Computational protocol: A case report of de novo missense FOXP1 mutation in a non-Caucasian patient with global developmental delay and severe speech impairment

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Protocol publication

[…] Exome sequencing was performed, following the protocol described in the SureSelect Library prep kit (post-pool version 4; Agilent Technologies, Inc., Santa Clara, CA). The DNA library was subjected to emulsion PCR (SOLiD™ EZ Bead™ Emulsifier kit; Life Technologies, Carlsbad, CA) to generate clonal DNA fragments on beads, followed by bead enrichment (SOLiD™ EZ Bead™ Enrichment kit; Life Technologies). Enriched template beads were sequenced on a SOLiD 5500xl sequencer as single-end, 60-bp reads (Life Technologies). The SOLiD 5500xl output reads were aligned against the human genome reference sequence (hg19) using LifeScope version 2.5.1 (Life Technologies) to generate BAM files. Variant calling was performed following the Best Practices specified in the Genome Analysis Toolkit (GATK, version 2.7.4), Picard (http://picard.sourceforge.net) and SAMtools , and only reads that mapped to a unique position in the reference genome were used.A total of 68,391 variants were detected in the patient. We first filtered out the variants with low-quality values generated by GATK output, resulting in a new total of 62,200 variants. To distinguish potentially pathogenic variants from other variants, we filtered out variants in our in-house references (57 exome samples), public data from dbSNP (http://www.ncbi.nlm.nih.gov/SNP/, version 137), and a 1000-genome database . After this filtering step was applied, 393 variants remained. We then used ANNOVAR software to filter out synonymous variants and intronic variants because they are less likely to be pathogenic , which resulted in 84 remaining variants. We then used SIFT , Polyphen2 , LRT , or MutationTaster software to predict the potential impact of an amino acid substitution on the function of human proteins, and we filtered out “benign” missense mutations as defined by the above-mentioned software. A total of 13 single-nucleotide variants and one frame-shift variant remained after this step (Table).We performed direct sequencing to evaluate these 14 candidate mutations using DNA obtained from the patient and her parents. Among these, two mutations (SPERT and GRP52) were not confirmed by direct sequencing. Ten of the mutations existed in at least one of the healthy parents, suggesting that they are unlikely to be pathogenic. Two of the mutations, FOXP1 and PRKAA1, were unique to the patient. […]

Pipeline specifications

Software tools LifeScope, GATK, Picard, SAMtools, ANNOVAR, SIFT, PolyPhen, MutationTaster
Databases dbSNP
Application WES analysis
Organisms Homo sapiens