Whole genome sequencing (WGS) is a powerful tool for postnatal genetic diagnosis, but relevant clinical studies in the field of prenatal diagnosis are limited. The present study aimed to prospectively evaluate the utility of WGS compared with chromosomal microarray (CMA) and whole exome sequencing (WES) in the prenatal diagnosis of fetal structural anomalies. We performed trio WGS (≈40-fold) in parallel with CMA in 111 fetuses with structural or growth anomalies, and sequentially performed WES when CMA was negative (CMA plus WES). In comparison, WGS not only detected all pathogenic genetic variants in 22 diagnosed cases identified by CMA plus WES, yielding a diagnostic rate of 19.8% (22/110), but also provided additional and clinically significant information, including a case of balanced translocations and a case of intrauterine infection, which might not be detectable by CMA or WES. WGS also required less DNA (100 ng) as input and could provide a rapid turnaround time (TAT, 18 ± 6 days) compared with that (31 ± 8 days) of the CMA plus WES. Our results showed that WGS provided more comprehensive and precise genetic information with a rapid TAT and less DNA required than CMA plus WES, which enables it as an alternative prenatal diagnosis test for fetal structural anomalies.

Background: Usher syndrome is a condition characterized by partial or total hearing loss and progressive pigmentary retinopathy. Usher syndrome type 1F is caused by biallelic loss‐of‐function variants in Protocadherin 15 (PCDH15), which encodes the PCDH15 protein that plays an important role in the morphogenesis and cohesion of stereocilium bundles and retinal photoreceptor cell maintenance and function.Methods: We report a child with bilateral nonsyndromic sensorineural hearing loss who received an inconclusive diagnosis based on clinical gene panel testing, which identified a paternal heterozygous nonsense variant (NM_033056.4: c.733C>T, p.R245*) in PCDH15. This variant has been described as a founder variant in the Ashkenazi Jewish population.Results: A novel deep‐intronic variant (NM_033056.4: c.705+3767_705+3768del) inherited from the patient's mother was identified by trio‐based whole‐genome sequencing (WGS). A minigene splicing assay revealed that c.705+3767_705+3768del results in aberrant retention of 50 or 68 bp of intron 7.Conclusion: Our genetic test results provided precise genetic counseling and prenatal diagnosis for this family, and our findings highlight the power of WGS for detecting deep‐intronic variants in patients with undiagnosed rare diseases. Additionally, this case expands the variant spectrum of the PCDH15 gene and our results support the extremely low carrier frequency of c.733C>T in the Chinese population.

Background As an adjunct to diagnostic exome sequencing and whole-genome sequencing, RNA sequencing (RNA-seq) has been demonstrated to improve diagnostic yield for Mendelian diseases. However, systematic evaluation of the associated experimental and computational processes and the establishment of robust and efficient practices for RNA diagnostics implemented in the clinic to analyse readily accessible whole blood samples are still required. Methods We simulated clinical conditions in which each patient’s sample is tested only once, and we evaluated the two typical experimental protocols (polyA-selection and rRNA depletion) by comparing the expression profiles, aberrant splicing events and monoallelic expression (MAE) identified from 11 patients in clinical settings with different bioinformatics software. Results We demonstrated that a higher proportion of unique reads from polyA-selection than rRNA depletion were mapped to exons or exon – intron junction regions (84.54% vs. 40.14%), resulting in more detectable OMIM genes (TPM > 1) in the blood (65.29% vs. 59.79%); thus, the rRNA depletion method requires a median of 258 more valid reads per gene to achieve the same level of gene quantification. Moreover, although the transcriptome profiling of protein-coding genes in the two methods is highly correlated, polyA-selection offers more sensitive detection of MAE variants and aberrant splicing under common filtering conditions in combination with DROP. Conclusions A combination of polyA+ and DROP is recommended when implementing blood-based RNA-seq for the diagnosis of Mendelian diseases in clinical practice, and filtering criteria for aberrant expression, aberrant splicing and MAE variants are suggested for reference.

Purpose: Cardiovascular diseases are the most common cause of death globally. In which atrioventricular block (AVB) is a common disorder with genetic causes, but the responsible genes have not been fully identified yet. To determine the underlying causative genes involved in cardiac AVB, here we report a three-generation Chinese family with severe autosomal dominant cardiac AVB that has been ruled out as being caused by known genes mutations. Methods: Whole-exome sequencing was performed in five affected family members across three generations, and co-segregation analysis was validated on other members of this family. Results: Whole-exome sequencing and subsequent co-segregation validation identified a novel germline heterozygous point missense mutation, c.49287C>A (p.N16429K), in the titin (TTN, NM_001267550.2) gene in all 5 affected family members, but not in the unaffected family members. The point mutation is predicted to be functionally deleterious by in-silico software tools. Our finding was further supported by the conservative analysis across species. Conclusion: Based on this study, TTN was identified as a potential novel candidate gene for autosomal dominant AVB; this study expands the mutational spectrum of TTN gene and is the first to implicate TTN mutations as AVB disease causing in a Chinese pedigree.

Background: A considerable number of patients suffering from neuromuscular disorders (NMD) are unable to receive an accurate diagnosis through initial genetic testing. It is imperative to develop a cost-effective diagnostic strategy that incorporates appropriate multi-omics techniques. Methods: This study included 33 NMD patients with negative results from whole-exome sequencing (WES). Whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) were performed concurrently to evaluate clinical utility. Additionally, eight diagnostic pathways were compared in terms of diagnostic rate, turnaround time, and cost. Results: Our implementation of parallel WGS and RNA-seq testing successfully validated the clinical utility of this strategy in the cohort of 33 NMD patients initially yielding negative results from WES. The combined utilization of both methods resulted in an additional diagnosis for 42% (15/33) of the patients, with WGS contributing to 36% and RNA-seq contributing to 6% of the diagnoses. The Integration of alternative splicing results derived from RNA-seq data into variant filtering significantly reduced the number of rare intronic variants requiring interpretation and provided compelling evidence to support the classification of variant pathogenicity based on functional impact. Our comprehensive analysis, comparing eight different diagnostic pathways, revealed the cost-effectiveness of parallel WGS and RNA-seq testing as a diagnostic approach for patients. Moreover, the analysis of rare genomic findings within our cases showcased their potential to inform patient care, aid treatment decisions, and expand the range of NMD mutations in diagnosing rare NMD cases. Conclusion: The integration of parallel WGS and RNA-seq testing represents a transformative diagnostic approach for NMD patients. The cost-effectiveness of this approach, coupled with its ability to improve diagnostic yield and interpretation efficiency, makes it a highly recommended strategy for clinical implementation to enhance the management and care of NMD patients.