Whole Exome Sequencing and Variant Analysis

The whole exome sequencing and data analysis procedures were previously reported (5 (link)–7 (link)). Briefly, we first extracted genomic DNA from the blood samples according to the standard procedure of the manufacturer (MagPure Buffy Coat DNA Midi KF Kit, Magen, China), and the qualified genomic DNA was then sequenced with PE100 + 100 on MGISEQ-2000. We applied the BGI MGIEasy V4 chip, which contains exons of all human genes and their adjacent ± 20 bp introns, to capture the targeted sequences. Bioinformatics processing and data analysis were then performed to explore the potential variants after we received the primary sequencing data. Several databases, including the 1,000 Genomes Project, HapMap, NCBI dbSNP, and a database of 200 normal Chinese adults, were used to filter and estimate all the SNVs and indels. Finally, Sanger sequencing was used to validate all mutations and potential pathogenic variants. The Human Gene Mutation Database (HGMD)¹ was introduced to screen previously reported mutations. To rule out the possibility of a polymorphism, the mutations were also blasted in the 1,000 Genomes Project,² ExAC,³ HapMap,⁴ ESP6500,⁵ NCBI dbSNP,⁶ GnomAD,⁷ and a database of 200 normal Chinese adults.