Mutation analysis of the CDKN2A promoter in Australian melanoma families.

Approximately 50% of all melanoma families worldwide show linkage to 9p21-22, but only about half of these have been shown to contain germ line CDKN2A mutations. It has been hypothesized that a proportion of these families carry mutations in the noncoding regions of CDKN2A. Several Canadian families have been reported to carry a mutation in the 5' UTR, at position -34 relative to the start site, which gives rise to a novel AUG translation initiation codon that markedly decreases translation from the wild-type AUG (Liu et al., 1999). Haplotype sharing in these Canadian families suggested that this mutation is of British origin. We sequenced 1,327 base pairs (bp) of CDKN2A, making up 1,116 bp of the 5' UTR and promoter, all of exon 1, and 61 bp of intron 1, in at least one melanoma case from 110 Australian families with three or more affected members known not to carry mutations within the p16 coding region. In addition, 431 bp upstream of the start codon was sequenced in an additional 253 affected probands from two-case melanoma families for which the CDKN2A mutation status was unknown. Several known polymorphisms at positions -33, -191, -493, and -735 were detected, in addition to four novel variants at positions 120, -252, -347, and -981 relative to the start codon. One of the probands from a two-case family was found to have the previously reported Q50R mutation. No family member was found to carry the mutation at position -34 or any other disease-associated mutation. For further investigation of noncoding CDKN2A mutations that may affect transcription, allele-specific expression analysis was carried out in 31 of the families with at least three affected members who showed either complete or "indeterminate" 9p haplotype sharing without CDKN2A exonic mutations. Reverse transcription polymerase chain reaction and automated sequencing showed expression of both CDKN2A alleles in all family members tested. The lack of CDKN2A promoter mutations and the absence of transcriptional silencing in the germ line of this cohort of families suggest that mutations in the promoter and 5' UTR play a very limited role in melanoma predisposition.

Second stage of a genome scan of schizophrenia: study of five positive regions in an expanded sample.

In a previous genome scan of 43 schizophrenia pedigrees, nonparametric linkage (NPL) scores with empirically derived pointwise P-values less than 0.01 were observed in two regions (chromosomes 2q12-13 and 10q23) and less than 0.05 in three regions (4q22-23, 9q22, and 11q21). Markers with a mean spacing of about 5 cM were typed in these regions in an expanded sample of 71 pedigrees, and NPL analyses carried out. No region produced significant genomewide evidence for linkage. On chromosome 10q, the empirical P-value remained at less than 0.01 for the entire sample (D10S168), evidence in the original 43 pedigrees was slightly increased, and a broad peak of positive results was observed. P-values less than 0.05 were observed on chromosomes 2q (D2S436) and 4q (D4S2623), but not on chromosomes 9q or 11q. It is concluded that this sample is most supportive of linkage on chromosome 10q, with less consistent support on chromosomes 2q and 4q. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:864-869, 2000.

Genome scan of schizophrenia.

OBJECTIVE: The goal of this study was to identify chromosomal regions likely to contain schizophrenia susceptibility genes. METHOD: A genomewide map of 310 microsatellite DNA markers with average spacing of 11 centimorgans was genotyped in 269 individuals--126 of them with schizophrenia-related psychoses--from 43 pedigrees. Nonparametric linkage analysis was used to assess the pattern of allele sharing at each marker locus relative to the presence of disease. RESULTS: Nonparametric linkage scores did not reach a genomewide level of statistical significance for any marker. There were five chromosomal regions in which empirically derived p values reached nominal levels of significance at eight marker locations. There were p values less than 0.01 at chromosomes 2q (with the peak value in this region at D2S410) and 10q (D10S1239), and there were p values less than 0.05 at chromosomes 4q (D4S2623), 9q (D9S257), and 11q (D11S2002). CONCLUSIONS: The results do not support the hypothesis that a single gene causes a large increase in the risk of schizophrenia. The sample (like most others being studied for psychiatric disorders) has limited power to detect genes of small effect or those that are determinants of risk in a small proportion of families. All of the most positive results could be due to chance, or some could reflect weak linkage (genes of small effect). Multicenter studies may be useful in the effort to identify chromosomal regions most likely to contain schizophrenia susceptibility genes.

CDKN2A/p16 is inactivated in most melanoma cell lines.

The CDKN2A gene maps to chromosome 9p21-22 and is responsible for melanoma susceptibility in some families. Its product, p16, binds specifically to CDK4 and CDK6 in vitro and in vivo, inhibiting their kinase activity. CDKN2A is homozygously deleted or mutated in a large proportion of tumor cell lines and some primary tumors, including melanomas. The aim of this study was to investigate the involvement of CDKN2A and elucidate the mechanisms of p16 inactivation in a panel of 60 cell lines derived from sporadic melanomas. Twenty-six (43%) of the melanoma lines were homozygously deleted for CDKN2A, and an additional 15 (25%) lines carried missense, nonsense, or frameshift mutations. All but one of the latter group were shown by microsatellite analysis to be hemizygous for the region of 9p surrounding CDKN2A. p16 was detected by Western blotting in only five of the cell lines carrying mutations. Immunoprecipitation of p16 in these lines, followed by Western blotting to detect the coprecipitation of CDK4 and CDK6, revealed that p16 was functionally compromised in all cell lines but the one that carried a heterozygous CDKN2A mutation. In the remaining 19 lines that carried wild-type CDKN2A alleles, Western blot analysis and immunoprecipitation indicated that 11 cell lines expressed a wild-type protein. Northern blotting was performed on the remaining eight cell lines and revealed that one cell line carried an aberrantly sized RNA transcript, and two other cell lines failed to express RNA. The promoter was found to be methylated in five cell lines that expressed CDKN2A transcript but not p16. Presumably, the message seen by Northern blotting in these cell lines is the result of cross-hybridization of the total cDNA probe with the exon 1beta transcript. Microsatellite analysis revealed that the majority of these cell lines were hemi/homozygous for the region surrounding CDKN2A, indicating that the wild-type allele had been lost. In the 11 cell lines that expressed functional p16, microsatellite analysis revealed loss of heterozygosity at the markers immediately surrounding CDKN2A in five cases, and the previously characterized R24C mutation of CDK4 was identified in one of the remaining 6 lines. These data indicate that 55 of 60 (92%) melanoma cell lines demonstrated some aberration of CDKN2A or CDK4, thus suggesting that this pathway is a primary genetic target in melanoma development.

A linkage study of schizophrenia to markers within Xp11 near the MAOB gene.

A sex chromosome locus for psychosis has been considered on the basis of some sex differences in genetic risk and expression of illness, and an association with X-chromosome anomalies. Previous molecular genetic studies produced weak evidence for linkage of schizophrenia to the proximal short arm of the X-chromosome, while some other regions were not ruled out. Here we report an attempt to expand the Xp findings in: (i) a multicenter collaboration focusing on 92 families with a maternal pattern of inheritance (Study I), and (ii) an independent sample of 34 families unselected for parental mode of transmission (Study II). In the multicenter study, a parametric analysis resulted in positive lod scores (highest of 1.97 for dominant and 1.19 for recessive inheritance at a theta of 0.20) for locus DXS7, with scores below 0.50 for other markers in this region (MAOB, DXS228, and ARAF1). Significant allele sharing among affected sibling pairs was present at DXS7. In the second study, positive lod scores were observed at MAOB (highest of 2.16 at a theta of 0.05 for dominant and 1.64 at a theta of 0.00 for recessive models) and ALAS2 (the highest of 1.36 at a theta of 0.05 for a recessive model), with significant allele sharing (P = 0.003 and 0.01, respectively) at these two loci. These five markers are mapped within a small region of Xp11. Thus, although substantial regions of the X-chromosome have been investigated without evidence for linkage being found, a locus predisposing to schizophrenia in the proximal short arm of the X-chromosome is not excluded.

Analysis of the CDKN2A, CDKN2B and CDK4 genes in 48 Australian melanoma kindreds.

Germline mutations within the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene and one of its targets, the cyclin dependent kinase 4 (CDK4) gene, have been identified in a proportion of melanoma kindreds. In the case of CDK4, only one specific mutation, resulting in the substitution of a cysteine for an arginine at codon 24 (R24C), has been found to be associated with melanoma. We have previously reported the identification of germline CDKN2A mutations in 7/18 Australian melanoma kindreds and the absence of the R24C CDK4 mutation in 21 families lacking evidence of a CDKN2A mutation. The current study represents an expansion of these efforts and includes a total of 48 melanoma families from Australia. All of these families have now been screened for mutations within CDKN2A and CDK4, as well as for mutations within the CDKN2A homolog and 9p21 neighbor, the CDKN2B gene, and the alternative exon 1 (E1beta) of CDKN2A. Families lacking CDKN2A mutations, but positive for a polymorphism(s) within this gene, were further evaluated to determine if their disease was associated with transcriptional silencing of one CDKN2A allele. Overall, CDKN2A mutations were detected in 3/30 (10%) of the new kindreds. Two of these mutations have been observed previously: a 24 bp duplication at the 5' end of the gene and a G to C transversion in exon 2 resulting in an M531 substitution. A novel G to A transition in exon 2, resulting in a D108N substitution was also detected. Combined with our previous findings, we have now detected germline CDKN2A mutations in 10/48 (21%) of our melanoma kindreds. In none of the 'CDKN2A-negative' families was melanoma found to segregate with either an untranscribed CDKN2A allele, an R24C CDK4 mutation, a CDKN2B mutation, or an E1beta mutation. The last three observations suggest that these other cell cycle control genes (or alternative gene products) are either not involved at all, or to any great extent, in melanoma predisposition.

Confirmation of susceptibility locus on chromosome 13 in Australian breast cancer families.

Two major genes determining predisposition to breast cancer, termed BRCA1 and BRCA2, have been mapped to the long arms of chromosomes 17 and 13, respectively. Each locus is believed to account for approximately 40% of cases of familial breast cancer. We used linkage and haplotype analysis with simple tandem repeat polymorphisms at chromosomal bands 17q21 and 13q12 to determine the contribution of the BRCA1 and BRCA2 genes to predisposition to breast cancer in four Australian breast cancer kindreds, one of which had two male cousins with breast cancer. Surprisingly all families segregated a haplotype of markers on 13q and showed positive lod scores supporting linkage to BRCA2. In addition, haplotype analysis identified an informative recombination between D13S260 and D13S171 in one affected individual, which refines the localisation of BRCA2 to between D13S260 and D13S267; a distance of 2-3 cM. Tumours of the stomach and cervix, as well as melanoma and leukaemia/lymphoma also occur in these pedigrees but the numbers are too low to determine whether they may be significantly associated with BRCA2 carrier status. Our results confirm the existence of BRCA2 on the long arm of chromosome 13 and support previous findings that this locus is likely to confer risk in families with affected males. Furthermore, our observations suggest that the BRCA2 gene may also contribute to the development of other neoplasma.

Deletion mapping of the short arm of chromosome 3 in Merkel cell carcinoma.

Little is known about the biology of Merkel cell carcinoma (MCC), also called small cell carcinoma of the skin. MCC has similarities with small cell lung cancer (SCLC): both are neuroendocrine malignancies with early metastasis to distant sites and a poor prognosis. Small cell lung cancer biopsies are known to have frequent losses on chromosome 3 in the region 3p21, yet MCCs have not been reported to have 3p deletions by karyotypic analysis. Considering the similarities between SCLC and MCC, we investigated 26 MCC tumours for loss of heterozygosity (LOH) on 3p. First, RFLP analysis was performed using PCR with nine primer sets from six loci. Second, 25 tumours were examined by microsatellite analysis for 3p markers D3S1289 and D3S1285 and SST on 3q. All 26 tumours were informative at one or more loci; of these, 18 (69%) demonstrated LOH for at least one marker on the short arm. For all informative loci the frequency of LOH was greater than 30% (range 33-75%). In a cell line derived from one tumour, it was possible to demonstrate rearrangement of chromosome 3 by in situ hybridisation. No LOH was seen in 15 informative cases for the 3q locus SST. A region 3p13-p21.1, centered on the marker D3S2, was deleted in all tumours demonstrating LOH, with a secondary deletion involving D3S30 detected in some tumours at 3p13. Our results indicate that LOH on 3p is a common occurrence in MCC; however, three tumours for which DNA was also available from a corresponding cell line suggest there may be a subset of MCC whose genesis is independent of deletions of 3p.

A genetic model of melanoma tumorigenesis based on allelic losses.

Previous karyotypic studies have indicated a possible series of non-random chromosomal events involved in progression of melanoma. We sought to verify and augment this model of melanocyte tumorigenesis by studying allelic deletions of markers mapping to these regions in 30 matched pairs of melanoma and constitutional DNA samples. Polymorphic loci on chromosomes 1, 7, 10, 11, 17, and 21 were analyzed and data combined with those previously obtained for chromosome arms 6q and 9p in the same series of tumours. The most frequent and earliest deletions were found on 9p (57%) and 10q (32%). With the exception of one case, no sample had loss of markers on another chromosome without concomitant loss of markers on 9p or 10q. Losses on 6q were also a frequent (31%) and early event whereas losses of loci on distal 1p (22%) or 11q (26%) occurred only in metastatic melanomas. A "background" rate (0-17%) of allele loss was seen on chromosomes 7, 17, and 21. These data strongly support the previous model based on karyotypic findings in melanocytic lesions. However, we have been able to further, augment that model by delimiting the regions of loss on 10q, to that distal to D10S254, and on 1p, to between D1S243 and D1S160.

Linkage analysis in familial melanoma kindreds to markers on chromosome 6p.

Malignant melanoma occurs as a familial cancer in 5%-10% of cases, where it segregates in a manner consistent with autosomal dominant inheritance. Evidence from cytogenetics, fine mapping studies of deletions in melanomas and recent linkage studies supports the location of a human melanoma predisposition gene on the short arm of chromosome 9. Evidence also exists for a melanoma gene on Ip, indicating genetic heterogeneity for melanoma predisposition. Previous studies have also reported findings suggestive of linkage of some melanoma families to the HLA region on the short arm of chromosome 6 (6p), indicating the possibility of even greater heterogeneity. To further define the possible effect of a gene within the HLA region on melanoma susceptibility, we have typed 7 simple tandem repeat polymorphisms (STRPs) from 6p in 16 Australian melanoma kindreds. Maximum 2-point LOD scores ranged from 1.13 (theta = 0.2) to 2.03 (theta = 0.15) for 4 contiguous markers flanking the HLA complex, and multi-point analysis gave a peak LOD score of 1.64, 24 centimorgans telomeric to D6S109. However, extended haplotype analysis of these markers showed that a region between D6S105 and HLAF segregated with melanoma in 5/16 families. These results are surprising given that the same cohort of families has previously been shown to be linked to chromosome 9. One interpretation of the current findings is that melanoma susceptibility in some families may result from a gene mapping within the HLA region of chromosome 6p.

Multiple endocrine neoplasia type 1 (MEN1) in two Asian families.

Multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disease characterized by neoplasia of the parathyroid glands, anterior pituitary and endocrine pancreas, is rarely reported in Asian populations. The MEN1 gene, mapped to chromosome 11q13 but yet to be cloned, has been found to be homogeneous in Caucasian populations through linkage analysis. Here, two previously unreported Asian kindreds with MEN1 are described; linkage analysis using microsatellite polymorphic markers in the MEN1 region was carried out. The first kindred, of Mongolian-Chinese origin, is a multigeneration family with over 150 living members, eight of whom are affected to date. The second kindred is of Chinese origin consisting of four affected members. Linkage to chromosome 11q13 was confirmed in both kindreds, supporting evidence for genetic homogeneity. A recombination in the larger kindred localizes the gene distal to marker D11S956, consistent with its placement from previous studies. We also show that it is feasible to use these markers for predictive testing, as four gene carriers were detected in 13 family members with unknown disease status in the first kindred.

Simple tandem repeat allelic deletions confirm the preferential loss of distal chromosome 6q in melanoma.

Karyotypic analysis, loss of somatic heterozygosity, microcell fusion and cDNA transfection studies have provided compelling evidence that at least one tumour suppressor gene for melanoma resides on chromosome 6. In an attempt to further define the regions to which these putative suppressor genes map, we have carried out loss of heterozygosity (LOH) studies on DNA from 25 fresh melanoma tumours for 9 simple tandem repeat (STR) polymorphism markers spanning chromosome 6. Four samples displayed LOH or homozygosity for all markers studied, indicating that they had lost one homologue of chromosome 6. An additional 3 samples showed LOH for all markers on 6q. Furthermore, 30 melanoma cell lines, for which there were no matching somatic DNA samples, were analyzed for hemizygosity of markers on 6q. One cell line had a homozygous deletion of all markers tested and a further 12 cell lines displayed only one allele for 3 or 4 contiguous markers, indicating that most, if not all of these samples were hemizygous for the region of 6q distal to D6S87. Overall, the rate of LOH on 6q in the 55 melanoma DNAs was 35%, and there were no losses of markers on 6p without concomitant loss of markers on 6q. Two of 5 samples derived from primary melanomas showed LOH, which indicates that LOH for the melanoma suppressor gene on 6q, which maps to a region that contains the SOD2 locus, is a frequent and early event in melanoma tumorigenesis.

The MLLT3 gene maps between D9S156 and D9S171 and contains an unstable polymorphic trinucleotide repeat.

MLLT3, one of the genes shown to be a translocation breakpoint partner for the acute lymphocytic leukemia (MLL) gene, has been mapped to 9p22. We have identified a polymorphic trinucleotide repeat within this gene that shows somatic instability. The inheritance pattern of this polymorphism in recombinant individuals from families previously typed for other chromosome 9 markers indicates that the gene lies in the interval bounded by D9S156 and D9S171.

Refined localization of the melanoma (MLM) gene on chromosome 9p by analysis of allelic deletions.

Various lines of evidence including linkage analysis, frequent homozygous and heterozygous deletions in melanoma DNAs, and the finding of a patient with multiple primary melanomas who harbours a 5p/9p translocation involving loss of several 9p markers, have indicated that the 9p22-p13 region harbours a gene important for the development of melanoma (MLM). We have used eight short tandem repeat polymorphism (STRP) markers mapping to this region to look for allelic losses in DNA from melanoma biopsies and cell lines. Heterozygous losses were found in 8/14 (57%) fresh melanoma biopsy DNAs with the smallest region of overlap (SRO) being between IFNA and D9S169. In addition, when DNA from 30 melanoma cell lines was studied, four cell lines (13%) were found to be homozygously deleted for various 9p markers. Two of these cell lines define the borders of overlapping homozygous deletions within a 4cM region of 9p21 between IFNA and D9S171. Moreover, a further 14 melanoma cell lines were hemizygous for the IFNA/D9S171/D9S126 region. These data support the hypothesis that the MLM gene acts as a tumour suppressor, and provide a refinement of its localization on 9p.

Haplotype analysis limits the position of the familial melanoma locus on 9p to the D9S169-D9S156 interval.

A gene for familial melanoma (MLM) has been mapped to 9p22-p13 by linkage analysis using simple tandem repeat polymorphisms (STRPs) at the IFNA and D9S126 loci. This localization is consistent with the finding of homozygous deletions of these markers in DNA from two melanoma cell lines, which suggest that the locus has the properties of a tumour suppressor gene. In an attempt to further define the position of the MLM locus we have typed 10 STRPs from the short arm of chromosome 9 in 15 Australian melanoma kindreds. Extended haplotype analysis of these markers and identification of recombinants in our pedigrees indicate that the MLM gene is flanked on the centromeric side by D9S169 and on the telomeric side by D9S156. These results limit the location of the MLM locus to an interval of about 16 centimorgans.

Confirmation of chromosome 9p linkage in familial melanoma.

Malignant melanoma occurs as a familial cancer in 5%-10% of cases where it segregates in a manner consistent with autosomal dominant inheritance. Evidence from cytogenetics, fine-mapping studies of deletions in melanomas, and recent linkage studies supports the location of a human melanoma predisposition gene on the short arm of chromosome 9. We have carried out linkage analysis using the 9p markers IFNA and D9S126 in 26 Australian melanoma kindreds. Multipoint analysis gave a peak lod score of 4.43, 15 cM centromeric to D9S126, although a lod score of 4.13 was also found 15 cM telomeric of IFNA. These data confirm the existence of a melanoma susceptibility gene on 9p and indicate that this locus most probably lies outside of the IFNA-D9S126 interval. No significant heterogeneity was found between families, when either pairwise or multipoint data were analyzed using HOMOG.

Linkage mapping of melanoma (MLM) using 172 microsatellite markers.

The incidence of malignant melanoma is currently increasing faster than any other cancer and in 5-12% of cases occurs in a familial context in which the disease cosegregates as an autosomal dominant trait. To identify the location of genes that predipose individuals to familial melanoma (MLM), we have carried out linkage analysis in three large Australian melanoma pedigrees using 172 microsatellite markers spread across all autosomes. Three additional smaller families were typed for 70 of the same markers. In five of the six families we found lod scores between 1.0 and 2.3, which may provide evidence for the location of melanoma genes in proximity to some of these markers. If this turns out to be the case, these data potentially demonstrate that MLM is genetically heterogeneous since there was no marker for which all families gave significantly high LODs. These data provide the foundation for an exclusion map for melanoma and, more importantly, high-light areas of the genome for others to substantiate the potential positions of some of the genes that may be responsible for susceptibility to MLM.