Gain-of-function mutation of KIT ligand on melanin synthesis causes familial progressive hyperpigmentation

Abstract

Familial progressive hyperpigmentation (FPH) is an autosomal-dominantly inherited disorder characterized by hyperpigmented patches in the skin, present in early infancy and increasing in size and number with age. The genetic basis for FPH remains unknown. In this study, a six-generation Chinese family with FPH was subjected to a genome-wide scan for linkage analysis. Two-point linkage analysis mapped the locus for FPH at chromosome 12q21.31-q23.1, with a maximum two-point LOD score of 4.35 (Ø = 0.00) at D12S81. Haplotype analysis confined the locus within an interval of 9.09 cM, flanked by the markers D12S1667 and D12S2081. Mutation profiling of positional candidate genes detected a heterozygous transversion (c. 107A-->G) in exon 2 of the KIT ligand (KITLG) gene, predicted to result in the substitution of a serine residue for an asparagine residue at codon 36 (p.N-->S). This mutant "G" allele cosegregated perfectly with affected, but not with unaffected, members of the FPH family. Function analysis of the soluble form of sKITLG revealed that mutant sKITLGN36S increased the content of the melanin by 109% compared with the wild-type sKITLG in human A375 melanoma cells. Consistent with this result, the tyrosinase activity was significantly increased by mutant sKITLGN36S compared to wild-type control. To our knowledge, these data provided the first genetic evidence that the FPH disease is caused by the KITLGN36S mutation, which has a gain-of-function effect on the melanin synthesis and opens a new avenue for exploration of the genetic mechanism of FPH.

Figure 1

Familial Progressive Hyperpigmentation in a Six-Generation Family (A) The affected individual, IV-1, manifests a typical clinical feature of FPH. The hyperpigmentation can be observed on his hands (a), palms (b), limbs (c), conjunctive face (d), neck (d), trunk (not shown), and soles (not shown). (B) Histological appearance of the specimen. Normal skin from an age-gender matched control (a) and hyperpigmented skin from a patient (b) are shown. Skin-biopsy specimens were stained with hematoxylin and eosin. Magnification: ×10. In hyperpigmented skin, there is a significant increase of the number of melanocytes and of the melanin content in the basal keratinocytes, as well as a slight increase in the size of melanocytes. (C) Pedigree structure and haplotype of the family. Markers are listed from top to bottom: centromere-D12S83-D12S326-D12S1708-D12S1667-D12S81-D12S351-D12S101-D12S2081-D12S346-D12S78-telomere. Black bars represent disease-carrying haplotypes. Question marks indicate that the genotype is not determined. Squares indicate male family members; circles indicate female family members; blackened symbols indicate affected members; open symbols indicate unaffected members.

Figure 2

Mutation Analysis of sKITLG in a Family with FPH and Effects of the Mutant sKITLG on Melanin Synthesis (A) Sequence trace of the WT allele, showing translation of asparagine residue at codon 36 (AAT). (B) Sequence trace of the mutant allele, showing the heterozygous c. 107A→G, which is predicted to result in the missense substitution of serine (AGT) for asparagine at codon 36 (p.N36S). (C) The soluble form of sKITLGN36S significantly increases melanin content in A375 cells, compared with the WT sKITLG. A375 cells were incubated with 100 ng/ml WT sKITLG and sKITLGN36S for 24 hr, respectively. Cell pellets were dissolved in buffer (1N NaOH, 10%DMSO) at 80°C for 2 hr and centrifuged for 10 min at 12,000 × g. Absorbance of melanin at 420 nm was measured with TECAN Safire2 (USA). A melanin standard curve was prepared with the use of synthetic melanin (Sigma). Melanin content was normalized to the cell number. Significance was determined according to a two-sided Student's t test performed with Excel software. The results are shown as mean ± SD (n = 6), and similar results were obtained when the experiments and measurements were repeated four times. Error bars indicate ± SD. (D) The soluble form of sKITLGN36S significantly increases the tyrosinase activity, compared with the WT sKITLG, in A375 cells. The A375 cells were treated as in melanin measurement for tyrosinase-activity analysis. The protein present in the supernatant was estimated by the method of Lowry, with BSA used as the standard, then 40 ug of protein was incubated in1 ml sodium phosphate buffer (pH7.4, containing 0.1% L-dopa) for 2 hr at 37°C. The absorbance was monitored at 475 nm with TECAN Safire2 (USA). Significance was determined according to a two-sided Student's t test performed with Excel software. The results are shown as mean ± SD (n = 10), and similar results were obtained when the experiments and measurements were repeated twice.