Search

Congenital melanocytic naevi (CMN) are pigmented birthmarks, known to be associated with an increased risk of neurological abnormalities and malignant melanoma. Current treatments for the cutaneous lesions and complications are limited for severely affected individuals, driving the research within this thesis into the aetiopathogenesis of this condition. The potential offered by studying rare diseases is also relevant to this project, particularly on the background of the rising incidence of melanoma in young people in the UK. The aims of this research were to expand the clinical description of the condition, to look for predisposing genetic alterations in the germline, to identify possible biomarkers for risk of complications and to investigate the lineage of naevus cells. To these ends we studied a large population of affected children to expand the phenotypic associations, investigating facial features, endocrinological status, pigmentary phenotype and growth, we undertook histopathological phenotyping of cutaneous and neurological lesions using immunohistochemistry and electron microscopy and employed array comparative genomic hybridisation and next generation sequencing for screening of germline haplotypes and mutations. Typical facial features were described in the majority of children with CMN, and the term CMN syndrome was proposed to encompass these. Post-natal growth was found to be rapid, leading to significantly increased BMI compared to the current UK population. Skewed anterior pituitary hormone measurements and increased parental thyroid abnormalities were suggestive of central hormonal dysfunction, however hormonal production from the cutaneous lesions could not be excluded. Histological phenotyping inferred that the primary abnormality underlying CMN may affect a population of stem cells, not necessarily destined to be melanocytes. An association between MC1R genotype and presence and severity of CMN was found using a candidate gene approach, and other germline candidates were identified. The results in this thesis have improved our understanding of the clinical and histological phenotype of CMN, and have identified genetic factors involved in the pathogenesis of the condition.

Congenital Melanocytic Naevus (CMN) syndrome is a rare sporadic disease which is characterised by large and/or multiple moles covering up to 80% of an individual's skin. Patients can have associated congenital neurological abnormalities, and are at risk of melanoma arising in the skin or brain. In 70% of cases the condition is caused by a postzygotic heterozygous missense mutation in the gene NRAS, most commonly NRAS c.(181C > A), p.(Q61K). NRAS encodes a GTPase signal transduction protein that is critical in the control of many key cell functions including regulation of proliferation, differentiation, and apoptosis. Pathogenic mutations in NRAS leading to amino acid changes in codon 61 result in constitutive activation of downstream signalling pathways. There is currently no treatment for CMN syndrome, with surgical removal of CMN having limited cosmetic effect on large or multiple naevi, no treatment available for most neurological abnormalities, and no successful treatment for melanoma in this context. Gene therapy offers the potential for reversing or attenuating the phenotype of CMN. The aim was to correct or attenuate the effects of the NRAS c.(181C > A) mutation in an in vitro cell culture model, in an allele-specific way which allows normal functionality of the wildtype allele. This has been successfully achieved through: i) culture and detailed functional of characterisation of paired NRAS c.(181C > A) mutant and wildtype melanoma cell lines, ii) design and optimisation of allele-specific knockdown of mutant c.(181C > A) NRAS gene expression using siRNA; iii) design and optimisation of mutant c.(181C > A) NRAS-specific CRISPR sgRNA. These results provide the first evidence of a potential genetic therapy for CMN syndrome, and in addition serve as proof of concept for the treatment of other mosaic diseases of the skin.