2.3 | PCR amplification and mutation screening
The primers used to amplify the eight coding exons of the EDA1gene in PCR were based on those used by Song et
al(Feng et al., 2018;
Song et al., 2009). PCR reactions were
elicited in a total volume of 50μl, each containing 100ng DNA, 4μl
dNTPs, 5ml 10 × TransStart Taq Buffer, 0.2μl of each primer, and 1.25 U
TransStart Taq DNA Polymerase (Thermo Fisher, USA). After denaturing at
95°C for 5 min, amplification was carried out as follows: 35 cycles at
95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and finally 72°C for 7 min.
Primers of exon 4 of the EDAR gene were as follows: F:
5’-GGCAAGAGTAGCTTCTGGAGAC-3’; R: 5’-GTTAATGGCCACTTAGGAGACAC-3’.
Amplification was tested by agarose gel electrophoresis and DNA was
sequenced by the Beijing Genomics Institute, Beijing, China. The
nucleotide sequence was analyzed using the BLAST database of the
National Center for Biotechnology Information
(https://blast.ncbi.nlm.nih.gov/Blast.cgi).
We identified the nucleotide variants in the EDA1 and EDARgenes and 100 unrelated population-matched controls.
2.4 |
Whole exome sequencing and Sanger sequencing
Whole exome sequencing was performed for the proband (Family 4), who did
not have any EDA1 mutations detected by PCR. Target enrichment
and amplification were performed via liquid-phase capture method with
testing kits from iGeneTech Bioscience (Beijing, China) Co., Ltd.
Illumina NovaSeq 6000 Genome Analyzer platform (Illumina, San Diego, CA,
USA) was used to sequence the exons from the targeted regions. With a
sequencing yield of more than 17,550 Mb raw bases, the samples achieved
a mean target depth of 138 ×. Reads were aligned to the Genome Reference
Consortium Human Build 37 (GRCh37/hg19) with the
Burrows-Wheeler Aligner.
Single-nucleotide variants and small indels were identified with
SAMtools and Genome Analysis Toolkit (GATK) and then annotated by
ANNOVAR. The candidate mutation of EDAR was verified with PCR,
followed by Sanger sequencing. PCR was performed, and the PCR products
were sequenced as described in Section 2.3. The reference sequence forEDAR was NM_022336.4. A cross-species alignment of the amino
acid sequence of EDAR was performed by Clustal Omega
(https://www.ebi.ac.uk/Tools/msa/clustalo).