinoma test by Castle Biosciences for its dataset biases, gene panel selection, and reported accuracy metrics, providing insight into broader challenges in the clinical genetic testing landscape. METHODS: Independent analyses of the DecisionDx®-SCC 40-GEP test data from Castle Biosciences were conducted. These included comparisons to clinical genetic testing standards, analysis of prevalence metrics against national cSCC rates, gene ontology of 34 genes for cSCC associations, and evaluation of accuracy metrics. RESULTS: The DecisionDx®-SCC met 11 of 44 CDC's ACCE criteria for clinical genetic testing. Its dataset showed a metastasis prevalence higher than the national average. Out of 34 genes, 15 had known associations with cSCC. Inconsistencies in accuracy metrics presentation were noted, particularly in moderate and high-risk stratifications. CONCLUSION: Analysis of DecisionDx®-SCC indicates potential biases and ambiguities, exacerbated by differences between FDA and CLIA standards. This highlights the need for systematic validation and a unified regulatory approach, stressing the necessity for precise and dependable genetic testing in patient care.

Improving the care of children with GENetic Rare disease: Observational Cohort study (GenROC)-a study protocol.

INTRODUCTION: Around 2000 children are born in the UK per year with a neurodevelopmental genetic syndrome with significantly increased morbidity and mortality. Often little is known about expected growth and phenotypes in these children. Parents have responded by setting up social media groups to generate data themselves. Given the significant clinical evidence gaps, this research will attempt to identify growth patterns, developmental profiles and phenotypes, providing data on long-term medical and educational outcomes. This will guide clinicians when to investigate, monitor or treat symptoms and when to search for additional or alternative diagnoses. METHODS AND ANALYSIS: This is an observational, multicentre cohort study recruiting between March 2023 and February 2026. Children aged 6 months up to 16 years with a pathogenic or likely pathogenic variant in a specified gene will be eligible. Children will be identified through the National Health Service and via self-recruitment. Parents or carers will complete a questionnaire at baseline and again 1 year after recruitment. The named clinician (in most cases a clinical geneticist) will complete a clinical proforma which will provide data from their most recent clinical assessment. Qualitative interviews will be undertaken with a subset of parents partway through the study. Growth and developmental milestone curves will be generated through the DECIPHER website (https://deciphergenomics.org) where 5 or more children have the same genetic syndrome (at least 10 groups expected). ETHICS AND DISSEMINATION: The results will be presented at national and international conferences concerning the care of children with genetic syndromes. Results will also be submitted for peer review and publication.

Circulating DNA genome-wide fragmentation in early detection and disease monitoring of hepatocellular carcinoma.

Genome-wide circulating cell-free DNA (ccfDNA) fragmentation for cancer detection has been rarely evaluated using blood samples collected before cancer diagnosis. To evaluate ccfDNA fragmentation for detecting early hepatocellular carcinoma (HCC), we first modeled and tested using hospitalized HCC patients and then evaluated in a population-based study. A total of 427 samples were analyzed, including 270 samples collected prior to HCC diagnosis from a population-based study. Our model distinguished hospital HCC patients from controls excellently (area under curve 0.999). A high ccfDNA fragmentation score was highly associated with an advanced tumor stage and a shorter survival. In evaluation, the model showed increasing sensitivities in detecting HCC using 'pre-samples' collected ≥4 years (8.3%), 3-4 years (20.0%), 2-3 years (31.0%), 1-2 years (35.0%), and 0-1 year (36.4%) before diagnosis. These findings suggested ccfDNA fragmentation is sensitive in clinical HCC detection and might be helpful in screening early HCC.

Identification of eight genes associated with recurrent patellar dislocation.

The inheritance of recurrent patellar dislocation (RPD) is known, but the susceptible gene remains unidentified. Here, we performed the first whole exome sequencing (WES) cohort study to identify the susceptible genes. The results showed eight genes were associated with this disease. Notably, the carboxypeptidase D (CPD) gene showed the highest relevance based on its gene function and tissue expression. Single-cell sequencing results indicate that the CPD gene is involved in the pathophysiological process of RPD through granulocytes. Implicated pathways include nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and Wnt/ß-catenin signaling, potentially influencing CPD's role in RPD pathogenesis. This study identified the susceptible gene and investigates the potential pathogenesis of RPD, which provided a new prospect for the understanding of RPD. Besides, it would offer the theoretical basis for disease prevention and genetic counseling.

Translation, cross-cultural adaptation, and preliminary validation of a patient-reported outcome measure for genetic counseling outcomes in Sweden.

Genetic counseling is key for understanding the consequences of hereditary and genetic diseases and, therefore, crucial for patients, their families, and healthcare providers. Genetic counseling facilitates individuals' comprehension, decision-making, and adaptation to hereditary diseases. This study focuses on the Swedish adaptation of the Genetic Counseling Outcome Scale-24 (GCOS-24), an internationally validated, patient-reported outcome measure (PROM) for quantifying patient empowerment in genetic counseling. This study aimed to translate and cross-culturally adapt the GCOS-24 to measure patient-reported outcome from genetic counseling in Sweden. The adaptation process was meticulously conducted, adhering to international guidelines, with cross-cultural adaptation, translation, and back translation, to ensure semantic, conceptual, and idiomatic equivalence with the original English version. Face validity and understandability was assured using qualitative cognitive interviews conducted with patient representatives, and by a committee of experts in the field. The psychometric properties of the Swedish version of GCOS-24 (GCOS-24swe) were evaluated using a robust sample of 374 patients. These individuals received genetic counseling by telephone or video, necessitated by the constraints of the COVID-19 pandemic. Participants responded to GCOS-24swe both before and after genetic counseling. The GCOS-24swe demonstrated face validity, good internal consistency (Cronbach's alpha = 0.86), significant responsiveness (Cohen's d = 0.65, p < 0.001), and good construct validity. The study's findings underscore the GCOS-24swe's potential as an effective instrument in both clinical practice and research within Sweden. It offers a valuable means for assessing patient empowerment, a key goal of genetic counseling. Additional psychometric assessment of test-retest reliability and interpretability would further enhance the utility of GCOS-24swe.

N6-methyladenosine-associated genetic variants in NECTIN2 and HPCAL1 are risk factors for abdominal aortic aneurysm.

Although N6-methyladenosine (m6A) modification has been implicated in the pathogenesis of abdominal aortic aneurysm (AAA), the relationship between m6A-associated single nucleotide polymorphisms (m6A-SNPs) and AAA remains unknown. This study used integrative multi-omics analysis and clinical validation approaches to systematically identify potential m6A-SNPs connected with AAA risk. We found that rs6859 and rs10198139 could modulate the expression of local genes, NECTIN2 and HPCAL1, respectively, which exhibited upregulation in AAA tissues, and their risk variants were significantly correlated with an increased susceptibility to AAA. Incorporating rs6859 and rs10198139 improved the efficiency of AAA risk prediction compared to the model considering only conventional risk factors. Additionally, these two SNPs were predicted to be located within the regulatory sequences, and rs6859 showed a substantial impact on m6A modification levels. Our findings suggest that m6A-SNPs rs6859 and rs10198139 confer an elevated risk of AAA, possibly by promoting local gene expression through an m6A-mediated manner.

Taking the risk. A systematic review of ethical reasons and moral arguments in the clinical use of polygenic risk scores.

Debates about the prospective clinical use of polygenic risk scores (PRS) have grown considerably in the last years. The potential benefits of PRS to improve patient care at individual and population levels have been extensively underlined. Nonetheless, the use of PRS in clinical contexts presents a number of unresolved ethical challenges and consequent normative gaps that hinder their optimal implementation. Here, we conducted a systematic review of reasons of the normative literature discussing ethical issues and moral arguments related to the use of PRS for the prevention and treatment of common complex diseases. In total, we have included and analyzed 34 records, spanning from 2013 to 2023. The findings have been organized in three major themes: in the first theme, we consider the potential harms of PRS to individuals and their kin. In the theme "Threats to health equity," we consider ethical concerns of social relevance, with a focus on justice issues. Finally, the theme "Towards best practices" collects a series of research priorities and provisional recommendations to be considered for an optimal clinical translation of PRS. We conclude that the use of PRS in clinical care reinvigorates old debates in matters of health justice; however, open questions, regarding best practices in clinical counseling, suggest that the ethical considerations applicable in monogenic settings will not be sufficient to face PRS emerging challenges.

Subcellular localisation of truncated MAGEL2 proteins: insight into the molecular pathology of Schaaf-Yang syndrome.

Schaaf-Yang syndrome (SYS) is an ultra-rare neurodevelopmental disorder caused by truncating mutations in MAGEL2 Heterologous expression of wild-type (WT) or a truncated (p.Gln638*) C-terminal HA-tagged MAGEL2 revealed a shift from a primarily cytoplasmic to a more nuclear localisation for the truncated protein variant. We now extend this analysis to six additional SYS mutations on a N-terminal FLAG-tagged MAGEL2. Our results replicate and extend our previous findings, showing that all the truncated MAGEL2 proteins consistently display a predominant nuclear localisation, irrespective of the C-terminal or N-terminal position and the chemistry of the tag. The variants associated with arthrogryposis multiplex congenita display a more pronounced nuclear retention phenotype, suggesting a correlation between clinical severity and the degree of nuclear mislocalisation. These results point to a neomorphic effect of truncated MAGEL2, which might contribute to the pathogenesis of SYS.

Assessing the impact of psychiatric genetic counseling on psychiatric hospitalizations.

Psychiatric genetic counseling (pGC) can improve patient empowerment and self-efficacy. We explored the relationship between pGC and psychiatric hospitalizations, for which no prior data exist. Using Population Data BC (a provincial dataset), we tested two hypotheses: (1) among patients (>18 years) with psychiatric conditions who received pGC between May 2010 and Dec 2016 (N = 387), compared with the year pre-pGC, in the year post-pGC there would be fewer (a) individuals hospitalized and (b) total hospital admissions; and (2) using a matched cohort design, compared with controls (N = 363, matched 1:4 for sex, diagnosis, time since diagnosis, region, and age, and assigned a pseudo pGC index date), the pGC cohort (N = 91) would have (a) more individuals whose number of hospitalizations decreased and (b) fewer hospitalizations post-pGC/pseudo-index. We also explored total days in hospital. Within the pGC cohort, there were fewer hospitalizations post-pGC than pre- pGC (p = 0.011, OR = 1.69), and total days in hospital decreased (1085 to 669). However, when compared to matched controls, the post-pGC/pseudo index change in hospitalizations among pGC cases was not statistically significant, even after controlling for the higher number of hospitalizations prior. pGC may lead to fewer psychiatric hospitalizations and cost savings; further studies exploring this are warranted.

Implementation of rapid genomic sequencing in safety-net neonatal intensive care units: protocol for the VIrtual GenOme CenteR (VIGOR) proof-of-concept study.

INTRODUCTION: Rapid genomic sequencing (rGS) in critically ill infants with suspected genetic disorders has high diagnostic and clinical utility. However, rGS has primarily been available at large referral centres with the resources and expertise to offer state-of-the-art genomic care. Critically ill infants from racial and ethnic minority and/or low-income populations disproportionately receive care in safety-net and/or community settings lacking access to state-of-the-art genomic care, contributing to unacceptable health equity gaps. VIrtual GenOme CenteR is a 'proof-of-concept' implementation science study of an innovative delivery model for genomic care in safety-net neonatal intensive care units (NICUs). METHODS AND ANALYSIS: We developed a virtual genome centre at a referral centre to remotely support safety-net NICU sites predominantly serving racial and ethnic minority and/or low-income populations and have limited to no access to rGS. Neonatal providers at each site receive basic education about genomic medicine from the study team and identify eligible infants. The study team enrols eligible infants (goal n of 250) and their parents and follows families for 12 months. Enrolled infants receive rGS, the study team creates clinical interpretive reports to guide neonatal providers on interpreting results, and neonatal providers return results to families. Data is collected via (1) medical record abstraction, (2) surveys, interviews and focus groups with neonatal providers and (3) surveys and interviews with families. We aim to examine comprehensive implementation outcomes based on the Proctor Implementation Framework using a mixed methods approach. ETHICS AND DISSEMINATION: This study is approved by the institutional review board of Boston Children's Hospital (IRB-P00040496) and participating sites. Participating families are required to provide electronic written informed consent and neonatal provider consent is implied through the completion of surveys. The results will be disseminated via peer-reviewed publications and data will be made accessible per National Institutes of Health (NIH) policies. TRIAL REGISTRATION NUMBER: NCT05205356/clinicaltrials.gov.

Genetic variant classification by predicted protein structure: A case study on IRF6.

Next-generation genome sequencing has revolutionized genetic testing, identifying numerous rare disease-associated gene variants. However, to impute pathogenicity, computational approaches remain inadequate and functional testing of gene variant is required to provide the highest level of evidence. The emergence of AlphaFold2 has transformed the field of protein structure determination, and here we outline a strategy that leverages predicted protein structure to enhance genetic variant classification. We used the gene IRF6 as a case study due to its clinical relevance, its critical role in cleft lip/palate malformation, and the availability of experimental data on the pathogenicity of IRF6 gene variants through phenotype rescue experiments in irf6-/- zebrafish. We compared results from over 30 pathogenicity prediction tools on 37 IRF6 missense variants. IRF6 lacks an experimentally derived structure, so we used predicted structures to explore associations between mutational clustering and pathogenicity. We found that among these variants, 19 of 37 were unanimously predicted as deleterious by computational tools. Comparing in silico predictions with experimental findings, 12 variants predicted as pathogenic were experimentally determined as benign. Even with the recently published AlphaMissense model, 15/18 (83%) of the predicted pathogenic variants were experimentally determined as benign. In comparison, mapping variants to the protein revealed deleterious mutation clusters around the protein binding domain, whereas N-terminal variants tend to be benign, suggesting the importance of structural information in determining pathogenicity of mutations in this gene. In conclusion, incorporating gene-specific structural features of known pathogenic/benign mutations may provide meaningful insights into pathogenicity predictions in a gene-specific manner and facilitate the interpretation of variant pathogenicity.

CRISPR activation to characterize splice-altering variants in easily accessible cells.

Genetic variants that affect mRNA splicing are a major cause of hereditary disorders, but the spliceogenicity of variants is challenging to predict. RNA diagnostics of clinically accessible tissues enable rapid functional characterization of splice-altering variants within their natural genetic context. However, this analysis cannot be offered to all individuals as one in five human disease genes are not expressed in easily accessible cell types. To overcome this problem, we have used CRISPR activation (CRISPRa) based on a dCas9-VPR mRNA-based delivery platform to induce expression of the gene of interest in skin fibroblasts from individuals with suspected monogenic disorders. Using this ex vivo splicing assay, we characterized the splicing patterns associated with germline variants in the myelin protein zero gene (MPZ), which is exclusively expressed in Schwann cells of the peripheral nerves, and the spastin gene (SPAST), which is predominantly expressed in the central nervous system. After overnight incubation, CRISPRa strongly upregulated MPZ and SPAST transcription in skin fibroblasts, which enabled splice variant profiling using reverse transcription polymerase chain reaction, next-generation sequencing, and long-read sequencing. Our investigations show proof of principle of a promising genetic diagnostic tool that involves CRISPRa to activate gene expression in easily accessible cells to study the functional impact of genetic variants. The procedure is easy to perform in a diagnostic laboratory with equipment and reagents all readily available.