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.