Sanger Sequencing Validation of Genetic Variants

Sanger sequencing was used to confirm the potential pathogenic variants detected by exome sequencing, including missense, nonsense, indels, and splice site variants. Primers used to amplify the sequence with each variant were either referred to in previous studies [13] (link),[15] (link),[17] (link) or designed by online tool Primer3 (http://frodo.wi.mit.edu/primer3/) (Table S1). The nucleotide sequences of amplicons were analyzed using an ABI BigDye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA) on an ABI 3130 Genetic Analyzer (Applied Biosystems). Sequencing results were aligned with consensus sequences from the National Center for Biotechnology Information (NCBI) human genome database (http://www.ncbi.nlm.nih.gov/), using the SeqManII program of the Lasergene package (DNAStar Inc. Madison, WI). Confirmed variants were further sequenced in the available family members and 96 unrelated control individuals. The descriptions of the variants followed the nomenclature recommended by the Human Genomic Variation Society (HGVS; http://www.hgvs.org/mutnomen/). The effects of missense variations were evaluated by the PolyPhen-2 [18] (link) and SIFT [19] (link) online tools, and the effects of intronic variants on splicing site changes were predicted by the Berkeley Drosophila Genome Project (BDGP; http://www.fruitfly.org/seq_tools/splice.html[20] (link).