Loss-of-function variants in GLMN are associated with generalized skin hyperpigmentation with or without glomuvenous malformation.

ABSTRACT

BACKGROUND: Inherited hyperpigmented skin disorders comprise a group of entities with considerable clinical and genetic heterogenicity. The genetic basis of a majority of these disorders remains to be elucidated. OBJECTIVES: This study aimed to identify the underlying gene for an unclarified disorder of autosomal-dominant generalized skin hyperpigmentation with or without glomuvenous malformation. METHODS: Whole-exome sequencing was performed in five unrelated families with autosomal-dominant generalized skin hyperpigmentation. Variants were confirmed using Sanger sequencing and a minigene assay was employed to evaluate the splicing alteration. Immunofluorescence and transmission electron microscopy (TEM) were used to determine the quantity of melanocytes and melanosomes in hyperpigmented skin lesions. GLMN knockdown by small interfering RNA assays was performed in human MNT-1 cells to examine melanin concentration and the underlying molecular mechanism. RESULTS: We identified five variants in GLMN in five unrelated families, including c.995_996insAACA(p.Ser333Thrfs*11), c.632 + 4delA, c.1470_1473dup(p.Thr492fs*12), c.1319G > A(p.Trp440*) and c.1613_1614insTA(Thr540*). The minigene assay confirmed that the c.632 + 4delA mutant resulted in abolishment of the canonical donor splice site. Although the number of melanocytes remained unchanged in skin lesions, as demonstrated by immunofluorescent staining of tyrosinase and premelanosome protein, TEM revealed an increased number of melanosomes in the skin lesion of a patient. The GLMN knockdown MNT-1 cells demonstrated a higher melanin concentration, a higher proportion of stage III and IV melanosomes, upregulation of microphthalmia-associated transcription factor and tyrosinase, and downregulation of phosphorylated p70S6 K vs. mock-transfected cells. CONCLUSIONS: We found that loss-of-function variants in GLMN are associated with generalized skin hyperpigmentation with or without glomuvenous malformation. Our study implicates a potential role of glomulin in human skin melanogenesis, in addition to vascular morphogenesis.

A group of skin conditions known as 'inherited hyperpigmented skin disorders' includes some diseases with different clinical and genetic traits. The genetic basis of the majority of these diseases is not understood. To identify the gene responsible for a disease that causes darker patches of skin (hyperpigmentation) with or without the abnormal growth of blood vessels and the presence of cells named glomus cells (a glomuvenous malformation), we used genetic techniques called whole-exome sequencing and Sanger sequencing in five unrelated families with this disease. We also used a technique called a 'minigene assay' to evaluate genetic alterations in a gene called GLMN, which encodes a protein called glomulin. Immunofluorescence and transmission electron microscopy (TEM) were used to determine the number of pigment-producing cells (called melanocytes) and melanosomes (where the pigment melanin is synthesized, stored and transported) in hyperpigmented skin lesions. We identified five different variants of the GLMN gene in five unrelated families. Although the number of melanocytes remained unchanged in skin lesions, TEM revealed an increased number of melanosomes. By 'switching off' the GLMN gene, we found that skin cells produced more pigment, as well as the proteins MITF and tyrosinase; they also showed a decrease in the phosphorylated protein p-p70S6 K. Overall, we found that loss-of-function mutations in GLMN caused skin hyperpigmentation with or without abnormal blood vessels. The results suggest there could be a potential role of the protein glomulin in human skin colour and blood vessel changes.