Background: CYP2C9 polymorphisms are associated with decreased S-warfarin clearance and lower maintenance dosage. Decreased expression of VKORC1 resulting from the ؊1639G>A substitution has also been implicated in lower warfarin dose requirements. We investigated the additional contribution of this polymorphism to the variance in warfarin dose. Methods: Sixty-five patients with stable anticoagulation were genotyped for CYP2C9 and VKORC1 with Tag-It™ allele-specific primer extension technology. Plasma Swarfarin concentrations and warfarin maintenance dose were compared among patients on the basis of the VKORC1 ؊1639G>A genotype. Results: Eighty percent of CYP2C9*1/*1 patients stabilized on <4.0 mg/day warfarin had at least 1 VKORC1 ؊1639A allele. Mean warfarin doses (SD) were 6.7 (3.3), 4.3 (2.2), and 2.7 (1.2) mg/day for patients with the VKORC1 ؊1639GG, GA, and AA genotypes, respectively. Steady-state plasma concentrations of S-warfarin were lowest in patients with the VKORC1 ؊1639AA genotype and demonstrated a positive association with the VKORC1 ؊1639G allele copy number (trend P ‫؍‬ 0.012). A model including VKORC1 and CYP2C9 geno-

Background: Heritable alterations in CDKN2A account for a subset of familial melanoma cases although no robust method exists to identify those at risk of being a mutation carrier. Methods: We set out to construct a model for estimating CDKN2A mutation carrier probability using a cohort of 116 consecutive familial cutaneous melanoma patients evaluated at Massachusetts General Hospital Pigmented Lesion Center between April 2001 and September 2004. Germline CDKN2A and CDK4 status on the familial melanoma cases and clinical features associated with mutational status were then used to build a multiple logistic regression model to predict carrier probability and performance of model on external validation. Results: From the 116 kindreds prone to melanoma in the Boston area, 13 CDKN2A mutation carriers were identified and 12 were subsequently used in the modeling. Proband age at diagnosis, number of proband primaries, and number of additional family primaries were most closely associated with germline mutations. The estimated probability of the proband being a mutation carrier based on the logistic

Coding mutations of the CDKN2A gene on chromosome 9p21 cosegregate with 25-60% of familial melanoma cases, but there remains a number of 9p21-linked kindreds that lack germline coding mutations of CDKN2A. We sequenced CDKN2A exons 1a, 2, 3, and the adjacent intronic regions in 167 melanoma-prone families (at least two affected first-degree relatives), and detected four splice site variations, three of which cosegregate with the disease. RT-PCR experiments verified that these three variants, including an AGgt to ATgt mutation that demonstrates a founder effect, do affect splicing. While an exon 1a splice donor site mutation incompletely abolishes splicing, the correctly spliced mRNA yields a protein (Q50P) that cannot effectively interact with CDK4. We also performed RT-PCR on mRNA from 16 melanoma-prone kindreds to search for cryptic splice sites deep within introns, but identified no splice variants. Meanwhile, we screened 139 affected families using allelespecific PCR for the recently discovered IVS2À105A4G mutation, but found only one family that possesses this alteration. We conclude that splice site mutations do predispose to disease in a subset of melanoma-prone kindreds. Characterization of additional splice site variants and other noncoding alterations of CDKN2A should allow us to detect a wider range of mutations in atrisk patients.

One of the most common melanoma-related CDKN2A mutations reported in North America is the V126D mutation. We examined nine markers surrounding CDKN2A in three American and four Canadian families carrying the V126D mutation. All seven families had a haplotype consistent with a common ancestor/founder for this mutation. In addition, the mutation appears to have originated 34–52 generations ago (1-LOD-unit support interval 13–98 generations). © 2001 Cancer Research Campaign http:///www.bjcancer.com

Background The presence of recurrent high-risk mutations in CDKN2A and CDK4 among melanoma-prone kindreds suggests that a high-throughput, multiplex assay could serve as an effective initial screening tool. Moreover, with the emergence of new melanoma risk single nucleotide polymorphisms (SNPs) through genome-wide association studies, a flexible platform that can easily accommodate these new risk alleles is needed for more accurate genetic risk profiling. To this end, we have developed a novel melanoma-associated mutation detection method using a multiplex bead-based assay. This assay is suitable for high-throughput CDKN2A and CDK4 genotyping and can be eventually adapted to multiple loci across various constituent populations. Methods Genomic DNA from a 1603 subjects (1005 in training set, 598 in validation set) were amplified by multiplex PCR using five primer sets followed by multiplex allele-specific primer extension for 39 different known germline variants. The products were then sorted on an xMAP™ (formerly Tag-It™) array and detected by use of the Luminex xMAP™ system. Genotypes were compared to previously-determined sequence data. Results In the Toronto training cohort, variants were detected in 145 samples, giving complete concordance between the bead assay and direct sequencing results. Analysis of the 598 samples from the GenoMEL validation set led to identification of 150/155 expected variants (96.77% concordance). Overall, the bead assay correctly genotyped 1540/1603 (96.07%) of all individuals in the study and 1540/1545 (99.68%) of individuals whose mutations were represented in the probe set. Out of a total of 62,512 SNP calls, 62,517 (99.99%) were correctly assigned. Conclusions In this initial evaluation, the multiplex bead-based assay for familial melanoma appears to be a highly accurate method for genotyping CDKN2A and CDK4 variants.

In contrast to the damage caused by far-UV, the damage caused by UVA (320-400 nm) is largely oxygen dependent, suggesting near-UV-mediated DNA damage involves reactive oxygen species. The DNA repair enzymes that recognize oxidized bases may, therefore, be an important part of the cell's near-UV defense repertoire. To evaluate the relative importance of Fpg (Fapy) glycosylase (an enzyme known to remove oxidized bases) and the DNA damage-inducible UvrABC excinuclease in recovery from near-UV-induced stress, we have constructed fpg- and uvrA- derivatives of Escherichia coli and tested the response (survival) of these strains to both UVA and far-UV radiation. Relative to control strains, the fpg- derivatives were found to be consistently more sensitive to the lethal effects of UVA, but not far-UV radiation. In contrast, uvrA- mutants were more sensitive than control strains to both UVA and far-UV radiation. Thymine dimers, known to be produced by far-UV and corrected by UvrABC, were not generated by the UVA fluences used in this study, suggesting that some other UVA-induced lesion(s) is recognized and repaired by this excinuclease.

Germline mutations in genes encoding several components of the retinoblastoma pathway have been linked with inherited predisposition to melanoma. Most commonly, such mutations involve CDKN2A, a cyclindependant kinase inhibitor of two kinases, CDK4 and CDK6, which phosphorylate the retinoblastoma protein (pRB) and thereby promote passage through the G 1 /S cell-cycle restriction point. Less frequently, germline mutations in the CDK4 gene have also been linked with an increased risk of melanoma. Despite the sequence and functional homology between CDK4 and CDK6, the role of germline mutations in CDK6 in melanoma predisposition is unknown. We detected no CDK6 mutations within the p16 (CDKN2A) binding domain in index cases from 60 melanoma-prone kindreds that lacked germline mutations in the coding regions of either CDKN2A or within the entire CDK4 coding region. We conclude that germline mutations in CDK6 do not make a signi®cant contribution to melanoma predisposition. Oncogene (2000) 19, 1849 ± 1852. Keywords: familial melanoma; CDK6Between 8 and 12% of melanoma cases arise in families with a genetic predisposition to the disease (Tucker and Goldstein, 1995). In a subset of these kindreds, germline coding mutations of the CDKN2A gene (on human chromosome 9p21) co-segregate with cases of melanoma (Hussussian et al., 1994;Walker et al., 1995;Dracopoli and Fountain, 1996;FitzGerald et al., 1996; NL and DH, unpublished). The CDKN2A gene product ± designated p16 ± is a cyclin-dependent kinase (CDK) inhibitor (CDKI). p16 plays a key regulatory role in the retinoblastoma (Rb) pathway at the G 1 /S cell-cycle restriction point by inhibiting the phosphorylation of Rb via CDK4/6 (Kato et al., 1993;Weinberg, 1995;Ruas and Peters, 1998). The genetic basis for melanoma in families and individuals that lack coding mutations of CDKN2A is not well understood at this time. Possible explanations include non-coding mutations of CDKN2A; alterations in other genes located at 9p21; and mutations of other major or minor melanoma predisposition genes located elsewhere in the human genome.Genetic alterations of two other members of the Rb pathway in addition to CDKN2A have been linked with an increased risk of melanoma. Individuals harbouring germline mutations in the Rb gene and who have retinoblastoma as children show an increased risk of melanoma as adults (Draper et al., 1986;Traboulsi et al., 1988;Eng et al., 1993;Bataille et al., 1995). In addition, three melanoma prone families have been reported to possess germline mutations in the CDK4 gene that cosegregate with the disease. Two of these families possess a cysteine rather than the wild-type arginine at amino acid 24 (R24C; Zuo et al., 1996). More recently, an additional mutation at position 24 (R24H) was identi®ed in a single French kindred (Sou®r et al., 1998). Moreover, the R24C (and presumably the R24H) mutant protein has a reduced a nity for p16, resulting in relaxation of control at the G1/S restriction point. CDK6 shows considerable amino acid homology with CDK4 ( Figure...

MLK-3 kinase is a widely expressed serine/ threonine kinase that bears multiple protein interaction domains and regulates signals mediated by the stress-responsive pathway. Thus, MLK-3 signaling affects numerous cellular processes, raising the possibility that MLK-3 might play a role in oncogenesis. In this report, we describe the fine mapping of the MLK-3 gene within the 11q13.1 chromosomal region. By integrating data from somatic cell hybrids and double color fluorescence in situ hybridization on metaphase chromosomes and DNA fibers. MLK-3 has been assigned approximately 1 Mb telomeric of PYGM, close to the D11S546 locus. Since the MEN1 susceptibility locus is also located within the 11q13.1 region, we have carried out Southern and Northern blot analyses, as well as protein truncation assays to establish whether abnormalities in MLK-3 lead to the development of this familial cancer syndrome. Our observations exclude MLK-3 as the MEN1 gene.