Identification of KITLG gene mutations
All subsequently detected variants were then filtered on the basis of population after filtering all variants, a novel heterozygous missense mutation c.104A > T (p.Asn35Ile) and a recurrent mutation c.101C > T (p.Thr34Ile) in the KITLG gene were revealed in the family 1 (Fig. 2a) and in the sporadic case respectively (Fig. 2b). These two mutations were not detected in the unaffected family members or 100 unrelated population-match controls (Fig. 2c). The variation c.102T > A (p.Thr34Thr) in family 1 is a synonymous mutation (Fig. 2a).
Prediction Of The Potential Impacts Of The Mutations
The mutation c.104A > T (p.Asn35Ile) and c.101C > T (p.Thr34Ile) was predicted to be “possibly damaging” and “deleterious” with Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://sift.jcvi.org/), respectively. 34Thr and 35Asn of KITLG, which polar, neutral, hydrophilic R-based amino acids had changed in to Ile, which non-polar, hydrophobic R-based amino acids, in the family 1 and a sporadic case with SWISS-MODEL (http://swiss model.expasy.org/).
Review Of The Previous Studies Documented Kitlg Gene Mutations
There were nine publications in PubMed (https://www.ncbi.nlm.nih.gov/pubmed) is related to pathogenic mutations of KITLG gene.
Mutations of KITLG gene is associated with autosomal dominant nonsyndromic deafness-69 (DFNA69, MIM 616697), Waardenburg syndrome-2 (WS2, MIM 193510), and FPHH. Seco Z et al.  reported mutations of KITLG, c.286_303delinsT (p.Ser96Ter), c.200_202del (p.His67_Cys68delinsArg), and c.310C > G (p.Leu104Val), cause asymmetric and unilateral hearing loss and Waardenburg Syndrome type 2 (WS2). Ogawa Y et al.  reported a patient with WS2 who had the unusual complication of large pigmented macules with homozygous KITLG mutation (c.94G > A, p.Arg32Cys). It was speculated that the mechanism of the mutation underlying WS2 leading to membrane incorporation and reducing secretion of KITLG occurs via a gain-of-function or dominant-negative effect. A de novo mosaic KITLG variant (NM_000899.3:c.329A > G; p.Asp110Gly) was found with a 6-year-old boy had congenital linear and mottled hyperpigmentation . However, all the phenotypes presented in these three publications with KITLG is not defined clearly to FPHH, therefor, we only summarize all the other KITLG mutations associated with FPHH here in this study.
Account the novel mutation (c.104A > T, p.Asn35Ile) we reported in this study, to date, eight different missense mutations in the KITLG gene responsible for FPHH have been identified (Supplemental Table S1, Fig. 4). Seven out of eight mutations were clustered in a short amino acid sequence (VTNNV, amino acid 33–37) in exon2 (Fig. 4), except c.337G༞A which in exon 4 of the KITLG gene. Most pathogenic mutations in FPHH occur within the VTNN domain of KITLG protein (amino acids 33–36), lies within the third b-strand of the protein. Only the p.Val37 change represents the first amino acid of the second a-helix (amino acids 37–46). So far, the reported mutations are only involved in 33V, 34T, 36N, 37V but not 35N. We first report the c.104A > T (p.Asn35Ile) mutation at 35N (Fig. 4) with FPHH patients.
Except diffuse hyper- and hypopigmentation, Vast CAL-like lesions had been detected as the most common skin problems present with FPHH patients. Vitiligo was found in one family. Sparse lateral eyebrows and malignancy (pharyngeal cancer, papillary thyroid cancer and melanoma) were found in two families. Short suture was found only with one family and mental retardation was not presented in these FPHH patients (Supplemental Table S1).