Exome Sequencing for Rare Skeletal Disorders

Genomic DNA was extracted from fetal amniocytes and peripheral blood leukocytes of the parents using the salt fractionation method (Magen, Guangdong, China). Exome capture was performed using xGen Exome Research panel v1 (Integrated DNA Technologies, Coralville, IA, USA), and sequencing was conducted on NovaSeq 6000 (Illumina, San Diego, CA). The sequences were aligned to a human reference sequence (NCBI Genome build GRCh37) using the Burrows–Wheeler Aligner. The Genome Analysis Toolkit pipeline was used to detect single‐nucleotide (SNPs) and indel polymorphisms. Prioritization was given to variants that were previously reported, considered LOF (nonsense, frameshift, or splice site mutations), or absent/rare in gnomAD. The candidate gene list was then narrowed down based on abnormal long bone morphology (Human Phenotype Ontology [HP]: 0011314). Variant interpretation was performed in accordance with the ACMG/AMP guidelines and ClinGen specifications (Richards et al., 2015 (link); Zhang et al., 2020 (link)).