A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma

Abstract

So far, two genes associated with familial melanoma have been identified, accounting for a minority of genetic risk in families. Mutations in CDKN2A account for approximately 40% of familial cases, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds. Here we report the whole-genome sequencing of probands from several melanoma families, which we performed in order to identify other genes associated with familial melanoma. We identify one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF). Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log of odds (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant. Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case-control sample. Likewise, it was similarly associated in an independent case-control sample from the United Kingdom. In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both. The variant allele was also associated with increased naevus count and non-blue eye colour. Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets. These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.

Figure 1. Co-segregation analysis of the MITF E318K variant in the family in which it was identified.

The pedigree shows individuals that have had melanoma (shaded circles or boxes), with the age of first melanoma diagnosis indicated in brackets and the number of melanomas that have occurred in the individual so far (for example, × 2 indicates two primary melanomas). If the number of melanomas is not stated, the individual has had a single melanoma. A diagonal line through the symbol indicates that the person is deceased. The genotype for the MITF E318K variant for individuals with an available DNA sample for testing is annotated ‘E318K’ if a carrier or wild type ‘WT’. Other cancer types are also indicated with the age of first diagnosis indicated in brackets if known. Br, breast; Mes, mesothelioma; MM, melanoma; Oes, oesophagus. The individual circled in Family 1 (FAM1) is the melanoma case in which the MITF E318K variant was discovered through whole-genome sequencing. See Supplementary Fig. 3 for pedigrees of all other families identified as carrying E318K. PowerPoint slide

Figure 2. E318K prevents MITF sumoylation and results in differential expression of MITF target genes.

a, His-tagged wild-type MITF or the indicated single or double point mutants were co-transfected with HA–SUMO1 in COS-7 cells or b, HA–SUMO was transfected alone into homozygous mutant E318K MITF melanoma cells (NAE). Single- and double-sumoylated forms of MITF are indicated by a dagger and double dagger, respectively. The doublet bands are caused by MAPK-mediated phosphorylation at serine 73 (ref. 30). c, UACC62 human melanoma cells were transfected with TRPM1-promoter constructs with indicated amounts of expression vector encoding wild-type or mutant forms of MITF. Fold induction is shown as the ratio to the average of no MITF transfection (0 ng). Data are mean ± s.d. of at least four independent experiments. d, Expression of MITF in two melanoma cell lines (HT144 and C32) engineered to inducibly express wild-type (WT) or mutant (E318K) MITF after treatment with tetracycline for 48 h (48), as determined by qRT–PCR. Performed in triplicate, error bars depict s.d. e, Expression of MITF target genes DCT (top left), MLANA (top right) and THBS1 (bottom left) determined by qRT–PCR in melanoma cell lines 48 h after induction of wild-type or E318K MITF. Gene expression is normalized to GAPDH and shown as fold change compared to uninduced cells. Performed in triplicate, error bars denote s.d. f, qRT–PCR analysis of total RNA isolated from UACC62 human melanoma cells, which were transfected with expression vector encoding wild-type or mutant forms of MITF. The expression level of each target gene was normalized to MITF mRNA. Fold induction is shown as the ratio to each mRNA expression with wild-type MITF. Data are mean ± s.d. of at least three independent experiments. *P < 0.05, **P < 0.01. PowerPoint slide