Characterization Of Molecular Defects For Genetic Skeletal Disorders By Whole Exome Sequencing

Objective Genetic skeletal disorders are consisted of highly genetic and clinicalheterogeneous disorders involving skeletal system development and function. In thecurrent study we apply whole exome sequencing (WES) to uncover the underlyingmolecular defects for multiple genetic skeletal disorders and assess the clinical utilityof WES in molecular diagnostics of genetic skeletal disorders.Methods Generally, a proband, an affected family member and a normal control fromeach of three pedigrees affected by syndactyly, congenital talipes equinovarus, anddistal arthrogryposis syndrome, respectively, were selected as study subjects.Genomic DNA were extracted from peripheral blood samples, using a GentraPuregene Kit (Qiagen, Germany). The genomic DNA library was prepared using theTruSeq DNA Sample Preparation Kit (Illumina, USA) in accordance with themanufacturer’s instructions. In solution exome enrichment was done using the TruSeqExome Enrichment kit (Illumina, USA), according to the manufacturer‘s instructions.The enriched DNA samples were sequenced via2×100paired-end sequencing using aHiseq2000Sequencing System (Illumina, USA). Illumina Sequencing Control v2.8,Illumina Off-Line Basecaller v1.8, and Illumina Consensus Assessment of SequenceAnd Variation v1.8software were used to produce100base pair (bp) sequence reads.Sequence reads were aligned to the human reference genome (hg19) usingBurrows-Wheeler Aligner with default parameters. Variants were identified usingGenome Analysis Toolkit (GATK) and VarScan software. Coverage analysis wasdetermined using the Picard software CalculateHsMetrics tool. Reads that matchedexonic regions, including exon-intron boundaries were analyzed. Single nucleotidepolymorphisms (SNPs) and insertion/deletion (indels) analysis was done usingdifferent filtering steps. Annovar was used to annotate the resulting list of variants.The variant detection frequency was set at a minimum of20%of the reads covering any aberration. A minimum coverage of10reads was set as the threshold for anyvariant to be considered a real mutation. In each case, all variants listed in the mostrecent version of the NCBI (National Center for Biotechnology Information) dbSNPdatabase（dbSNP137）were excluded, as well as silent mutations. Unreportednon-synonymous amino acid variants were analyzed by MutationTaster(http://www.mutationtaster.org), Polyphen-2(http://genetics.bwh.harvard. edu/pph2)and SIFT (http://sift.jcvi.org) to assess any potentially damaging effects. Variants withlow SIFT score (<0.05) and high PolyPhen-2Score and MutationTaster score (＞0.85)were further subjected to advanced filtering procedures. Combining OMIMannotation knowledge, GO analysis, and pathway analysis, most likely causal variantswere prioritized and were subsequently validated by Sanger sequencing.Results In total, we performed whole exome sequencing in eight subjects from thethree pedigrees, generating on average80.3075million reads with86.85%of themhaving a phred quality score of30. Whole exome sequencing also revealed that anaverage66.99%of target sequences with a phred quality score of30was obtainedwith an average target coverage of62.34%. Among86,899.5±14,325.5singlenucleotide variants (SNVs) detected in each subject,10,505±1,034.3SNVs occurredin exonic region that were predicted to have unfavorable functional impacts. Onaverage, each subject had1,251±108low frequency SNVs (＜0.01for dominant and＜0.05for recessive). Of three pedigrees with different genetic skeletal disorders, wesuccessfully identified the causal variants for each condition. A nonsynonymoussubstitution c.302G>T (p.R101L) in GJA1, c.4717G>T (p.D1573Y) in FLNB, andc.533T>G (p.F178C) in TNNI2were identified in syndactyly, congenital talipesequinovarus, and distal arthrogryposis syndrome, respectively. All of three mutationswere validated by Sanger sequencing, and predicted to severely damage the normalfunctions of individual protein with multiple bioinformatic tools. What’s more, eachof the three mutations was found to cosegregate with the conditions in each pedigree.GJA1mutated in syndactyly type3(SD3; MIM#186100) as well as oculodentodigital dysplasia (ODDD; MIM#164200). ODDD is characterized by craniofacial, neurologic,limb and ocular abnormalities, while SD3is a rare congenital distal limbmalformation characterized by complete and bilateral syndactyly between the4th and5th fingers with feet unaffected. It’s suggested that both syndromes are part of thesame spectrum. However, the patient in our study was characterized with bilateralsyndactyly both in hand and feet and absence of craniofacial, neurologic, and ocularabnormalities. Mutations in FLNB were believed to be the molecular defect for aspectrum of conditions including Larsen syndrome (LS; MIM#150250),Atelosteogenesis I and III (AOI; MIM#108720and AOIII; MIM#108721), andBoomerang Dysplasia (BD; MIM#112310). In addition, it’s observed that there’s aassociation between mutation site and phenotype severity. The novel c.4717G>T(p.D1573Y) occurred in repeat14of Rod domain1, a domain involved inactin-binding of FLNB. Compared to the clinical presentations observed inLS-AO-BD spectrum of conditions, our patient and her affected family membersshown only very mild phenotypes, indicating the high expression heterogeneity ofFLNB mutation in human diseases. Distal arthrogryposes (DA) are a group ofautosomal dominant disorders that mainly involve the distal parts of the limbs. Elevensubtypes of distal arthrogryposes have been described to date. DA is alsocharacterized with phenotype overlap between different types of conditions and highgenetic heterogeneity. It’s reported that DA type2B (DA2B; MIM#601680) is causedby mutations in TNNI2. The novel mutation of c.533T>G (p.F178C) detected in thisstudy is believed to contribute to the patient’s clinical features including multiplecongenital contracture, club foot, and small mandible.Conclusion It’s demonstrated that WES, in combination with appropriate sampleselection, is effective in delineation of causal variants for genetic skeletal disorders.With this approach, we successfully identified the causal variants in three familieswith different genetic skeletal disorders.