Germline mutations within melanoma susceptibility genes are present only in minority of melanoma patients and it is expected that additional genes will be discovered with next generation sequence technology and whole-exome sequencing (WES).
Herein we performed WES on a cohort of 96 unrelated Polish patients with melanoma diagnosed under the age of 40 years who all screened negative for the presence of
We selected 21 potentially deleterious variants in 20 genes (
Although most of the changes seemed to be neutral we could not exclude an association between variants in
Melanoma is one of the most aggressive human malignancies. The mean survival time for metastatic patients is 6 months when standard chemotherapy is used or up to 12 months if used in combination with the BRAF enzyme inhibitor vemurafenib. The incidence of melanoma has been increasing dramatically in the Caucasian population since the 1950s [
In Poland the number of affected individuals has increased by 60% over the past 11 years and has reached over 2,600 new cases identified annually. The etiology of melanoma is complex, involving both genetic and environmental factors. Significant heritability of this malignancy was observed in large twin study [
Family studies provide strong support for heterogeneous mechanisms involved in a polygenic inherited susceptibility to melanoma that includes both inherited high-risk and lowrisk alleles. To date only one major melanoma susceptibility gene:
Given the unique homogenous structure of the Polish population, we undertook an exome screen to determine if there existed one or more novel high/moderate risk gene(s) for MM, which may be associated with specific founder effects. Herein, we performed whole-exome sequencing (WES) on a cohort of 96 unrelated Polish patients with melanoma diagnosed under the age of 40 years. All of whom had previously been screened for the present of causative
In the subsequent verification step, we evaluated recurrent truncating mutations identified by WES in large case-control study of 1,200 unselected MM patients and 6,200 control subjects from Poland.
In the first step of this study, we have performed whole exome sequencing in a cohort of 96 unrelated patients with early-onset (< 40 years) melanoma who all screened negative for CDKN2A-causative variants (65 females; mean age, 30.8 years; range, 15 to 40 years and 31 males; mean age, 30.9 years; range, 18 to 39 years). Half of the patients (48 cases) were also characterized by a familial aggregation of melanoma among first- or second-degree relatives.
We verified the role of variants revealed by WES using a large case control-study of 1,230 unselected melanoma patients (760 females; mean age, 53.9 years; range, 15 to 92 years and 470 males; mean age, 55 years; range, 18 to 84 years) from Poland. 748 unselected MM cases (464 women; mean age, 53.7 years; 284 men) comprising 62.3% of the total group were diagnosed between 2002 and 2006 and identified from cancer registries in five Polish cities (Szczecin, Opole, Bialystok, Zielona Gora, and Gorzow Wielkopolski). The registries capture more than 95% of all diagnosed melanomas. The remaining 482 (37.7%) unselected MM cases (296 females, 186 males) were diagnosed in the city of Szczecin between the years 2010 and 2016.
The control group consisted two non-overlapping cohorts of healthy adults. All mutations that were confirmed by Sanger sequencing were genotyped in a group of 1,696 healthy adults (943 women, 753 men) with no cancers diagnosed in their families. The healthy adults were assessed as having a negative cancer family history (first- and seconddegree relatives included) after answering questionnaires about their family's medical history, which was part of a population-based study of the 1.5 million residents of West Pomerania aimed at identifying familial aggregations of malignancies performed recently by our center. During the interview, the goals of the study were explained, informed consent was obtained, genetic counseling was given and a blood sample was taken for DNA analysis. Individuals affected with any malignancy or with cancers diagnosed among first- or second-degree relatives were excluded from our study control group.
Variants with significantly different allele distribution among cases and the first cohort of controls, described above, were additionally genotyped in a second cohort of 5,000 healthy adults (1,775 men; mean age, 58 years and 3,225 females; mean age, 60 years) who underwent genetic counselling and were diagnosed as having no genetically increased risk of cancer on the basis of molecular examination and a negative cancer family history. During the interview, the goals of the study were explained, informed consent was obtained, genetic counseling was given and a blood sample was taken for DNA analysis. Individuals affected with any malignancy or with cancers diagnosed among firstor second-degree relatives were excluded from our study control group.
Overall, patient participation rates exceeded 75%. All patients and control subjects were of European ancestry and ethnic Poles.
DNA has been isolated using standard methods from peripheral blood leukocytes taken from participants in Department of Genetics and Pathology in Szczecin.
The Illumina Nexter Rapid Capture Expanded Exome kit (target region size=62 MB, coding exons, untranslated regions (UTRs) and miRNAs included; Illumina, San Diego, CA) was used for capturing sequence target regions. The kit captured 62 Mbp of the human genome covering coding exons in CCDS and RefSeq databases as well as exons annotated by GENCODE project. The captured regions for each sample were barcoded and every two samples were pooled and used for paired-end sequencing for 100 cycles (generating 100 bp reads) on a single lane of Illumina HiSeq2000’s flow-cell. The 100 million reads for each exome sequence for each individual were aligned to the reference sequence of the human genome using Burrows-Wheeler transform algorithm [
We considered only those genes with a minimum of two patients as candidates for further studies.
We employed disease-network approach to select those genes which are functionally more related to cancer pathogenesis (such as DNA repair and cell cycle control pathway genes) using the ToppGene algorithm [
Each variant identified by WES in the discovery phase and selected for validation was confirmed by Sanger direct sequencing. Sequencing reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing kit (Life Technologies, Foster City, CA). according to the manufacturer's protocol. Sequencing products were analyzed on the ABI Prism 3500XL Genetic Analyzer (Life Technologies). All sequences were compared reference sequences for variant detection using Mutation Surveyor software (SoftGenetics).
DNA was isolated from 5 to 10 mL of peripheral blood. The validated variants were genotyped using a TaqMan assay (Applied Biosystems/Life Technologies) and the Light-Cycler Real-Time PCR 480 system (Roche Life Science, Indianapolis, IN). The primer and probe sequences are available upon request. Laboratory technicians were blinded to casecontrol status. The overall genotyping call rate was 99.3%. The presence of the mutation was confirmed by Sanger sequencing as described above.
All genotype comparisons between cases and controls were performed using Fisher exact test, and two-tailed p-values were calculated. OR values were calculated for each comparison together with their 95% confidence intervals. To exclude recessive trait of inheritance we performed additional genotyping of parents and siblings of patients with identified mutations from the cohort of 96 early-onset MM cases.
The Polish study was approved by the ethics committee of Pomeranian Medical University (Szczecin, Poland). All study subjects provided a signed consent form for participation in the study.
Using GATK algorithm, we detected 2,272,282 indels in the total data set for our cohort of early-onset melanoma patients, after prioritization we identified 1,012 new and 218 previously reported frameshift mutations (523 new variants in sporadic early-onset cases and 489 in familial cases; 137 previously reported variants in sporadic cases and 81 in familial cases). Using SCALPEL algorithm, we detected 2,297 variants (1,151 of them in sporadic early-onset cases, 1,146 in familial cases). These comprised 3′ UTR and 5′ UTR variants (452 and 56, respectively), 228 downstream variants, 88 in frame deletions, 40 intergenic variants, 1,000 intron variants, 11 intron variants and NMD transcript variants, 55 intron variants and non-coding transcript variants, 57 non-coding transcript exon variants and noncoding transcript variants, five regulatory regions variants, three splice acceptor variants, 60 splice region variants and intron variants, two transcription factor variants, one splice region variant and intron variant and noncoding transcript variant, 161 upstream gene variants and 74 frameshift mutations.
The frameshift mutations were selected for further interrogation as they were most likely to reveal new genes of interest with respect to melanoma development.
All the frameshift mutations were filtered by examining the quality control values, minor allele frequencies and intense gene databases/literature data analysis supported by the ToppGene algorithm. We selected 21 potentially deleterious variants in 20 genes, which after confirmation by Sanger sequencing were evaluated in the association study. These included the genes
The variants in these genes were further investigated in those families where trios were available to determine if they were recessively or dominantly inherited. The results of this aspect of the study revealed that there were some genetic variants that were homozygous in the proband and in one or other of the parents. Further evaluation revealed that these variants were unlikely to be associated with disease since either the unaffected parents were both homozygous for the allele and the proband heterozygous or the proband was homozygous for the variant and one of the parents were homozygous and the other heterozygous for that specific allele. Genotyping of the parents of homozygous carriers of identified mutations revealed no case of homozygous proband and both parents being the carriers of the mutations. Together, this suggested that those variants were unlikely to be associated with any change in melanoma risk (
Additional evidence about the pathogenicity of the identified variants was provided by a replication study where a set of 1,200 melanoma patient DNA samples was compared to a similarly large series of control DNA samples taken from healthy subjects (
The results revealed that most of the changes were neutral but although we found no statistically significant differences between cases and controls, 10 genes cannot be excluded as being associated with melanoma risk—due to genetic heterogeneity and the reduced penetrance of this disease. A greater level of confidence in the pathogenicity of the variant was observed if the homozygous state was associated with a greater difference in risk compared to being in the heterozygote state. Unfortunately, many of the variants occurred within the study populations at much lower frequency to be reliably associated with disease risk. Potential candidates included
From 2008 to 2013, the approach of finding melanoma susceptibility genes centered on genome-wide association studies (GWAS). The results of several of these studies aimed at identifying susceptibility loci for melanoma have been reported [
Low-penetrance variants cannot be categorically associated with familial clusters, for many reasons that include molecular interactions between low/moderate penetrance genes, which could potentially be responsible for early-onset disease.
Recently, WES has been developed which overcomes many of the problems of GWAS [
Given the homogeneity of the Polish population we expected to find a limited number of recurrent founder mutations. However, we found large numbers of germline DNA alterations which appeared causative as they were predicted to result in truncated protein products, but none appeared to be novel high-risk melanoma gene in either sporadic familial early-onset patients. We observed known recurrent truncating mutations in genes predisposing to other cancers, such as
Taken together the results from this study with respect to the Polish population suggests that there is extensive genetic heterogeneity within melanoma families from Poland that can not be explained by a few recurrent highly penetrant causal variants. Large multi-center studies are needed for further investigation of possible involvement of
Conflict of interest relevant to this article was not reported.
This study was supported by the Polish National Science Centre (grant number 2014/13/B/NZ2/03888).
Allele distribution among carriers and their relatives in trios-available families
Gene (mutation) | Position | Proband | Father | Mother | Brother | 2nd brother | Sister |
---|---|---|---|---|---|---|---|
96860761 | het mut |
0 |
het mut | 0 | 0 | 0 | |
het mut | het mut | hom wt |
0 | 0 | 0 | ||
152697964 | het mut | het mut | hom wt | 0 | 0 | 0 | |
het mut | 0 | het mut | 0 | 0 | 0 | ||
het mut | het mut | hom wt | 0 | 0 | 0 | ||
225192859 | het mut | hom wt | het mut | het mut | het mut | 0 | |
hom mut |
hom mut | hom mut | 0 | 0 | 0 | ||
40141200 | het mut | het mut | hom wt | 0 | 0 | 0 | |
60497504 | het mut | het mut | hom wt | hom wt | 0 | 0 | |
90966617 | het mut | het mut | hom wt | 0 | 0 | 0 | |
het mut | het mut | hom wt | 0 | 0 | 0 | ||
79211976 | het mut | hom wt | hom mut | 0 | 0 | 0 | |
het mut | hom wt | het mut | 0 | 0 | 0 | ||
7921995 | het mut | hom wt | het mut | 0 | 0 | 0 | |
het mut | het mut | hom wt | 0 | 0 | 0 | ||
40841469 | het mut | het mut | hom wt | 0 | 0 | 0 | |
het mut | hom wt | het mut | 0 | 0 | 0 | ||
43537404 | het mut | het mut | hom wt | 0 | 0 | 0 | |
het mut | het mut | 0 | 0 | 0 | hom wt | ||
40515573 | het mut | het mut | hom wt | 0 | 0 | 0 | |
8392543 | het mut | het mut | hom wt | 0 | 0 | 0 | |
939436 | hom mut | het mut | hom mut | 0 | 0 | 0 | |
hom mut | het mut | hom mut | 0 | 0 | 0 | ||
hom mut | het mut | hom mut | 0 | 0 | 0 | ||
hom mut | hom mut | het mut | 0 | 0 | 0 | ||
35627312 | het mut | hom mut | hom wt | 0 | 0 | 0 | |
het mut | hom mut | hom wt | 0 | 0 | 0 | ||
het mut | 0 | hom wt | het mut | 0 | 0 | ||
het mut | 0 | het mut | 0 | 0 | 0 | ||
het mut | 0 | 0 | 0 | 0 | het mut | ||
23136057 | het mut | het mut | hom mut | 0 | 0 | 0 | |
het mut | hom mut | 0 | het mut | het mut | 0 | ||
het mut | hom mut | het mut | 0 | 0 | 0 | ||
het mut | hom wt | het mut | 0 | 0 | 0 | ||
156979266 | het mut | hom wt | het mut | 0 | 0 | 0 | |
het mut | het mut | hom wt | 0 | 0 | 0 | ||
het mut | hom wt | hom mut | 0 | 0 | 0 | ||
het mut | 0 | hom wt | het mut | 0 | 0 | ||
145847527 | het mut | hom wt | hom mut | 0 | 0 | 0 | |
hom mut | het mut | hom mut | 0 | 0 | 0 | ||
218331057 | het mut | hom wt | hom mut | 0 | 0 | 0 | |
89867987 | hom mut | hom mut | het mut | 0 | 0 | het mut |
Heterozygous mutation,
Not examined,
Homozygous wild type,
Homozygous mutation. In all cases parents and siblings were not diagnosed with melanoma.
Prevalence of the validated potentially protein-truncating variants among cases and controls
Gene/mutation | POS |
Genotype | No. (%) |
Odds ratio | Significance level p-value | |
---|---|---|---|---|---|---|
MM | CONTROLS | |||||
79211976 | ALLELE X |
1,058 (91.0) | 1,399 (90.4) | |||
Heterozygote XY | 102 (8.8) | 143 (9.2) | 0.943 | 0.667 | ||
ALLELE Y |
4 (0.3) | 5 (0.3) | 1.058 | 0.933 | ||
7921995 | ALLELE X | 1,160 (97.8) | 1,603 (98.5) | |||
Heterozygote XY | 23 (1.9) | 23 (1.4) | 1.382 | 0.277 | ||
ALLELE Y | 3 (0.3) | 2 (0.1) | 2.073 | 0.425 | ||
96860761 | ALLELE X | 789 (89.2) | 761 (86.4) | |||
Heterozygote XY | 96 (10.8) | 114 (12.9) | 0.945 | 0.698 | ||
ALLELE Y | 0 | 6 (0.7) | 0.086 | 0.095 | ||
145847527 | ALLELE X | 1,008 (95.0) | 1,720 (94.1) | |||
Heterozygote XY | 47 (4.4) | 104 (5.7) | 0.771 | 0.149 | ||
ALLELE Y | 2 (0.2) | 3 (0.2) | 1.138 | 0.888 | ||
939436 | ALLELE Y | 101 (9.0) | 194 (11.9) | |||
Heterozygote XY | 1,003 (90.2) | 1,415 (86.9) | 0.594 | 0.219 | ||
ALLELE X | 8 (0.7) | 19 (1.2) | 0.809 | 0.629 | ||
225192859 | ALLELE X | 919 (87.0) | 1,561 (87.1) | |||
Heterozygote XY | 134 (12.6) | 221 (12.3) | 1.030 | 0.801 | ||
ALLELE Y | 7 (0.7) | 10 (0.6) | 1.189 | 0.726 | ||
23136057 | ALLELE X | 805 (88.0) | 1,447 (87.1) | |||
Heterozygote XY | 107 (11.7) | 204 (12.3) | 0.943 | 0.643 | ||
ALLELE Y | 5 (0.5) | 11 (0.6) | 0.817 | 0.709 | ||
4168367 | ALLELE X | 1,210 (99.0) | 1,501 (99.5) | |||
Heterozygote XY | 12 (1.0) | 7 (0.5) | 2.127 | 0.114 | ||
43537404 | ALLELE X | 1,110 (93.7) | 907 (93.1) | |||
Heterozygote XY | 74 (6.2) | 63 (6.5) | 0.960 | 0.817 | ||
ALLELE Y | 1 (0.1) | 4 (0.4) | 0.204 | 0.156 | ||
152697964 | ALLELE X | 1,135 (94.9) | 3,874 (94.9) | |||
Heterozygote XY | 59 (4.9) | 200 (4.9) | 1.007 | 0.964 | ||
ALLELE Y | 2 (0.2) | 6 (0.1) | 1.138 | 0.874 | ||
40841469 | ALLELE X | 1,144 (95.9) | 1,631 (96.2) | |||
Heterozygote XY | 48 (4.0) | 65 (3.8) | 1.053 | 0.791 | ||
ALLELE Y | 1 (0.1) | 0 ( | 4.276 | 0.374 | ||
35627312 | ALLELE X | 943 (79.4) | 834 (79.5) | |||
Heterozygote XY | 229 (19.3) | 204 (19.4) | 0.993 | 0.946 | ||
ALLELE Y | 15 (1.3) | 11 (1.1) | 1.206 | 0.639 | ||
40515573 | ALLELE X | 1,142 (97.2) | 1,640 (96.4) | |||
Heterozygote XY | 32 (2.7) | 54 (3.2) | 0.851 | 0.476 | ||
ALLELE Y | 1 (0.1) | 7 (0.4) | 0.205 | 0.139 | ||
218331057 | ALLELE X | 1,126 (98.6) | 1,020 (98.1) | |||
Heterozygote XY | 16 (1.4) | 20 (1.9) | 0.811 | 0.536 | ||
60497504 | ALLELE X | 1,159 (96.3) | 6,216 (97.3) | |||
Heterozygote XY | 45 (3.7) | 175 (2.7) | 1.379 | 0.059 | ||
ALLELE Y | 0 | 1 (0.03) | 1.787 | 0.722 | ||
90966617 | ALLELE X | 1,053 (95.2) | 4,595 (94.8) | |||
Heterozygote XY | 49 (4.4) | 233 (4.8) | 0.918 | 0.593 | ||
ALLELE Y | 4 (0.4) | 18 (0.4) | 0.970 | 0.956 | ||
40141200 | ALLELE X | 1,132 (93.0) | 6,137 (93.1) | |||
Heterozygote XY | 82 (6.7) | 445 (6.7) | 0.999 | 0.994 | ||
ALLELE Y | 3 (0.3) | 13 (0.2) | 1.251 | 0.727 | ||
156979266 | ALLELE X | 1,144 (95.6) | 1,018 (95.0) | |||
Heterozygote XY | 53 (4.4) | 53 (5.0) | 0.890 | 0.558 | ||
74520191 | ALLELE X | 1,207 (99.0) | 1,774 (99.0) | |||
Heterozygote XY | 12 (1.0) | 18 (1.0) | 0.980 | 0.957 | ||
8392543 | ALLELE X | 1,084 (95.0) | 4,658 (96.0) | |||
Heterozygote XY | 59 (5.2) | 191 (3.9) | 1.327 | 0.064 | ||
ALLELE Y | 1 (0.1) | 2 (0.04) | 2.148 | 0.532 | ||
89867987 | ALLELE X | 703 (76.4) | 843 (77.4) | |||
Heterozygote XY | 186 (20.2) | 200 (18.4) | 0.897 | 0.337 | ||
ALLELE Y | 31 (3.4) | 46 (4.2) | 0.725 | 0.213 |
Pos-genomic nucleotide position (Genome assembly GRCh38.p2),
Allele X- major allele,
Allele Y- minor allele.