Screening of germline mutations in the CDKN2A and CDKN2B genes in Swedish families with hereditary cutaneous melanoma

BACKGROUND: Approximately 10% of human cutaneous melanomas occur in families in which several members are affected. The familial predisposition to this disease is often associated with dysplastic nevus syndrome, a condition in which afflicted family members have multiple dysplastic nevi (atypical moles). The chromosome region 9p21 and markers on chromosomes 1p and 6p have been linked to melanoma susceptibility. The tumor suppressor genes CDKN2A and CDKN2B have been mapped to the 9p21 region, and genetic analyses have revealed the presence of germline CDKN2A alterations in melanoma families. The reported frequencies of such alterations, however, vary among these families. PURPOSE: The present investigation was carried out to determine the frequencies of CDKN2A and CDKN2B germline gene mutations among members in a population-based cohort of Swedish melanoma families (i.e., melanoma kindreds). METHODS: DNA was prepared from blood samples obtained from 181 individuals belonging to 100 melanoma kindreds. The polymerase chain reaction (PCR) technique, followed by single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses, were used to identify the types and frequencies of mutations in exons 1, 1beta, 2, and 3 of the CDKN2A gene and in exons 1 and 2 of the CDKN2B gene. RESULTS: CDKN2A gene aberrations were independently identified by both SSCP and nucleotide-sequence analyses. Nucleotide-sequence analysis identified a single point mutation leading to a substitution of leucine for proline in codon 48 of exon 1 in a family with a history of melanoma and several other cancers. A second abnormality, leading to an insertion of an extra arginine residue at codon number 113 of exon 2, was seen in four separate families. The CDKN2A exon-3 coding region had the wild-type sequence in all samples. No germline mutations were found in the alternative exon 1beta of the CDKN2A gene or in exons 1 and 2 of the CDKN2B gene. CONCLUSIONS: The present investigation demonstrates that CDKN2A germline gene mutations were observed in 7.8% of the 64 Swedish melanoma kindreds that each included at least two first-degree relatives with melanoma and dysplastic nevus syndrome. No CDKN2A exon 1beta or CDKN2B mutations were identified. The critical genes responsible for the inheritance of a susceptibility to develop melanoma among family members in this population have yet to be identified.     

Haplotype analysis of two recurrent CDKN2A mutations in 10 melanoma families: evidence for common founders and independent mutations

Germ-line mutations in CDKN2A have been shown to predispose to cutaneous malignant melanoma. We have identified 2 new melanoma kindreds which carry a duplication of a 24bp repeat present in the 5' region of CDKN2A previously identified in melanoma families from Australia and the United States. This mutation has now been reported in 5 melanoma families from 3 continents: Europe, North America, and Australasia. The M53I mutation in exon 2 of CDKN2A has also been documented in 5 melanoma families from Australia and North America. The aim of this study was to determine whether the occurrence of the mutations in these families from geographically diverse populations represented mutation hotspots within CDKN2A or were due to common ancestors. Haplotypes of 11 microsatellite markers flanking CDKN2A were constructed in 5 families carrying the M53I mutation and 5 families carrying the 24bp duplication. There were some differences in the segregating haplotypes due primarily to recombinations and mutations within the short tandem-repeat markers; however, the data provide evidence to indicate that there were at least 3 independent 24bp duplication events and possibly only 1 original M53I mutation. This is the first study to date which indicates common founders in melanoma families from different continents.     

BRCA2 germline mutations in Swedish breast cancer families

Life-threatening situations in sarcoidosis are extremely rare. They may be due to failure of vital organs--lungs, heart, kidney, liver and brain--and usually due to irreversible fibrosis. Respiratory failure follows irreversible pulmonary fibrosis and the development of cor pulmonale. Cardiac sarcoidosis is more sinister for it may be silent, ill-recognised with sudden death or high morbidity. It needs sophisticated techniques to uncover this latent iceberg. Renal failure may be due to granulomatous interstitial nephritis and/or nephrocalcinosis. Hepatic failure is due to intrahepatic cholestasis, portal hypertension and bleeding oesophageal varices. Neurosarcoidosis carries a mortality of 10 per cent, over twice that of sarcoidosis overall. The treatment of each situation is discussed including organ transplantation.     

Analysis of the p16 gene, CDKN2, in 17 Australian melanoma kindreds

CDKN2 has been implicated as a melanoma susceptibility gene in some kindreds with a family history of this disease. Mutation analysis of CDKN2 in 17 familial melanoma Australian kindreds revealed a paucity of exon mutations and none of the previously described disease-related mutations. One novel germline mutation was found in exon one, Arg24Pro, which segregates with melanoma in 1/17 kindreds. Two previously described polymorphisms, Ala148Thr and a base change at nucleotide 540 were detected and one novel polymorphism in the untranslated region of exon 3 (nucleotide 580) was also found. Together with other recent reports, these findings provide support for CDKN2 as a susceptibility locus for familial melanoma but suggest that other loci are involved in some hereditary melanoma kindreds.     

Molecular characterization of germline mutations in neurofibromatosis 2 in two families

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder that predisposes patients to central nervous system tumors. It is caused by mutations in the NF2 tumor suppressor gene, which is located on chromosome 22q12. We studied 2 multigenerational NF2 families (three members of family 1 and the proband of the family) by gene mutation analysis and clinical assessment. One member of family 1 had a 169 C-->T point mutation at codon 57 of exon 2 and had a severe phenotype. His father had a silent 1113 C-->T point mutation at codon 371 of exon 11 and had a normal phenotype. The proband of family 2 had a deletion at nucleotide 720 G (codon 240) of exon 8. This led to a frameshift and termination at codon 250, and a severe NF2 phenotype. Our results indicate that clinical abnormalities can be present in carriers. Nonsense and frameshift mutations in the NF2 tumor suppressor gene are associated with phenotypes. The clinical abnormalities can develop at a young age.     

Mutations in the CDKN2A (p16INK4a) gene in microdissected sporadic primary melanomas

The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from alpha-D-glucose 1-phosphate and D-glucose were cloned from a basidiomycete, Grifola frondosa. Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns. Consistent with the novelty of TSase, there were no homologous proteins registered in the data-bases. Recombinant TSase with a histidine tag at the NH2-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G. frondosa strain. Incubation of alpha-D-glucose 1-phosphate and D-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that the equilibrium lay far in the direction of trehalose synthesis.     

Frequency of mutation and deletion of the tumor suppressor gene CDKN2A (MTS1/p16) in hepatocellular carcinoma from an Australian population

The tumor suppressor gene CDKN2A (MTS1/p16), located on chromosome 9p21, is inactivated in a variety of tumors including melanomas and tumors of the biliary tract, pancreas, and stomach. The aim of the present study was to determine whether this gene is inactivated in hepatocellular carcinoma (HCC). Twenty-three primary HCCs and four HCC cell lines were examined. Loss of heterozygosity (LOH) analysis was performed using eight polymorphic markers immediately surrounding CDKN2A, and showed a contiguous region of loss, with the two most commonly deleted markers being D9S1604, located between the p16 and p15 genes, at which 7 of 13 informative tumors (54%) showed loss, and D9S171, with 4 of 14 LOH (29%). Exons 1, 2, and 3 of CDKN2A were amplified by polymerase chain reaction to detect homozygous deletions, and single-strand conformation polymorphism (SSCP) analysis was performed to screen for mutations. No homozygous deletions were detected in any sample. SSCP and sequence analysis showed the same nucleotide change at codon 148 in four tumors. This has been reported elsewhere as a polymorphism. One of these four tumors also contained a mutation at codon 119, resulting in the substitution of an acidic amino acid for a basic one. It is concluded that CDKN2A is infrequently deleted or mutated in HCC. The region of allelic loss upstream from CDKN2A might result in inactivation of regulatory sequences important in the expression of this gene; alternatively, a second tumor suppressor gene may be present in the region 9p21-22, proximal to CDKN2A. These possibilities require further investigation.     

Astrocytomas and choroid plexus tumors in two families with identical p53 germline mutations

Germline p53 mutations carry an increased risk of development of breast cancer, soft tissue and osteosarcomas, brain tumors, leukemia and adrenocortical carcinomas. Cerebral neoplasms are usually of astrocytic lineage and occur in 40% of affected families. This report presents clinical, neuropathological and molecular genetic data from 2 families in France with an identical p53 germline mutation in codon 248 (CGG->TGG; Arg->Trp) and a clustering of CNS tumors. The youngest patient in each family developed a malignant choroid plexus tumor while several young adults of both kindred succumbed to low-grade astrocytoma, anaplastic astrocytoma or glioblastoma. The only non-neural neoplasm was an adrenocortical carcinoma in a boy aged 4 years who developed an anaplastic choroid plexus papilloma 2 years later. Of 2 previously reported inherited choroid plexus tumors, 1 occurred in a family which also carried a germline mutation in codon 248. It remains to be shown whether this unusual pattern of CNS tumors is due to an organ-specific effect of this particular p53 mutation or whether it reflects the genetic background of the affected families.     

p53 tumour suppressor gene mutations in benign prostatic hyperplasia and prostate cancer

OBJECTIVE: Seminal vesicle cysts combined with ipsilateral renal agenesis represent a rare urological anomaly. We searched the literature to review the clinical presentation, diagnosis and therapeutic treatment options of this anomaly. METHODS: A pooled analysis was performed of 52 cases of seminal vesicle cysts combined with ipsilateral renal agenesis, including our own observation. The evaluation included: patient age at diagnosis, race, laterality (R/L), presence of ureteral remnant in the cyst, presenting symptoms, diagnostic examinations, treatment and outcome. RESULTS: The mean age at diagnosis was 30.2 years. The majority presented in the 2nd, 3rd and 4th decade of their lives. Only 2 patients (4%) were of African origin, all others were Caucasians. The distribution R:L was 2:1. Ureteral remnants were present in 14 patients (27%). The most common symptoms were: dysuria (37%), frequency (33%), perineal pain (29%), epididymitis (27%), pain following ejaculation (21%) and scrotal pain (13%). Infertility was found in 9 patients (17%). The cyst was palpable by digital rectal examination in 79%. All patients underwent intravenous urography, and 88% underwent cystoscopy. Other frequently performed investigations are: ultrasonography (27%), CT scanning (27%), vasovesiculography (46%) and urethrocystography (23%). The final treatment was open surgery in 74%, aspiration in 6%, transurethral deroofing of the cyst in 6% and spontaneous rupture in 4%. In 6% no treatment was given and in 4% the treatment is unknown. All patients were free of symptoms after open exploration. The success rates after transurethral deroofing and aspiration were 75 and 30% respectively. CONCLUSION: Seminal vesicle cysts combined with ipsilateral renal agenesis are a rare urological anomaly, occurring in men in the 2nd to 4th decade of their life. They present with symptoms of bladder irritation and obstruction and with pain in the perineum and scrotum. Epididymitis is frequently found. The diagnostic work-up consists of a digital rectal examination, transrectal and abdominal ultrasonography, CT scan and a cystoscopy. Open surgery and transurethral deroofing of the cyst give excellent results (100 and 75% cure respectively). Aspiration of the cyst should only be used for diagnostic purposes.     

CDKN2A mutations in multiple primary melanomas

BACKGROUND: Germ-line mutations in the CDKN2A tumor-suppressor gene (also known as p16, p16INK4a, and MTS1) have been linked to the development of melanoma in some families with inherited melanoma. Whether mutations in CDKN2A confer a predisposition to sporadic (nonfamilial) melanoma is not known. In some patients with sporadic melanoma, one or more additional primary lesions develop, suggesting that some of these patients have an underlying genetic susceptibility to the cancer. We hypothesized that this predisposition might be due to germ-line CDKN2A mutations. METHODS: We used the polymerase chain reaction, single-strand conformation polymorphism analysis, and direct DNA sequencing to identify germ-line mutations in the CDKN2A gene in patients with multiple primary melanomas who did not have family histories of the disease. A quantitative yeast two-hybrid assay was used to evaluate the functional importance of the CDKN2A variants. RESULTS: Of 33 patients with multiple primary melanomas, 5 (15 percent; 95 percent confidence interval, 4 percent to 27 percent) had germ-line CDKN2A mutations. These included a 24-bp insertion at the 5' end of the coding sequence, three missense mutations (Arg24Pro, Met53Ile, and Ser56Ile), and a 2-bp deletion at position 307 to 308 (resulting in a truncated CDKN2A protein). In three families, CDKN2A mutations identical to those in the probands were found in other family members. In two families with mutations, we uncovered previously unknown evidence of family histories of melanoma. CONCLUSIONS: Some patients with multiple primary melanomas but without family histories of the disease have germ-line mutations of the CDKN2A gene. The presence of multiple primary melanomas may signal a genetic susceptibility to melanoma not only in the index patient but also in family members, who may benefit from melanoma-surveillance programs.     

Mutational analysis of CDKN2 (MTS1/p16ink4) in human breast carcinomas

The CDKN2 gene that encodes the cell cycle regulatory protein cyclin-dependent kinase-4 inhibitor (p16) has recently been mapped to chromosome 9p21. Frequent homozygous deletions of this gene have been documented in cell lines derived from different types of tumors, including breast tumors, suggesting that CDKN2 is a tumor suppressor gene involved in a wide variety of human cancers. To determine the frequency of CDKN2 mutations in breast carcinomas, we screened 37 primary tumors and 5 established breast tumor cell lines by single-strand conformation polymorphism analysis. In addition, Southern blot analysis was performed on a set of five primary breast carcinoma samples and five breast tumor cell lines. Two of the five tumor cell lines revealed a homozygous deletion of the CDKN2 gene, but no mutations were observed in any of the primary breast carcinomas. These results suggest that the mutation of the CDKN2 gene may not be a critical genetic change in the formation of primary breast carcinoma.     

Lack of germ-line mutations of CDK4, p16(INK4A), and p15(INK4B) in families with glioma

Gliomas are tumors of the central nervous system that may be inherited in some patients. The gene(s) responsible for the clustering of gliomas in families have not yet been identified. Molecular studies of sporadic high-grade gliomas have revealed mutations or deletions of the genes encoding the protein kinase inhibitors p16(INK4A) and p15(INK4B) in a large proportion of tumors. Moreover, those tumors without deletions frequently display gene amplification and/or over-expression of mRNA encoding the protein kinase cdk4. We hypothesized that germ-line mutations in the p16(INK4A), p15(INK4B), or CDK4 genes might contribute to some cases of familial gliomas. To address this issue, we analyzed 36 kindreds with a predisposition to glial tumors. Genomic DNA from index members of these families was screened by PCR-single-strand conformational polymorphism analysis. We did not detect any functional mutations in the p16(INK4A), p15(INK4B), or CDK4 genes, although two individuals did have a previously described A140T polymorphism in p16(INK4A). Thus, despite the association between the sporadic forms of high-grade glioma and abnormalities of p16(INK4A), p15(INK4B), or CDK4, we found no evidence that germ-line mutations in the coding region of these three genes predispose to inherited glial tumors.     

Frequency of homozygous deletion at p16/CDKN2 in primary human tumours

BACKGROUND: Carcinoma of the breast is characterized by a variable course with prognosis dependent on disease stage at presentation. Paradoxically, some patients with early malignancy demonstrate disease progression within a short time. The role of tumor markers in the management of carcinoma of the breast is controversial. While CA15-3 is the most widely used tumor marker in carcinoma of the breast, its role in the management of patients with early disease is controversial. STUDY DESIGN: Since 1986, all patients presenting to our unit with carcinoma of the breast have had serial CA15-3 levels measured. This study evaluates the role of serial CA15-3 levels in the management of a consecutive series of 168 patients with Stage I disease at presentation. RESULTS: The mean preoperative CA15-3 levels at presentation were significantly elevated in patients with Stage I disease compared with patients with benign disease. Sixteen patients had either locoregional (five patients) or metastatic recurrence (11 patients). CA15-3 levels were not elevated in patients with locoregional disease and were significantly elevated in patients with bony metastases and gave a mean lead time of 6.3 months over bone scintigraphy. CONCLUSIONS: Serial CA15-3 measurements are an efficient and cost-effective method of monitoring disease progression and have advantages over conventional investigations in patients with early carcinoma of the breast.     

p16 (CDKN2/MTS1) gene deletions are rare in prostatic carcinomas in the United States and Japan

PURPOSE: The incidence of clinically apparent prostatic carcinoma is much higher in the United States than in Japan. Alterations in the p16 tumor suppressor gene have been identified in various tumor types, including cultured prostatic carcinoma cell lines. We studied the possible deletions of either exon 2 or 3 of this gene in primary clinical prostatic carcinomas from Japan and the United States. MATERIALS AND METHODS: Genomic DNA was extracted from 36 formalin-fixed, paraffin-embedded clinical prostatic carcinomas from Japan and 27 carcinomas from the United States. Exons 2 and 3 of the p16 gene were amplified using comparative multiplex polymerase chain reactions (PCR) and then analyzed for possible deletions of either exon. RESULTS: Two out of 36 (5.6%) carcinomas from Japan clearly demonstrated deletion of p16 exon 2, but this deletion was not detected in any of the 27 carcinomas from the United States. CONCLUSIONS: Although slightly higher in Japan than in the United States, the frequency of p16 exon deletions in clinical prostatic carcinomas is very low, and probably is not important in the development of this neoplasm.     

Homozygous deletions of methylthioadenosine phosphorylase (MTAP) are more frequent than p16INK4A (CDKN2) homozygous deletions in primary non-small cell lung cancers (NSCLC)

Homozygous deletions of the tumor suppressor gene p16INK4A and deficiency of methylthioadenosine phosphorylase (MTAP), both located on chromosome 9p21, have been independently reported in non-small cell lung cancer (NSCLC). To determine the frequency of co-deletion of these two genes, we investigated 50 samples of primary NSCLC using a quantitative PCR-ELISA. All specimens were fixed in formalin, paraffin embedded and stored until assayed. Histologic subtypes included 25 adenocarcinomas (50%), 21 squamous cell carcinomas (42%) and four large cell carcinomas (8%). Homozygous deletions of MTAP exon 8 could be detected in 19 of 50 NSCLC samples (38%). Adenocarcinoma (11 of 25, 44%) showed a higher deletion frequency than squamous cell carcinoma (six of 21, 29%). In contrast, homozygous p16INK4A deletions were detected in only nine of 50 (18%) samples using specific primers for p16INK4A exon 1alpha. No difference between the histological subtypes and p16INK4A deletion frequency was observed. We further investigated the ten samples with MTAP deletions but intact p16INK4A exon 1alpha with primers specific for p16INK4A exon 3, the exon nearest to MTAP exon 8. Interestingly, none of the ten samples had deletion of the p16INK4A exon 3 coding region. Fine mapping analysis performed in ten samples showed a frequent breakpoint between MTAP exon 4 and exon 5. In addition, p16 protein expression could not be detected in five out of six samples with intact p16 but deleted MTAP locus. These data show a high frequency of homozygous MTAP deletions in NSCLC which is associated with detectable co-deletion of p16INK4A in only half of the cases. This result suggests the existence either of another tumor suppressor gene telomeric of p16INK4A or of deletions involving 3'-untranslated (3'-UTR) regulatory regions of p16INK4A that can interfere with its expression or function.     

DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to therapeutic manipulation.     

Two Li-Fraumeni syndrome families with novel germline p53 mutations: loss of the wild-type p53 allele in only 50% of tumours

We describe two Li-Fraumeni syndrome families. Family A was remarkable for two early childhood cases of adrenocortical tumours, family B for a high incidence of many characteristic cancers, including a childhood case of choroid plexus tumour. Using direct sequencing, we analysed exons 5-9 of the p53 gene in constitutional DNA of individuals from both families and found two novel germline mutations in exon 5. In family A, we detected a point substitution in codon 138 (GCC to CCC), which resulted in the replacement of the alanine by a proline residue. Family B harboured a single-base pair deletion in codon 178 (CAC to -AC), resulting in a frameshift and premature chain termination. Three out of six tumours examined from both families, a renal cell carcinoma, a rhabdomyosarcoma and a breast cancer, showed loss of heterozygosity and contained only the mutant p53 allele. The remaining three neoplasms, both adrenocortical tumours and the choroid plexus tumour retained heterozygosity. Immunohistochemistry with anti-p53 antibody confirmed accumulation of p53 protein in tumours with loss of heterozygosity, while the remaining tumours were p53 negative. These results support the view that complete loss of activity of the wild-type p53 need not be the initial event in the formation of all tumours in Li-Fraumeni individuals.