3. Methods
3.1 Sample collection
Blood samples from the couples and the gastrocnemius muscle from the fetus corpse were collected, and genomic DNA was extracted from these tissue using the QIAamp DNA Blood Mini kit (Qiagen, 51106) following the manufacturer’s protocol. Informed consent was obtained from the couples.
3.2 Next-generation sequence
The genomic DNA (5μg) was randomly fragmented and purified by magnetic beads.DNA fragments were ligated with adaptor and captured by oligo probes from the IDT XGen Exome Research Panel (IDT, Lowa,USA) with targeting 19,396 genes approximately. The captured DNA libraries were subjected to NovaSeq6000 sequencer for sequencing according to the manufacturer’s instructions (Illumina, San Diego, USA). All reads were mappedagainstthe hg19 from UCSC database by Burrows-Wheeler Aligner (BWA, v0.5.9-r16) [Li, & Durbin, 2010]. Follow by data annotation, variants were called by PriVar toolkit [Lu et al.,2013], then the clinical significance of the variants were annotated[Yang et al.,2013]. The candidate variants were verified using PCR amplification, and the products were sequenced using 3500XL Genetic Analyzer (Applied Biosystems, Foster City, USA) according to the manufacturer’s instructions.
3.3PCR and Sanger sequencing
3.3 PCR and Sanger sequencing
The specific amplification primers of c.790C>T (Foward primer: TCCTCATTAACAAGTCATCGG, Reverse primer: GTGAACCGAGATCATGCC) and c.296G>T (Foward primer: TCCTCATTAACAAGTCATCGG, Reverse primer: GTGAACCGAGATCATGCC) were designed using Primer Premier v5.0.
The target segments were amplified applying 2X PCR MasterMix kits (Tiangen Biotech) on ABI9700 PCR instrument (Life technology, USA), and then sequenced on ABI3500 Genetic Analyzer (Life technology, USA).
3.4 Mutation prediction.
Potential disease-causing was predicted using mutation prediction tool Mutation Taster (http://www.mutationtaster.org/).
3.5 Results
From the Sanger sequencing results, we revealed a novel compound heterozygous variant contained a nonsense mutation(c.790C > T:p.(Arg264*) )and a missense mutation(c.296G > T:p.(Cys99Phe)), which was absent in relevant databases. Meanwhile, the pregnant woman carries a missense mutationc.296G >T in exon 3, and the husband carries a nonsense mutation c.790C > T in exon 7. With Mutation Taster, we found that the two mutations were nonsense-mediated mRNA decay and missense mutation respectively, and their corresponding protein might be affected.