Sen Zhao

Objective: While RNAseq has enhanced variant interpretation in postnatal cases, its potential in the prenatal setting remains underexplored. This study investigates the utility of RNAseq in prenatal diagnostics by analyzing the expression profiles of cultured chorionic villus samples (cCVS) and amniotic fluid (cAF) samples. Methods: We performed RNAseq on 25 prenatal samples (10 cCVS and 15 cAF) and compared their expression profiles with those of postnatal tissues—blood and skin fibroblasts. Results: To evaluate the clinical relevance of gene expression in these samples, we curated a list of genes associated with fetal‐onset genetic disorders ( n = 375). Using this curated list as a reference, our analysis revealed that cAF samples have the highest proportion of highly expressed genes (60%), surpassing cCVS (54%), fibroblasts (54%), and blood (34%). Differential expression analysis identified unique gene expression patterns in cCVS and cAF samples, reflecting their distinct tissue origins. Specifically, cAF samples showed elevated expression of genes involved in fetal kidney development, whereas cCVS samples were enriched for genes related to trophoblast function. Conclusions: These findings demonstrate that prenatal RNAseq reliably detects clinically relevant gene expressions, offering insights into prenatal conditions and fetal organ development. This underscores the potential of prenatal RNAseq as a valuable tool for supporting genetic variant interpretation.

Congenital scoliosis (CS) is a type of vertebral malformation for which the etiology remains elusive. The notochord is pivotal for vertebrae development, but its role in CS is still understudied. Here, we generated a zebrafish knockout of ptk7a, a planar cell polarity (PCP) gene that is essential for convergence and extension (C&E) of the notochord, and detected congenital scoliosis-like vertebral malformations (CVMs). Maternal zygotic ptk7a mutants displayed severe C&E defects of the notochord. Excessive apoptosis occurred in the malformed notochord, causing a significantly reduced number of vacuolated cells, and compromising the mechanical properties of the notochord. The latter manifested as a less-stiff extracellular matrix along with a significant reduction in the number of the caveolae and severely loosened intercellular junctions in the vacuolated region. These defects led to focal kinks, abnormal mineralization, and CVMs exclusively at the anterior spine. Loss of function of another PCP gene, vangl2, also revealed excessive apoptosis in the notochord associated with CVMs. This study suggests a new model for CS pathogenesis that is associated with defects in notochord C&E and highlights an essential role of PCP signaling in vertebrae development.

Summary: Despite rapid advancements in clinical sequencing, over half of diagnostic evaluations still lack definitive results. RNA-seq has shown promise in research settings for bridging this gap by providing essential functional data for accurate interpretation of diagnostic sequencing results. However, despite advanced research pipelines, clinical translation of diagnostic RNA-seq has not yet been realized. We have developed and validated a clinical diagnostic RNA-seq test in a CLIA laboratory for individuals with suspected genetic disorders who have existing or concurrent comprehensive DNA diagnostic testing. This diagnostic RNA-seq test processes patient RNA samples from fibroblasts or blood and derives clinical interpretations based on the analytical detection of outliers in gene expressions and splicing patterns. The clinical validation involves 150 samples, including benchmark, negative, and positive samples. We developed provisional expression and splicing benchmarks using short-read and long-read RNA-seq data from the HG002 lymphoblastoid sample produced by the Genome in a Bottle Consortium. Our validation data achieved analytical sensitivity and specificity higher than 99% against the benchmarks. For clinical validation, we first established reference ranges for each gene and junction based on expression distributions from our control data. We then evaluated the clinical performance of our outlier-based pipeline using positive samples with previously identified diagnostic findings from the Undiagnosed Diseases Network project. Our pipeline identified 19 of 20 positive findings in both fibroblast and blood samples and highlighted the limitations of the test. Our study provides a paradigm and necessary resources for independent laboratories to validate a clinical RNA-seq test.

Congenital vertebral malformation, affecting 0.13–0.50 per 1000 live births, has an immense locus heterogeneity and complex genetic architecture. In this study, we analyze exome/genome sequencing data from 873 probands with congenital vertebral malformation and 3794 control individuals. Clinical interpretation identifies Mendelian etiologies in 12.0% of the probands and reveals a muscle-related disease mechanism. Gene-based burden test of ultra-rare variants identifies risk genes with large effect sizes (ITPR2, TBX6, TPO, H6PD, and SEC24B). To further investigate the biological relevance of the genetic association signals, we perform single-nucleus RNAseq on human embryonic spines. The burden test signals are enriched in the notochord at early developmental stages and myoblast/myocytes at late stages, highlighting their critical roles in the developing spine. Our work provides insights into the developmental biology of the human spine and the pathogenesis of spine malformation.

Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity, affecting millions of adolescents worldwide, but it lacks a defined theory of etiopathogenesis. Because of this, treatment of AIS is limited to bracing and/or invasive surgery after onset. Preonset diagnosis or preventive treatment remains unavailable. Here, we performed a genetic analysis of a large multicenter AIS cohort and identified disease-causing and predisposing variants of SLC6A9 in multigeneration families, trios, and sporadic patients. Variants of SLC6A9, which encodes glycine transporter 1 (GLYT1), reduced glycine-uptake activity in cells, leading to increased extracellular glycine levels and aberrant glycinergic neurotransmission. Slc6a9 mutant zebrafish exhibited discoordination of spinal neural activities and pronounced lateral spinal curvature, a phenotype resembling human patients. The penetrance and severity of curvature were sensitive to the dosage of functional glyt1. Administration of a glycine receptor antagonist or a clinically used glycine neutralizer (sodium benzoate) partially rescued the phenotype. Our results indicate a neuropathic origin for “idiopathic” scoliosis, involving the dysfunction of synaptic neurotransmission and central pattern generators (CPGs), potentially a common cause of AIS. Our work further suggests avenues for early diagnosis and intervention of AIS in preadolescents.

Clinical exome sequencing (CES) has shown great utility in the diagnosis of Mendelian disorders. CES can unravel secondary findings (SFs) unrelated to the primary diagnosis but with potential health implications. The American College of Medical Genetics and Genomics (ACMG) has published a guideline for reporting secondary findings and recently updated an ACMG SF v3.0 list comprising 73 genes. Several studies have been performed to explore the prevalence of SFs. However, the data were limited in the Chinese population. In this study, we evaluated the genetic data of 2987 individuals from the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO) study group in accordance with the ACMG SF v3.0 list. The detected variants were evaluated using the ACMG classification guidelines, HGMD, and ClinVar database. Totally, 157 (157/2987, 5.3%) individuals had reportable variants within genes associated with cancer, cardiovascular, metabolic, and miscellaneous phenotypes. We identified 63 known pathogenic (KP) variants in 72 individuals (72/2987, 2.4%) and 96 expected pathogenic (EP) variants in 105 individuals (3.5%). Forty-five individuals carried SFs in v3.0 newly added genes, which accounted for 1.5% of our cohort. Our findings could contribute to existing knowledge of secondary findings in different ethnicities and indicate the necessity for clinicians to update the SFs gene list.

Background & Aims: Genetic factors underlie a substantial proportion of paediatric liver diseases. Hereditary liver diseases have considerable genetic heterogeneity and variable clinical manifestations, which bring great challenges to clinical and molecular diagnoses. In this study, we investigated a group of paediatric patients with varying degrees of liver dysfunction using a hierarchical genetic testing strategy.Methods: We first applied a panel encompassing 166 known causal genes of liver disease. We then used exome sequencing (ES) in those patients whose cases remained undiagnosed to identify the genetic aetiology of their symptoms.Results: In total, we enrolled 131 unrelated paediatric patients with liver disease of Chinese Han ethnicity. We first applied targeted gene sequencing of 166 genes to all patients and yielded a diagnostic rate of 35.9% (47 of 131). Eighty‐four patients who remained undiagnosed after target gene sequencing were subjected to ES. As a result, eight (8/84, 9.5%) of them obtained molecular diagnoses, including four patients suspected of abnormal bilirubin metabolism and four idiopathic cases. Non‐typical genetic findings, including digenic inheritance and dual molecular diagnosis, were also identified. Through a comprehensive assessment of novel candidate variants of uncertain disease association, 11 patients of the remaining undiagnosed patients were able to obtain likely molecular diagnoses.Conclusions: Our study presents evidence for the diagnostic utility of sequential genetic testing in a cohort of patients with paediatric liver disease. Our findings expand the understanding of the phenotypic and mutational spectrum underlying this heterogeneous group of diseases.

Background: Brain arteriovenous malformations (BAVMs) are abnormal vessels that are apt to rupture, causing life-threatening intracranial hemorrhage (ICH). The estimated prevalence of BAVMs is 0.05% among otherwise healthy individuals. In this study, we aim to investigate the mutational spectrum of syndromic genes in sporadic BAVM. Methods: We recruited a cohort of 150 patients with BAVM and performed whole-exome sequencing on their peripheral blood DNA. To explore the mutational spectrum of syndromic genes in sporadic brain arteriovenous malformation, we selected six genes according to the Online Mendelian Inheritance in Man (OMIM) and literature. All variants in the six candidate genes were extracted and underwent filtering for qualifying variants. Results: There are a total of four patients with rare variants in hereditary hemorrhagic telangiectasia-related genes. In addition, we identified two patients have the variant of RASA1 gene in our database, which are also rare mutations that are absent from population databases. However, we did not find any patients with GNAQ mutations in our database. Conclusions: In conclusion, we demonstrated that variants in syndromic vascular malformations play important roles in the etiology of sporadic BAVM.