MITF E318K: A rare homozygous case with multiple primary melanoma

MITF E318K moderates melanoma risk. Only five MITF E318K homozygous cases have been reported to date, one in association with melanoma. This novel report uses 3D total‐body‐photography (TBP) to describe the dermatological phenotype of a homozygous MITF E318K individual. The case, a 32‐year‐old male, was diagnosed with his first of six primary melanomas at 26 years of age. Five melanomas were located on the back and one in the groin. Two were superficial spreading. Three arose from pre‐existing naevi and one was a rare naevoid melanoma. 3D‐TBP revealed a high naevus count (n = 162) with pigmentation varying from light to dark. Most naevi generally (n = 90), and large (>5 mm diameter) and clinically atypical naevi specifically were located on the back where sun damage was mild. In contrast, naevi count was low (n = 25 total) on the head/neck and lower limbs where sun damage was severe. Thus, melanoma location correlated with naevi density, rather than degree of sun damage. In addition to the MITF E318K homozygosity, there was heterozygosity for four other moderate‐risk variants, which may contribute to melanoma risk. Further research is warranted to explore whether melanomas in E318K heterozygous and other homozygotes coincide with regions of high naevi density as opposed to sun damage. This could inform future melanoma screening/surveillance.

MITF E318K is described almost exclusively in heterozygotes, with homozygosity so rare the frequency is reported as zero (Karczewski et al., 2020).To date, only five MITF E318K homozygous cases have ever been reported: one individual with multiple primary melanoma (MPM) and a high melanocytic naevi count (Bassoli et al., 2018); two individuals with Waardenburg syndrome (Pang et al., 2019); two individuals with endocrine tumours where one had sporadic, metastatic pheochromocytoma and the other had paraganglioma secondary to a SDHB-mutation (Castro-Vega et al., 2016).
This study is novel in that we use 3D total-body-photography (TBP) (WB360 imaging system-Canfield Scientific Inc., Parsippany, NJ, USA) to characterise genotype-phenotype correlations, specifically in relation to the dermatological landscape, in an MITF E318K homozygote carrier with MPM.Artificial intelligence software generated a 3D-avatar of the participant which captured naevus counts, composition (pigmentation and size) and location.Clinically atypical naevi were classified by a dermatologist (HPS) based on size (>5 mm diameter), boarder irregularity and abnormal pigmentary characteristics.Degree of sun damage across body sites was also determined from the 3D-avatar and classified as absent, mild, moderate or severe by a dermatologist (HPS) using a method previously described (Betz-Stablein et al., 2021).The innate typology angle (ITA), a numerical value which accurately correlates with biologically efficient doses of UV radiation (i.e.doses required to induce sunburn) (Del Bino & Bernerd, 2013) (CIELAB 1976 System), was calculated using VEC-TRA mirror software algorithmic L* and b* wavelength data (Gribbin et al., 2022).Other phenotypic characteristics including hair and eye colour were also recorded.
The participant, a 32-year-old male, presented to the Dermatology Department at the Princess Alexandra Hospital with a history of six early-onset primary cutaneous melanomas confirmed on histopathological reports.Initial diagnosis was at 26 years of age with a melanoma arising within a pre-existing naevus (Breslow thickness 0.6 mm) located on the left scapula.Two subsequent melanomas were diagnosed 6 months later; one on the upper left scapula arising within a dysplastic naevus and the second on the left lateral groin classified as a rare naevoid melanoma (Breslow thickness 0.4 mm and 0.6 mm, respectively).One month later, a fourth melanoma was identified on the sacrum (Breslow thickness 0.6 mm).Fifteen months thereafter, a fifth melanoma in situ was identified on the right interscapular back arising in association with a pre-existing naevus.This fifth melanoma was captured using VECTRA 3D-TBP, showing two timepoints 1-year apart, visually demonstrating subtle morphologic change with increased reddish colouration (Figure 1a,b).
A sixth superficial-spreading melanoma (Breslow thickness 0.5 mm) was identified 2 years later (30 years of age), and located on the left anterior superior iliac spine.Due to participant privacy, no further imaging data could be included.3D-TBP at timepoint 2 revealed the participant had a high naevus count (n = 162; n = 127 naevi ≥ 2-5 mm, 35 naevi > 5 mm (diameter)) which varied in degrees of pigmentation from light to dark; numerous scars from excisions (n = 44 excisions of which n = 6 were melanomas) were also evident.While the naevus count in our participant is greater than the general population, it is less than the average naevus count reported in heterozygote MITF E318K carriers (n = 238) (Sturm et al., 2014).The largest proportion of naevi were located on the participant's back (n = 71 ≥ 2 mm; n = 19 > 5 mm diameter) where sun damage was mild; Figure 1c,d shows comparison of the back between two timepoints, 1 year apart, with multiple large (>5 mm diameter) and clinically atypical naevi observed.Interestingly, severe sun damage was evident on the subject's head/neck and lower limbs (see Table S2), but naevi were comparably fewer (n = 1 on head/neck and n = 24 on lower limbs) (Table S3).No freckling was observed.The ITA classified the participant's skin as 'light' (41° < ITA < 55°) inferring the participant is highly sensitive to UV-radiation.
The participant had brunette hair, brown eyes and reported European ancestry.Family history revealed that the participant's father was diagnosed with melanoma at 62 years of age.His mother

Significance
Only five individuals with the rare, MITF E318K homozygous variant have ever been reported.Using 3D totalbody-photography, the dermatological landscape of an MITF E318K homozygous individual (diagnosed six earlyonset multiple primary melanomas) was evaluated.Imaging analysis found melanoma sites were in areas of mild or no sun damage; furthermore, clinically atypical naevi and high counts of large naevi (>5 mm) were observed in areas of mild sun damage.Thus, melanoma development and naevi density do not correlate with severity of sun damage in this individual.Genotype-phenotype correlations derived from this research may help clinicians to personalise screening for individuals at high-risk of melanoma.
was reportedly diagnosed with breast cancer at 18 years of age, and his paternal grandfather was reportedly diagnosed with metastatic colorectal cancer at approximately 40 years of age.Family members were not available for participation and imaging.Although there have been reports of pancreatic cancer (Ghiorzo et al., 2013) and renal cell carcinoma in association with MITF E318K (Bertolotto et al., 2011;Lang et al., 2021), a recent meta-analysis did not find any increased risk for non-cutaneous cancers in E318K carriers (Guhan et al., 2020).assay (Sturm et al., 2014) was performed.participant was negative for pathogenic variants in CDKN2A, the most frequently mutated familial melanoma gene.However, sequencing other highpenetrance melanoma genes (e.g.POT1, POLE, BAP1, ACD, TERT and CDK4) was beyond the scope of this study.We did genotype known melanoma susceptibility variants in other moderate and low penetrance genes to identify additional variants which could be modifying risk.Genes analysed included MC1R, TYR, OCA2/HER2, TYRP1, SLC24A5, SLC45A2, ASIP, BNC2, DCT, IRF4, KITLG, MITF, POMC, SLC24A4, TPCN2 and MTAP.The analysis of these genes has been previously described (Rayner et al., 2020).

Genotyping was performed on the
Genotyping results (Table 1) identified the MITF E318K homozygosity and also revealed heterozygosity for MC1R 'R' common red hair colour (RHC) variant p.R151C (genotype R/wt) associated with a twofold increased melanoma risk (Cust et al., 2012).Of relevance, a recent report shows that MC1R genotypes modify melanoma risk in MITF E318K heterozygous carriers (Courtney et al., 2023).
Additional variants identified in this participant included: one pigmentation variant in TYR (rs1126809) shown to modify melanoma susceptibility (Rayner et al., 2019), and MTAP (rs7023329) associated with increased naevus counts (Yang et al., 2010).The participant also carried SLC45A2 (rs16891982) and SLC24A5 (rs1425564), which are melanoma risk alleles (Reis et al., 2020).No functional studies have been conducted on any of these variants to date.Remaining variants identified in known melanoma risk genes (Table 1) were common in the general population (minor allele frequency >0.05), classified as benign by the American College of Medical Genetics (ACMG criteria), and not previously associated with melanoma susceptibility.
Comparison of phenotypic characteristics of all reported MITF p.E318K homozygotes to date is described in

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.The data are not publicly available due to privacy or ethical restrictions.

E TH I C S S TATEM ENT
Ethics was approved at The University of Queensland and Princess Alexandra Hospital (HREC/09/QPAH/2009001590).

PATI ENT CO N S ENT
Consent for the publication of de-identified patient imaging data or any identifiable material was obtained by the authors prior to article submission to Pigment Cell and Melanoma Research stating that the Illumina Infinium Human-CoreExome-24 Microarray; three MC1R RHC 'r' variants (V60L, R163Q and D294H) were not captured on the array; thus, a TaqMan F I G U R E 1 MITF (E318K) homozygote (a) Image of participant's back at timepoint 1 (November 2017): Clinically atypical naevi observed and scarring evident from previous excisions; (b) Image of participant back timepoint 2 (July 2018): A melanoma in situ arising within a pre-existing naevus (highlighted in red); four more scars evident from excisions between timepoints; (c) Dermoscopy timepoint 1 (November 2017); (d) Dermoscopy timepoint 2 (July 2018).Please note subtle morphologic change with increased reddish colouration between timepoints.Genotype report from Infinium Global Screening Array v2.0 SNPs and reported variant impact.
Exome sequencing was funded by an Australian Skin and Skin Cancer Research Centre ECF grant (AML and BBS).This participant was originally recruited to the Brisbane Naevus Morphology Study (BNMS), funded by an NHMRC (APP1062935) and Centre of Research Excellence for the Study of Naevi (APP1099021).This work was also made possible by a Metro South Health Research Support Scheme Program Grant (RSS_2021_028).This research was carried out at the Translational Research Institute (TRI), Woolloongabba, Queensland Australia.CO N FLI C T O F I NTER E S T S TATEM ENTHPS is a shareholder of MoleMap NZ Limited and e-derm consult GmbH and undertakes regular teledermatological reporting for both companies.HPS is a Medical Consultant for Canfield Scientific Inc., Blaze Bioscience Inc., MoleMap Australia Pty Limited, and a Medical Advisor for First Derm.No other authors have conflicts to declare.

Table S1 .
The only naevus count in areas of mild sun damage.Furthermore, five of the six melanomas were located on the subject's back where total and clinically atypical naevus counts were high, but sun damage was mild.The sixth melanoma developed in the groin where no sun damage was recorded.Thus, the site of melanoma location does not correlate with severity of sun damage.Genotyping revealed the subject was positive for four additional moderate, melanoma risk variants and thus genetic background, particularly variants in related pathways, may further modify melanoma risk in this individual.We Medicine Fellowship.E.J.M. and C.K.W. are funded by a Research Training Stipend form the Australian Department of Education.K.L. holds an NHMRC Postgraduate Research Scholarship (GIN2013961).