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).