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.     

Molecular and phenotypic characteristics of seven novel mutations causing branched‐chain organic acidurias

Specific mitochondrial enzymatic deficiencies in the catabolism of branched‐chain amino acids cause methylmalonic aciduria (MMA), propionic acidemia (PA) and maple syrup urine disease (MSUD). Disease‐causing mutations were identified in nine unrelated branched‐chain organic acidurias (BCOA) patients. We detected eight previously described mutations: p.Asn219Tyr, p.Arg369His p.Val553Glyfs*17 in MUT, p.Thr198Serfs*6 in MMAA, p.Ile144_Leu181del in PCCB, p.Gly288Valfs*11, p.Tyr438Asn in BCKDHA and p.Ala137Val in BCKDHB gene. Interestingly, we identified seven novel genetic variants: p.Leu549Pro, p.Glu564*, p.Leu641Pro in MUT, p.Tyr206Cys in PCCB, p.His194Arg, p.Val298Met in BCKDHA and p.Glu286_Met290del in BCKDHB gene. In silico and/or eukaryotic expression studies confirmed pathogenic effect of all novel genetic variants. Aberrant enzymes p.Leu549Pro MUT, p.Leu641Pro MUT and p.Tyr206Cys PCCB did not show residual activity in activity assays. In addition, activity of MUT enzymes was not rescued in the presence of vitamin B12 precursor in vitro which was in accordance with non‐responsiveness or partial responsiveness of patients to vitamin B12 therapy. Our study brings the first molecular genetic data and detailed phenotypic characteristics for MMA, PA and MSUD patients for Serbia and the whole South‐Eastern European region. Therefore, our study contributes to the better understanding of molecular landscape of BCOA in Europe and to general knowledge on genotype–phenotype correlation for these rare diseases.     

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.     

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.     

Identification of novel mutations in the human ornithine transcarbamylase (OTC) gene of Korean patients with OTC deficiency and transient expression of the mutant proteins in vitro

The urea cycle plays key roles to prevent the accumulation of toxic nitrogenous compound and synthesize arginine de novo. Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of urea cycle, which is inherited in an X-linked manner. This study was undertaken to characterize molecular defects in Korean patients with OTC deficiency. With direct sequence analysis of OTC gene of 26 unrelated Korean patients with OTC deficiency, 23 different mutations were identified. Among these mutations, eleven were novel mutations. The novel mutations were p.Leu9X, p.Arg26Pro, p.Gly100Arg, p.Met205Thr, p.Lys221Asn, p.Asp249Gly, p.Phe281Ser, p.Val323Met, c.571delC, c.853delC, and c.796-805del. All the novel mutations in this study were tested in 100 normal alleles. In vitro expression study of some of novel missense mutations elucidated the correlation of genotype and phenotype of the OTC deficiency.     

The spectrum of BRCA1 and BRCA2 pathogenic sequence variants in Middle Eastern,North African,and South European countries

BRCA1 BRCA2 mutational spectrum in the Middle East, North Africa, and Southern Europe is not well characterized. The unique history and cultural practices characterizing these regions, often involving consanguinity and inbreeding, plausibly led to the accumulation of population‐specific founder pathogenic sequence variants (PSVs). To determine recurring BRCA PSVs in these locales, a search in PUBMED, EMBASE, BIC, and CIMBA was carried out combined with outreach to researchers from the relevant countries for unpublished data. We identified 232 PSVs in BRCA1 and 239 in BRCA2 in 25 of 33 countries surveyed. Common PSVs that were detected in four or more countries were c.5266dup (p.Gln1756Profs), c.181T>G (p.Cys61Gly), c.68_69del (p.Glu23Valfs), c.5030_5033del (p.Thr1677Ilefs), c.4327C>T (p.Arg1443Ter), c.5251C>T (p.Arg1751Ter), c.1016dup (p.Val340Glyfs), c.3700_3704del (p.Val1234Glnfs), c.4065_4068del (p.Asn1355Lysfs), c.1504_1508del (p.Leu502Alafs), c.843_846del (p.Ser282Tyrfs), c.798_799del (p.Ser267Lysfs), and c.3607C>T (p.Arg1203Ter) in BRCA1 and c.2808_2811del (p.Ala938Profs), c.5722_5723del (p.Leu1908Argfs), c.9097dup (p.Thr3033Asnfs), c.1310_1313del (p. p.Lys437Ilefs), and c.5946del (p.Ser1982Argfs) for BRCA2. Notably, some mutations (e.g., p.Asn257Lysfs (c.771_775del)) were observed in unrelated populations. Thus, seemingly genotyping recurring BRCA PSVs in specific populations may provide first pass BRCA genotyping platform.     

Vacuolating megalencephalic leukoencephalopathy with subcortical cysts: functional studies of novel variants in MLC1

Nine new unrelated patients presenting vacuolating myelinopathy with subcortical cysts were identified and analyzed for variations in the MLC1 gene. We detected 12 mutations (p.Leu37fs, p.Met80Val, p.Leu83Phe, p.Pro92Ser, p.Ser93Leu, p.Ile108fs, p.Gly130Arg, p.Cys171fs, p.Glu202Lys, p.Ser269Tyr, p.Ala275Asn, and p.Leu310_311insLeu) of which nine were novel. In one patient we did not detect mutations. Using a heterologous system, three new missense variants (p.Glu202Lys, p.Ser269Tyr, and p.Ala275Asn) and a single leucine insertion (p.Leu310insLeu)--lying in a stretch of seven leucines--were functionally assayed by determining total protein levels and mutant protein expression at the plasma membrane. No correlation was observed between mutation, clinical features, and plasma membrane expression of mutant protein.     

Mutations in MFSD8/CLN7 are a frequent cause of variant‐late infantile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCL) are a group of genetically heterogeneous neurodegenerative disorders. The recent identification of the MFSD8/CLN7 gene in a variant‐late infantile form of NCL (v‐LINCL) in affected children from Turkey prompted us to examine the relative frequency of variants in this gene in Italian patients with v‐LINCL. We identified nine children harboring 11 different mutations in MFSD8/CLN7. Ten mutations were novel and included three nonsense (p.Arg35Stop, p.Glu381Stop, p.Arg482Stop), four missense (p.Met1Thr, p.Gly52Arg, p.Thr294Lys, p.Pro447Leu), two splice site mutations (c.863+3_4insT, c.863+1G>C), and a 17‐bp deletion predicting a frameshift and premature protein truncation (c.627_643del17/p.Met209IlefsX3). The clinical phenotype, which was similar to that of the Turkish v‐LINCL cases, was not influenced by type and location of the mutation nor the length of the predicted residual gene product. As well as identifying novel variants in MFSD8/CLN7, this study contributes to a better molecular characterization of Italian NCL cases, and will facilitate medical genetic counseling in such families. The existence of a subset of v‐LINCL cases without mutations in any of the known NCL genes suggests further genetic heterogeneity. © 2009 Wiley‐Liss, Inc.     

Ten novel mutations in VMD2 associated with Best macular dystrophy (BMD)

Mutations in the vitelliform macular dystrophy 2 (VMD2) gene encoding besrtophin are responsible for Best macular dystrophy (BMD), a juvenile-onset autosomal dominant disorder of the central retina. Here, we report ten novel VMD2 mutations identified in clinically diagnosed BMD patients. The heterozygous alterations include nine missense mutations (c.32A>T, c.76G>C, c.85T>C, c.122T>C, c.122T>C, c.310G>C, c.722C>A, c.880C>G, c.893T>C) resulting in amino acid changes (respectively: Asn11Ile, Gly26Arg, Tyr29His, Leu41Pro, Trp102Arg, Asp104His, Thr241Asn, Leu294Val and Phe298Ser) located within four previously defined hotspot regions of the gene. In addition, a silent exonic mutation (c.624G>A) was identified in a two generation BMD pedigree. To determine a possible pathogenic effect of this variant, the consequences on splicing behaviour and potential exonic splice enhancer (ESE) motifs were analyzed. Finally, a 1-bp deletion (c.779delC) resulting in a frameshift mutation (Pro260fsX288) was found in exon 7, representing the first case of a potential frameshift mutation that affects the N-terminal half of the VMD2 protein. Besides a dominant negative effect which is likely attributable to the identified missense mutations, the deletion mutation suggests haploinsufficiency as an infrequent disease-causing mechanism in BMD.     

Functional Characterization and Classification of Frequent Low‐Density Lipoprotein Receptor Variants

Familial hypercholesterolemia (FH) is an autosomal‐dominant disorder mostly caused by mutations in the low‐density lipoprotein receptor (LDLR) gene leading to increased risk for premature cardiovascular diseases. According to functional studies, LDLR mutations may be classified into five classes. The main objective of this study was to characterize seven LDLR variants previously detected in FH patients. Analysis by flow cytometry and confocal microscopy of LDLR activity demonstrate that all the studied variants are pathogenic. Among the mutations located in β‐propeller, p.Trp577Gly and p.Ile624del were classified as class 2, whereas p.Arg416Trp and p.Thr454Asn as class 5. p.Phe800Glyfs*129 (located in the cytoplasmic domain), p.Cys155Tyr (located in the binding domain), and p.Asn825Lys (inside FxNPxY motif) were classified as class 2, 3, and 4, respectively. The results also show that LDLR activity of these class 4 and 5 variants is not completely abolished, showing a milder phenotype. We have also determined that statin response is more efficient lowering total cholesterol in heterozygous patients carrying p.Ile624del (class 2) compared with p.Arg416Trp and p.Thr454Asn (class 5) variants. In conclusion, these findings emphasize the importance of characterizing LDLR pathogenic variants to provide an indisputable FH diagnosis and to gain insight into the statin response depending on the LDLR class mutation.     

Screening for mutations in the peripheral myelin genes PMP22, MPZ and Cx32 (GJB1) in Russian Charcot-Marie-Tooth neuropathy patients

Charcot-Marie-Tooth disease (CMT) and related inherited peripheral neuropathies, including Dejerine-Sottas syndrome, congenital hypomyelination, and hereditary neuropathy with liability to pressure palsies (HNPP), are caused by mutations in three myelin genes: PMP22, MPZ and Cx32 (GJB1). The most common mutations are the 1.5 Mb CMT1A tandem duplication on chromosome 17p11.2-p12 in CMT1 patients and the reciprocal 1.5 Mb deletion in HNPP patients. We performed a mutation screening in 174 unrelated CMT patients and three HNPP families of Russian origin. The unrelated CMT patients included 108 clinically and electrophysiologically diagnosed CMT1 cases, 32 CMT2 cases, and 34 cases with unspecified CMT. Fifty-nine CMT1A duplications were found, of which 58 belonged to the CMT1 patient group. We found twelve distinct mutations in Cx32, six mutations in MPZ, and two mutations in PMP22. Of these respectively, eight, five, and two lead to a CMT1 phenotype. Eight mutations (Cx32: Ile20Asn/Gly21Ser, Met34Lys, Leu90Val, and Phe193Leu; MPZ: Asp134Gly, Lys138Asn, and Thr139Asn; PMP22: ValSer25-26del) were not reported previously. Phenotype-genotype correlations were based on nerve conduction velocity studies and mutation type.     

Mutations of APC and MYH in unrelated Italian patients with adenomatous polyposis coli

The analysis of APC and MYH mutations in adenomatous polyposis coli patients should provide clues about the genetic heterogeneity of the syndrome in human populations. The entire coding region and intron-exon borders of the APC and MYH genes were analyzed in 60 unrelated Italian adenomatous polyposis coli patients. APC analysis revealed 26 point mutations leading to premature termination, one missense variant and one deletion spanning the entire coding region in 32 unrelated patients. Novel truncating point mutations included c.1176_1177insT (p.His393_PhefsX396), c.1354_1355del (p.Val452_SerfsX458), c.2684C>A (p.Ser895X), c.2711_2712del (p.Arg904_LysfsX910), c.2758_2759del (p.Asp920_CysfsX922), c.4192_4193del (p.Ser1398_SerfsX1407), c.4717G>T (p.Glu1573X) and a novel cryptic APC exon 6 splice site. MYH analysis revealed nine different germline variants in nine patients, of whom five were homozygotes or compound heterozygotes. The mutations included 4 novel MYH missense variants (c.692G>A, p.Arg231His; c.778C>T, p.Arg260Trp; c.1121T>C, p.Leu374Pro; and c.1234C>T, p.Arg412Cys) affecting conserved amino acid residues in the ENDO3c or NUDIX domains of the protein and one novel synonymous change (c.672C>T, p.Asn224Asn). Genotype-phenotype correlations were found in carriers of APC mutations but not in carriers of biallelic MYH mutations, except for a negative correlation with low number of polyps. A distinctive characteristic of patients negative for APC and MYH mutations was a significantly (p<0.0001) older age at diagnosis compared to patients with APC mutations. Moreover, the proportion of cases with an attenuated polyposis phenotype was higher (p = 0.0008) among patients negative for APC and MYH mutations than among carriers of APC or biallelic MYH mutations.     

Inhibitory influence of amino acids on secretagogues induced exocrine secretion in isolated perfused rat pancreas

Influence of amino acids upon pancreatic exocrine secretion has been investigated in the isolated perfused pancreas of rats. Arg produced significant and dose-related inhibition of pancreatic juice flow, protein output and amylase output evoked by CCK-PZ (1.25 pM). The secretory response evoked by CCK-PZ was inhibited by other amino acids (Ala, Asp, Asn, Gly, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Val, in each 20 mM). A similar inhibitory pattern was observed using 10 mixed amino acids of 2 mM each (Pro, Phe, Thr, Met, Lys, Asp, Leu, Trp, Val, Gly). Gly at a concentration of 20 mM produced significant inhibition of exocrine secretion evoked by ACh (50 nM) or GRP (36 pM). The inhibitory response induced by amino acids could not be repeated by using exogenous insulin (1 microM) and glucagon (280 nM). The inhibitory response was also not changed by increased extracellular Ca (5 or 10 mM). However, Gly (20 mM) produced inhibition of exocrine secretion evoked by Ca reintroduction into a pancreas which was pretreated with A 23187. It was suggested that the inhibitory effects of some amino acids on exocrine secretion are mainly caused by suppression of Ca influx in a stimulus-secretion coupling process.     

The analysis of myotonia congenita mutations discloses functional clusters of amino acids within the CBS2 domain and the C‐terminal peptide of the ClC‐1 channel

Myotonia congenita (MC) is a skeletal‐muscle hyperexcitability disorder caused by loss‐of‐function mutations in the ClC‐1 chloride channel. Mutations are scattered over the entire sequence of the channel protein, with more than 30 mutations located in the poorly characterized cytosolic C‐terminal domain. In this study, we characterized, through patch clamp, seven ClC‐1 mutations identified in patients affected by MC of various severities and located in the C‐terminal region. The p.Val829Met, p.Thr832Ile, p.Val851Met, p.Gly859Val, and p.Leu861Pro mutations reside in the CBS2 domain, while p.Pro883Thr and p.Val947Glu are in the C‐terminal peptide. We showed that the functional properties of mutant channels correlated with the clinical phenotypes of affected individuals. In addition, we defined clusters of ClC‐1 mutations within CBS2 and C‐terminal peptide subdomains that share the same functional defect: mutations between 829 and 835 residues and in residue 883 induced an alteration of voltage dependence, mutations between 851 and 859 residues, and in residue 947 induced a reduction of chloride currents, whereas mutations on 861 residue showed no obvious change in ClC‐1 function. This study improves our understanding of the mechanisms underlying MC, sheds light on the role of the C‐terminal region in ClC‐1 function, and provides information to develop new antimyotonic drugs.     

Identification and characterization of three novel mutations in the CASQ1 gene in four patients with tubular aggregate myopathy

Here, we report the identification of three novel missense mutations in the calsequestrin‐1 (CASQ1) gene in four patients with tubular aggregate myopathy. These CASQ1 mutations affect conserved amino acids in position 44 (p.(Asp44Asn)), 103 (p.(Gly103Asp)), and 385 (p.(Ile385Thr)). Functional studies, based on turbidity and dynamic light scattering measurements at increasing Ca²⁺ concentrations, showed a reduced Ca²⁺‐dependent aggregation for the CASQ1 protein containing p.Asp44Asn and p.Gly103Asp mutations and a slight increase in Ca²⁺‐dependent aggregation for the p.Ile385Thr. Accordingly, limited trypsin proteolysis assay showed that p.Asp44Asn and p.Gly103Asp were more susceptible to trypsin cleavage in the presence of Ca²⁺ in comparison with WT and p.Ile385Thr. Analysis of single muscle fibers of a patient carrying the p.Gly103Asp mutation showed a significant reduction in response to caffeine stimulation, compared with normal control fibers. Expression of CASQ1 mutations in eukaryotic cells revealed a reduced ability of all these CASQ1 mutants to store Ca²⁺ and a reduced inhibitory effect of p.Ile385Thr and p.Asp44Asn on store operated Ca²⁺ entry. These results widen the spectrum of skeletal muscle diseases associated with CASQ1 and indicate that these mutations affect properties critical for correct Ca²⁺ handling in skeletal muscle fibers.     

Amino-terminal sequence of baboon type C virus p30.

The NH₂-terminal amino acid sequence of a baboon Type C virus p30 was determined to be: ProLeuArgThrValAsnAgrThrValGlnTyrTrp XPhe. This is identical, assuming that X = Pro, to the terminal 14 residues of the endogenous feline virus, RD 114, p30,     

Identification of eight novel glucokinase mutations in Italian children with maturity-onset diabetes of the young

Maturity-onset diabetes of the young (MODY) is a clinically heterogeneous group of disorders characterized by early onset non-insulin-dependent diabetes mellitus, autosomal dominant inheritance, and primary defect in the function of the beta cells of the pancreas. Mutations in the glucokinase (GCK) gene account for 8%-56% of MODY, with the highest prevalences being found in the southern Europe. While screening for GCK mutations in 28 MODY families of Italian origin, we identified 17 different mutations (corresponding to 61% prevalence), including eight previously undescribed ones. The novel sequence variants included five missense mutations (p.Lys161Asn c.483G>C in exon 4, p.Phe171Leu c.511T>C in exon 5 and p.Thr228Ala c.682A>G, p.Thr228Arg c.683C>G, p.Gly258Cys c.772G>T in exon 7), one nonsense mutation (p.Ser383Ter c.1148C>A in exon 9), the splice site variant c.1253+1G>T in intron 9, and the deletion of 12 nucleotides in exon 10 (p.Ser433_Ile436del c.1298_1309del12). Our study indicates that mutations in the GCK/MODY2 gene are a very common cause of MODY in the Italian population and broadens our knowledge of the naturally occurring GCK mutation repertoire.     

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.     

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.