Variants from 234 genes were selected from the whole exome sequencing dataset of the 298 probands with eoHM. Low-certainty variant positions with a depth of coverage less than 10 were ignored. Then variants unlikely to be pathogenic were filtered out in the following order. (1) Single nucleotide polymorphisms (SNPs) and short indels in the exome region were filtered against data from dbSNP, 1000 Human Genome Project, Exome Variant Server, and Exome Aggregation Consortium (ExAC, Cambridge, MA, USA;
http://exac.broadinstitute.org [in the public domain], accessed November 2015) excluding minor allele frequency (MAF) ≥ 1/6000 (according to a prevalence of approximately 1/3000 of retinal degeneration
34 and an allele frequency of a variant in AD genes not exceeding 1/6000 in the general population). (2) We excluded variants in noncoding region as well as the synonymous variants that did not affect splice sites according to the Berkeley Drosophila Genome Project (BDGP;
http://www.fruitfly.org/ [in the public domain]).
35 (3) We excluded variants predicted to be benign by two online tools: PolyPhen-2 (
http://genetics.bwh.harvard.edu/pph2/ [in the public domain])
36 and SIFT (
http://sift.jcvi.org [in the public domain]).
37 (4) We excluded variants not consistent with hereditary patterns, which were only one hit heterozygous variants in AR genes and homozygous variants in AD genes. (5) We excluded variants not consistent with the mutation types or mutant regions of the diseasing-causing mutations in corresponding genes based on the Human Gene Mutation Database (HGMD,
http://www.hgmd.cf.ac.uk/ac/index.php [in the public domain]) and previous studies. These included (a) truncation and the glycine replaced missense variants in
COL2A1 (MIM 120140) and
COL11A1 (MIM 120280), which are well known to be pathogenic; several missense mutations in
COL2A1 were also considered to be pathogenic; other variants in the two genes were excluded; (b)
CACNA1F (MIM 300110), where all mutations listed in HGMD were hemizygous; all heterozygous variants were excluded; (c)
GUCY2D (MIM 600179), mainly described as AR retinal disease genes,
38 except that mutations at codons 837, 838, and 849 caused AD retinal disease
39–41; (d) most of the causative mutations in
DMD (MIM 300377), which were truncation or splice mutations. Only a few missense mutations, which were located in the N-terminal or C-terminal domains, were reported as pathogenic.
42 (6) A total of 312 patients were included as ethnicity-matched relative controls; they had ocular diseases other than high myopia and retinal degeneration. Whole exome sequencing was also performed on the 312 individuals, using the same sequencing platform as used for the 298 probands with eoHM. The variants were excluded if they were found in the 312 controls.