Functional,structural, and genetic evaluation of 20 CDKN2A germ line mutations identified in melanoma‐prone families or patients

Germline mutations of the CDKN2A gene are found in melanoma‐prone families and individuals with multiple sporadic melanomas. The encoded protein, p16^INK4A, comprises four ankyrin‐type repeats, and the mutations, most of which are missense and occur throughout the entire coding region, can disrupt the conformation of these structural motifs as well as the association of p16^INK4a with its physiological targets, the cyclin‐dependent kinases (CDKs) CDK4 and CDK6. Assessing pathogenicity of nonsynonymous mutations is critical to evaluate melanoma risk in carriers. In the current study, we investigate 20 CDKN2A germline mutations whose effects on p16^INK4A structure and function have not been previously documented (Thr18_Ala19dup, Gly23Asp, Arg24Gln, Gly35Ala, Gly35Val, Ala57Val, Ala60Val, Ala60Arg, Leu65dup, Gly67Arg, Gly67_Asn71del, Glu69Gly, Asp74Tyr, Thr77Pro, Arg80Pro, Pro81Thr, Arg87Trp, Leu97Arg, Arg99Pro, and [Leu113Leu;Pro114Ser]). By considering genetic information, the predicted impact of each variant on the protein structure, its ability to interact with CDK4 and impede cell proliferation in experimental settings, we conclude that 18 of the 20 CDKN2A variants can be classed as loss of function mutations, whereas the results for two remain ambiguous. Discriminating between mutant and neutral variants of p16^INK4A not only adds to our understanding of the functionally critical residues in the protein but provides information that can be used for melanoma risk prediction. Hum Mutat 0, 1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Population-based prevalence of CDKN2A and CDK4 mutations in patients with multiple primary melanomas

The presence of multiple primary cutaneous melanomas (MPM) has been advocated as guidance to identifying melanoma families. Frequencies of CDKN2A mutations in materials of sporadic MPM cases from pigmented lesion clinics vary between 8 and 15%. Patients with MPM have therefore been regarded as good candidates for CDKN2A mutational screening. We describe a population-based study where all persons in Norway diagnosed with MPM between 1953 and 2004 (n = 738 alive per April 2004) were invited to participate. Three-hundred-and-ninety patients (52.8%) responded confidentially. Mutations in CDKN2A were found in 6.9% of the respondents. Eighty-one MPM patients (20.8%) reported that they belonged to melanoma families, and 17 (21.0%) of these harboured a CDKN2A mutation, compared to 3.2% of the nonfamilial cases. The probability of finding a CDKN2A mutation increased when the patients had three or more melanomas, or a young age of onset of first melanoma. We identified five novel CDKN2A variants (Ala57Gly, Pro81Arg, Ala118Val, Leu130Val, and Arg131Pro) and four that previously have been reported in melanoma families (Glu27X, Met53Ile, Arg87Trp, and Ala127Pro). A large deletion (g.13623_23772del10150) encompassing exon 1alpha and the 5' part of exon 2 was detected in six patients with a family history of melanoma. Three patients, belonging to the same family, had the CDK4 Arg24His mutation. The frequency of CDKN2A mutations was lower than previously reported in other studies, an observation which probably is due to the population-based design of our study.     

Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India

We report the clinical features and molecular characterization of 23 patients with cyanosis due to NADH‐cytochrome b5 reductase (NADH‐CYB5R) deficiency from India. The patients with type I recessive congenital methemoglobinemia (RCM) presented with mild to severe cyanosis only whereas patients with type II RCM had cyanosis associated with severe neurological impairment. Thirteen mutations were identified which included 11 missense mutations causing single amino acid changes (p.Arg49Trp, p.Arg58Gln, p.Pro145Ser, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, p.Ala179Thr, p.Thr238Met, and p.Val253Met), one stop codon mutation (p.Trp236X) and one splice‐site mutation (p.Gly76Ser). Seven of these mutations (p.Arg50Trp, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, and p.Thr238Met) were novel. Two mutations (p.Gly76Ser and p.Trp236X) were identified for the first time in the homozygous state globally causing type II RCM. We used the three‐dimensional (3D) structure of human erythrocyte NADH‐CYB5R to evaluate the protein structural context of the affected residues. Our data provides a rationale for the observed enzyme deficiency and contributes to a better understanding of the genotype–phenotype correlation in NADH‐CYB5R deficiency.     

New founder germline mutations of CDKN2A in melanoma-prone families and multiple primary melanoma development in a patient receiving levodopa treatment

Germline mutations in the CDKN2A gene have been shown to predispose individuals to cutaneous malignant melanoma. Here, we describe three melanoma-prone families and one isolated patient affected by multiple melanoma who carried a tandem germline mutation of CDKN2A at the nucleotide level, [c.339G>C;c.340C>T], [p.Leu113Leu;p.Pro114Ser]. We also describe three other melanoma-prone families that carried a missense germline CDKN2A mutation, c.167G>T, p.Ser56Ile. All these families and patients resided in southeast France. We analyzed six 9p21 markers where the CDKN2A gene is located and found that carrier haplotypes for both mutations were consistent with two respective common founder ancestors. In one family, we identified two fourth-degree relatives homozygous for the Ser56Ile mutation, indicating a possible consanguinity. Furthermore, we observed that a carrier of the founder CDKN2A [p.Leu113Leu;p.Pro114Ser] mutation as well as two MC1R moderate-risk variants, [p.Arg151Cys(+)p.Arg163Gln] developed 22 primary melanomas in the three years that followed initiation of levodopa therapy for Parkinson's disease. This observation suggests that there is a need for reconsideration of the hypothesis that levodopa may play a role in melanoma development, at least when in the context of a high-risk genetic background.     

Functional consequences and structural interpretation of mutations of human choline acetyltransferase

Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyzes synthesis of acetylcholine from acetyl-CoA (AcCoA) and choline in cholinergic neurons. Mutations in CHAT cause potentially lethal congenital myasthenic syndromes associated with episodic apnea (ChAT-CMS). Here, we analyze the functional consequences of 12 missense and one nonsense mutations of CHAT in 11 patients. Nine of the mutations are novel. We examine expression of the recombinant missense mutants in Bosc 23 cells, determine their kinetic properties and thermal stability, and interpret the functional effects of 11 mutations in the context of the atomic structural model of human ChAT. Five mutations (p.Trp421Ser, p.Ser498Pro, p.Thr553Asn, p.Ala557Thr, and p.Ser572Trp) reduce enzyme expression to less than 50% of wild-type. Mutations with severe kinetic effects are located in the active-site tunnel (p.Met202Arg, p.Thr553Asn, and p.Ala557Thr) or adjacent to the substrate binding site (p.Ser572Trp), or exert their effect allosterically (p.Trp421Ser and p.Ile689Ser). Two mutations with milder kinetic effects (p.Val136Met and p.Ala235Thr) are also predicted to act allosterically. One mutation (p.Thr608Asn) below the nucleotide binding site of CoA enhances dissociation of AcCoA from the enzyme-substrate complex. Two mutations introducing a proline residue into an α-helix (p.Ser498Pro and p.Ser704Pro) impair the thermal stability of ChAT.     

Mutation screening of the CDKN2A promoter in melanoma families

Germline mutations of CDKN2A, at 9p21, are responsible for predisposition to melanoma in some families. However, evidence of linkage to 9p21 has been demonstrated in a significant proportion of kindreds with no detectable mutations in CDKN2A. It is possible that mutations in noncoding regions may be responsible for predisposition to melanoma in these families. We have analyzed approximately 1 kb of the CDKN2A promoter upstream of the start codon in an attempt to identify causal mutations in 107 melanoma families. Four sequence variants were detected. Two of these (A-191G and A-493T) did not segregate with disease and were present in a control population at a comparable frequency, indicating that they are unlikely to predispose to melanoma. The A-493T variant appeared to be in linkage disequilibrium with the previously described CDKN2A polymorphism Ala148Thr. The variant G-735A was detected in the control population, but segregation of this variant with melanoma within families could not be discounted. The fourth variant (G-34T), located in the 5' UTR, creates an aberrant initiation codon. This variant appeared to segregate with melanoma and was not detected in a control population. G-34T has recently been identified in a subset of Canadian melanoma families and was concluded to be associated with predisposition to melanoma. The creation of an aberrant initiation site in the 5' UTR may have an important role in carcinogenesis in a small percentage of families; however, mutations in the CDKN2A promoter appear to have a limited role in predisposition to melanoma.     

A large Norwegian family with inherited malignant melanoma, multiple atypical nevi, and CDK4 mutation

Mutations in two loci encoding cell-cycle-regulatory proteins have been shown to cause familial malignant melanoma. About 20% of melanoma-prone families bear a mutation in the CDKN2A locus, which encodes two unrelated proteins, p16INK4A and p14ARF. Mutations in the other locus, CDK4, are much rarer and have been linked to the disease in only three families worldwide. In the 1960s, a large Norwegian pedigree with multiple atypical nevi and malignant melanomas was identified. Subsequently, six generations and more than 100 family members were traced and 20 cases of melanoma verified. In this article, we report that CDK4 codon 24 is mutated from CGT to CAT (Arg24His) in this unusually large melanoma kindred. Intriguingly, one of the family members had ocular melanoma, but the CDK4 mutation could not be detected in archival tissue samples from this subject. Thus, the case of ocular melanoma in this family was sporadic, suggesting an etiology different from that of the skin tumors. The CDK4 mutation in the Norwegian family was identical to that in melanoma families in France, Australia, and England. Haplotype analysis using microsatellite markers flanking the CDK4 gene and single-nucleotide polymorphisms within the gene did not support the possibility that there was a common founder, but rather indicated at least two independent mutational events. All CDK4 melanoma families known to date have a substitution of amino acid 24. In addition to resulting from selection pressure, this observation may be explained by the CG dinucleotide of codon 24 representing a mutational hot spot in the CDK4 gene.     

Intronic sequence variants of the CDKN2A gene in melanoma pedigrees

Germ-line mutations of the tumor-suppressor gene CDKN2A predispose individuals to melanoma in families worldwide. However, coding mutations of CDKN2A have not been detected in a significant proportion of those affected. The identification of a disease-associated intronic mutation of CDKN2A in UK families, which has proved to be the most common CDKN2A mutation as yet identified in this population, has highlighted the possibility that additional causal mutations may lie within the intronic sequence of the gene. In this article, we describe the comprehensive screening of 109 English and 26 Australian melanoma pedigrees for intronic mutations of CDKN2A. In total, 24 sequence variants were identified across the two introns of the gene. We show evidence that two of the CDKN2A intronic variants (IVS1 + 1104 C > A and IVS1 - 1104 C > G) predispose to melanoma. IVS1 + 1104 was shown to result in the aberrant splicing of both p16(INK4a) and p14(ARF) mRNA. Overall, however, the proportion of English melanoma families with these variants is small.     

Mutations in EDAR account for one-quarter of non-ED1-related hypohidrotic ectodermal dysplasia

Hypohidrotic ectodermal dysplasia (HED) is characterized by abnormal development of the eccrine sweat glands, hair, and teeth. The X-linked form of the disease, caused by mutations in the ED1 gene, represents the majority of HED cases. Autosomal-dominant and -recessive forms occur occasionally and result from mutations in at least two genes: EDAR and EDARADD. These different forms are phenotypically indistinguishable. To better assess the implication of the EDAR gene in HED, we screened for mutations in 37 unrelated HED families or sporadic cases with no detected mutations in the ED1 gene. We identified 11 different mutations, nine of which are novel variants, in two familial and seven sporadic cases. Seven of the 11 are recessive mutations (c.140G>A (p.Cys47Tyr), c.266G>A (p.Arg89His), c.329A>C (p.Asp110Ala), c.442T>C (p.Cys148Arg), c.1208C>T (p.Thr403Met), c.1302G>T (p.Trp434Cys) and c.528+1G>A), and the other four are probably dominant (c.1129C>T (p.Leu377Phe), c.1237A>C (p.Thr413Pro), c.1253T>C (p.Ile418Thr), and c.1259G>A (p.Arg420Gln)). Our study demonstrates that EDAR is implicated in about 25% of non-ED1 HED, and may account for both autosomal-dominant and -recessive forms. The correlation between the nature and location of EDAR mutations and their mode of inheritance is discussed. A genotype-phenotype relationship was evaluated, since such data could be helpful for genetic counseling.     

Five novel somatic CDKN2/p16 mutations identified in melanoma, glioma and carcinoma of the pancreas. Mutations in brief no. 170. Online

Germ-line mutations in the p16/CDKN2 gene are known to predispose to melanoma. This gene belongs to a family of cyclin-dependent kinase inhibitors and blocks G1-S progression. The occurrence of p16/CDKN2 germline mutations in 12 Icelandic melanoma kindreds (kindreds with two or more cases of melanoma or melanoma, pancreas and/or glioma cases) was examined. No germ-line mutation was found, however five mutations not previously discribed in solid tumours were identified, Pro48Leu, Ala57Val, Gly89Asp, Leu117Met, Tyr129Stop.     

Prevalence of p16 and CDK4 germline mutations in 48 melanoma-prone families in France. The French Familial Melanoma Study Group [published erratum appears in Hum Mol Genet 1998 May;7(5):941]

Germline mutations in the p16 and CDK4 genes have been reported in a subset of melanoma pedigrees, but their prevalence is not well known. We searched for such germline mutations in 48 French melanoma-prone families selected according to two major criteria: families with at least three affected members (n = 20) or families with two affected members, one of them affected before the age of 50 (n = 28), and one additional minor criterion. Sixteen different p16 germline mutations were found in 21 families, while one germline mutation, Arg24His, was detected in the CDK4 gene. The frequency of p16 gene mutation in our sample (44%) is among the highest rates yet reported and the CDK4 mutation is the second mutation detected in this gene worldwide. In summary, our results show frequent involvement of the p16 gene in familial melanoma and confirm the role of the CDK4 gene as a melanoma- predisposing gene.      

Identification and functional analysis of novel variants of the human melanocortin 1 receptor found in melanoma patients

The melanocortin 1 receptor, a Gs protein‐coupled receptor expressed in epidermal melanocytes, is a major determinant of skin pigmentation and phototype and an important contributor to melanoma risk. MC1R activation stimulates synthesis of black, strongly photoprotective eumelanin pigments. Several MC1R alleles are associated with red hair, fair skin, increased sensitivity to ultraviolet radiation, and increased skin cancer risk. The MC1R gene is highly polymorphic, but only a few naturally occurring alleles have been functionally characterized, which complicates the establishment of accurate correlations between the signaling properties of mutant alleles and defined cutaneous phenotypes. We report the functional characterization of six MC1R alleles found in Spanish melanoma patients. Two variants (c.152T>C, p.Val51Ala and c.865T>C, p.Cys289Arg) have never been described, and the others (c.112G>A, p.Val38Met; c.122C>T, p.Ser41Phe; c.383T>C, p.Met128Thr; and c.842A>G, p.Asn281Ser) have not been analyzed for function. p.Asn281Ser corresponds to a functionally silent polymorphism. The other mutations are associated with varying degrees of loss of function (LOF), from moderate decreases in coupling to the cAMP pathway (p.Val38Met and p.Val51Ala) to nearly complete absence of functional coupling (p.Ser41Phe, p.Met128Thr, and p.Cys289Arg). The LOF p.Met128Thr and p.Cys289Arg mutants are trafficked to the cell surface, but are unable to bind agonists efficiently. Conversely, LOF of p.Val38Met, p.Ser41Phe, and p.Val51Ala is due to reduced cell surface expression as a consequence of retention in the endoplasmic reticulum (ER). Therefore, LOF of MC1R alleles is frequently associated with aberrant forward trafficking and accumulation within the ER or with inability to bind properly the activatory ligand. Hum Mutat 30:1–12, 2009. © 2009 Wiley‐Liss, Inc.     

Mutation analysis of the PLOD1 gene: an efficient multistep approach to the molecular diagnosis of the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA)

The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA) is an inheritable connective tissue disorder characterized by a deficiency of lysyl hydroxylase due to mutations in PLOD1. We describe a mutation analysis strategy for the PLOD1 gene using either cDNA or gDNA or a combination thereof, which allows for reliable, time-effective and efficient mutation detection in patients with EDS VIA. We report the results obtained in 9 index patients from 12 unrelated families: three patients were homozygous for three novel mutations (p.Ile454IlefsX2, p.Ala667Thr, and p.His706Arg), four patients were homozygous for the common duplication of exons 10-16, one patient was compound heterozygous for the common duplication and p.Ile454IlefsX2, and one patient was homozygous for p.Arg319X.     

CDKN2A mutations in Spanish cutaneous malignant melanoma families and patients with multiple melanomas and other neoplasia

The CDKN2A gene has been implicated in cutaneous malignant melanoma (CMM) in about 40% of families with linkage to chromosome 9p21, while a small proportion of families have mutations in the CDK4 gene. In order to estimate the importance of these genes in the predisposition to CMM in Spanish families and patients we have analysed, by SSCA, a total of 56 subjects belonging to 34 CMM families, and nine patients with multiple CMM and other neoplasia. We have detected germline CDKN2A mutations in six out of the 34 families (17%). A frameshift mutation (358delG) and four missense mutations (G59V, G101W (two cases), D84Y, and R87W) were identified. Five CMM patients from different families (14%) carried the A148T variant, which is known not to affect p16 activity. No mutations were detected in the patients with multiple CMM or other neoplasms. We have not found mutations either in exon 1 beta of the CDKN2A gene or in exon 2A of CDK4. Linkage analysis of the 9p21 region showed exclusion for one of the families for CMM and for four families for CMM/dysplastic naevi. This study indicates a small role for CDKN2A in Spanish CMM families and suggests that other genes are also responsible for CMM predisposition.     

Allelic heterogeneity of the carbohydrate sulfotransferase-6 gene in patients with macular corneal dystrophy

Allelic heterogeneity of the carbohydrate sulfotransferase-6 gene in patients with macular corneal dystrophy.Macular corneal dystrophy (MCD) is an autosomal recessive disorder characterized by grayish white opacities in the cornea. It is caused by mutations in the carbohydrate sulfotransferase-6 (CHST6) gene, which codes for the enzyme corneal N-acetylglucosamine-6-sulfotransferase. This enzyme catalyzes the sulfation of keratan sulfate, an important component of corneal proteoglycans. We screened 31 patients from 26 families with MCD for mutations in the coding region of the CHST6 gene. Twenty-six different mutations were identified, of which 14 mutations are novel. The novel mutations are one nonsense mutation found in one patient (Trp2Ter), one frameshift (insertion plus deletion) mutation in two patients (His335fs), and 12 missense mutations (Leu3Met, Ser54Phe, Val56Arg, Ala73Thr, Ser98Leu, Cys165Trp, Ser167Phe, Phe178Cys, Leu193Pro, Pro204Arg, Arg272Ser, and Arg334Cys) in 11 patients. These data demonstrate a high degree of allelic heterogeneity of the CHST6 gene in patient populations with MCD from Southern India, where this disease may have a relatively higher prevalence than in outbred communities.     

High prevalence of the G101W germline mutation in the CDKN2A (P16(ink4a)) gene in 62 Italian malignant melanoma families.

CDKN2A germline mutation frequency estimates are commonly based on families with several melanoma cases. When we started counseling in a research setting on gene susceptibility analysis in northern and central Italy, however, we mostly found small families with few cases. Here we briefly characterize those kindred, estimate CDKN2A/CDK4 mutation test yields, and provide indications on the possibility of implementing formal DNA testing for melanoma-prone families in Italy. In September 1995 we started genetic counseling in a research setting at our Medical Genetics Center. Screening for CDKN2A/CDK4 mutations was performed on families with two melanoma patients, one of whom was younger than 50 years at onset, the other complying with one of the following: 1) being a first-degree relative, 2) having an additional relative with pancreatic cancer, or 3) having multiple primary melanomas. Sixty-two of 67 (80%) melanoma cases met our criteria. Four previously described CDKN2A mutations (G101W, R24P, V126D, and N71S) were found in 21 of the 62 families (34%) with a high prevalence of G101W (18/21). The percentage of families with two melanoma cases/family harboring a mutation was low (7%, 2/27), but rose to 45% (9/20) if one of the melanoma patients carried multiple melanomas or if pancreatic cancer was present in that family. In the 15 families with three melanoma cases the presence of a mutation was higher (67%, 10/15) and reached 100% in the 4 families with four or more melanoma cases. Our results suggest that CDKN2A/CDK4 counseling-based mutational analysis may be reasonably efficient also for families with two melanoma cases, if one patient carries multiple melanomas or if pancreatic cancer is present in the family.     

CDKN2A (P16(INK4a)) and CDK4 mutation analysis in 131 Australian melanoma probands: effect of family history and multiple primary melanomas.

Mutation analysis of two genes involved in melanoma susceptibility (CDKN2A/p16(INK4a) and CDK4) was undertaken in 131 probands with a family history of melanoma. Screening of all three exons of CDKN2A and exon 2 of CDK4 by single-strand conformation polymorphism (SSCP) analysis and/or direct sequencing identified a total of 10 different CDKN2A germline mutations, including 6 not previously described in the germline. All but one has been previously proven to, or is likely to, affect the structure and function of p16(INK4a). The incidence of CDKN2A mutation was 8.4% (11/131), but was significantly higher in families with three or more cases of melanoma (10/66, 15.1%) than in those in which only two relatives were affected (1/65, 1.5%). The incidence of CDKN2A mutation was also higher in families with three or more cases of melanoma and at least one member with multiple primary melanomas (6/19, 31.6%) than in similar families without multiple primary melanomas (4/47, 8.5%). One novel CDK4 variant of uncertain significance was found in a kindred that also carries a CDKN2A mutation. Genes Chromosomes Cancer 25:339-348, 1999.     

Fifteen novel mutations in PKLR associated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK

Pyruvate kinase (PK) deficiency is a rare disease but an important cause of hereditary nonspherocytic hemolytic anemia. The disease is caused by mutations in the PKLR gene and shows a marked variability in clinical expression. We report on the molecular characterization of 38 PK‐deficient patients from 35 unrelated families. Twenty‐nine different PKLR mutations were detected, of which 15 are reported here for the first time. Two novel deletions are reported: c.142_159del18 is the largest in‐frame deletion described thus far and predicts the loss of six consecutive amino acids (p.Thr48_Thr53del) in the N‐terminal domain of red blood cell PK. The other deletion removes nearly 1.5 kb of genomic DNA sequence (c.1618+37_2064del1477) and is one of a few large deletional mutants in PKLR. In addition, 13 novel point mutations were identified: one nonsense mutant, p.Arg488X, and 12 missense mutations, predicting the substitution of a single amino acid: p.Arg40Trp, p.Leu73Pro, p.Ile90Asn, p.Gly111Arg, p.Ala154Thr, p.Arg163Leu, p.Gly165Val, p.Leu272Val, p.Ile310Asn, p.Val320Leu, p.Gly358Glu, and p.Leu374Pro. We used the three‐dimensional (3D) structure of recombinant human tetrameric PK to evaluate the protein structural context of the affected residues. In addition, in selected patients red blood cell PK antigen levels were measured by enzyme‐linked immunosorbent assay (ELISA). Collectively, the results provided us with a rationale for the observed enzyme deficiency and contribute to both a better understanding of the genotype‐to‐phenotype correlation in PK deficiency as well as the enzyme's structure and function. Hum Mutat 0, 1–8, 2008. © 2008 Wiley‐Liss, Inc.     

Functional characterization of missense variants in the creatine transporter gene (SLC6A8): improved diagnostic application

Creatine transporter deficiency is an X-linked mental retardation disorder caused by mutations in the creatine transporter gene (SLC6A8). So far, 20 mutations in the SLC6A8 gene have been described. We have developed a diagnostic assay to test creatine uptake in fibroblasts. Additionally, we expanded the assay to characterize novel SLC6A8 missense variants. A total of 13 variants were introduced in the SLC6A8 cDNA by site-directed mutagenesis. All variants were transiently transfected in SLC6A8-deficient fibroblasts and tested for restoration of creatine uptake in deficient primary fibroblasts. Thus, we proved that nine variants (p.Gly87Arg, p.Phe107del, p.Tyr317X, p.Asn336del, p.Cys337Trp, p.Ile347del, p.Pro390Leu, p.Arg391Trp, and p.Pro554Leu) are pathogenic mutations and four variants (p.Lys4Arg, p.Gly26Arg, p.Met560Val, and p.Val629Ile) are nonpathogenic. The present study provides an improved diagnostic tool to classify sequence variants of unknown significance.     

Mutational analysis of the N-ras, p53, p16INK4a, CDK4, and MC1R genes in human congenital melanocytic naevi

Eighteen human congenital melanocytic naevi (CMN) from 17 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 and for sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis. In addition, all lesions were screened for deletions and point mutations in the tumour suppressor genes p53 and p16INK4a (CDKN2A) by combined multiplex PCR/SSCP analysis. Positive screening data were specified by sequencing of the corresponding PCR product. Activating point mutations in the N-ras gene (nine CAA (Gln) to AAA (Lys) transversions and one CAA (Gln) to CGA (Arg) transition at codon 61) were detected at high frequency (56%). Furthermore, three missense mutations (V92M) and two silent mutations (CGA (Arg) to CGG (Arg), codon 213, exon 6) were found in the MC1R and p53 genes, respectively. No mutations were found in p16 or CDK4. The activated N-ras oncogene, which is also found in human cutaneous melanomas, may constitute a potential risk factor for melanoma formation within CMN.