Exclusion of an extracolonic disease modifier locus on chromosome 1p33-36 in a large Swiss familial adenomatous polyposis kindred.

Familial adenomatous polyposis (FAP), an autosomal dominantly inherited colorectal cancer predisposition syndrome, displays considerable inter- and intrafamilial phenotypic heterogeneity, which represents a major problem in genetic counselling of APC mutation carriers. The Min mouse model indicated a putative disease modifier locus on chromosome 4, which is syntenic to human chromosome 1p35-36. This finding was subsequently supported by parametric and nonparametric linkage analyses in FAP families, however, without identifying functional variants in candidate genes. Recently, germline mutations in the base-excision repair gene MYH (1p33-34) have been described in patients with multiple adenomas, pointing to a possible role as disease modifier in FAP. Here, we present critical reassessment of one of the largest FAP kindreds published, which was previously used in linkage mapping of 1p35-36. In this family, all affected members harbour the same APC germline mutation (5945delA), but display marked phenotypic variability, in particular regarding the occurrence of extracolonic disease that segregates in several branches of the family tree. Using updated clinical information, additional mutation carriers and polymorphic markers, fine mapping of the critical region as well as mutation analysis of the MYH gene were performed. These investigations allowed us to significantly exclude (i) the 1p33-36 region as a modifier locus and (ii) MYH as a modifier gene for extracolonic disease in this FAP kindred. Our results do not eliminate 1p33-36 from suspicion in other families, but clearly indicate that in our family linkage analysis of further putative candidate regions is necessary to identify a disease modifier locus in FAP.

Severe hypophosphatasia: characterization of fifteen novel mutations in the ALPL gene.

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterization of ALPL gene mutations in a series of 11 families from various origins affected by perinatal and infantile hypophosphatasia. Sixteen distinct mutations were found, fifteen of them not previously reported: M45V, G46R, 388-391delGTAA, 389delT, T131I, G145S, D172E, 662delG, G203A, R255L, 876-881delAGGGGA, 962delG, E294K, E435K, and A451T. This confirms that severe hypophosphatasia is due to a large spectrum of mutations in Caucasian populations.

Expression of putative anticancer targets in familial adenomatous polyposis and its association with the APC mutation status.

Several substances interfering with colorectal carcinogenesis may reduce or prevent adenoma formation in familial adenomatous polyposis (FAP), an inherited predisposition to colorectal cancer. This study determined the expression of genes coding for putative anticancer targets (COX-2, iNOS, MMP-7, ODC, PKCbeta, PPARgamma, RXRalpha, RXRbeta, RXRgamma) in FAP patients to provide one of the rationales for the design of chemotherapy and -prevention strategies. Gene expression was assessed by TaqMan analysis in colonic tissue of 9 FAP patients with mutations in the APC gene (APCpos), 5 FAP patients without identified genetic defect (APCneg), and 3 healthy individuals. Among the examined genes, PKCbeta and MMP-7 were most consistently altered in adenoma tissue relative to matched mucosa. Intriguingly, ODC was clearly overexpressed in polyps from APCpos but not APCneg patients. Furthermore, PKCbeta, MMP-7, ODC, and COX-2 as well as all RXRs displayed altered expression in apparently healthy FAP mucosa as opposed to that of healthy individuals. Our data suggests PKCbeta and MMP-7 to be the most suited as anticancer targets among the genes studied.

Nontruncating APC germ-line mutations and mismatch repair deficiency play a minor role in APC mutation-negative polyposis.

Familial adenomatous polyposis, an autosomal-dominantly inherited colorectal cancer predisposition syndrome, is caused by germ-line mutations in the adenomatous polyposis coli (APC) gene. Despite the use of different screening methods, studies worldwide fail to identify APC mutations in 20-50% of all familial adenomatous polyposis patients (APC mutation-negatives). In this study, missense mutations in the coding region of the APC gene, which would have been missed by the protein truncation test, as well as mutations in the APC promoter and the 3' untranslated region, were determined by the single nucleotide polymorphism discovery assay and direct DNA sequencing in 31 mutation-negative polyposis patients. Seventeen gene alterations were identified, whereof four (12.9%) represent possibly pathogenic germ-line mutations: silent A290T (promoter) and A8822G (3' untranslated region) as well as missense R99W and E1317Q (coding region). The 27 remaining, truly APC mutation-negative polyposis patients displayed a significantly later age at diagnosis compared with APC mutation carriers (46.1 versus 35.2 years; P < 0.01). APC mutation-negative individuals with >100 colonic polyps were more likely to present with extracolonic disease (P < 0.05) than those with <100. Assessment of microsatellite instability (MSI), a hallmark of mismatch repair deficiency, in 68 tumors from 21 truly APC mutation-negative patients, identified 4 (5.9%) unstable tubulo-villous adenomas (3 MSI-High and 1 MSI-Low), stemming from 4 (19%) unrelated individuals and likely to be caused by hMLH1 promoter hypermethylation. In conclusion, only a small proportion of APC germ-line mutation carriers is missed by the protein truncation test, and mismatch repair deficiency does not seem to substantially contribute to tumor development in APC mutation-negative polyposis patients.

Relationship between APC genotype, polyp distribution, and oral sulindac treatment in the colon and rectum of patients with familial adenomatous polyposis.

PURPOSE: Familial adenomatous polyposis is an inherited colorectal cancer syndrome characterized by the presence of multiple adenomatous colorectal polyps. Molecular studies have revealed that germline mutations in the APC gene are the underlying cause of the disease. The nonsteroidal anti-inflammatory agent sulindac has been shown to reduce the number of colorectal adenomas. Most sulindac trials in the large bowel have focused on the distal colon and relatively little is known about its effect on the proximal colon. Moreover, it is unknown whether the site of the APC mutation affects the efficacy of sulindac. METHODS: This study investigated whether there were regional differences in the effect of sulindac on the colon and whether response to sulindac was dependent on the site of mutation in the APC gene. In an open prospective study 17 patients with familial adenomatous polyposis were treated with 300 mg oral sulindac daily for four months followed by a washout phase of six months. Ten of the patients had an intact colon and seven had rectal stumps only. The number, size, and the degree of dysplasia of the adenomas were evaluated by colonoscopy at entry, end of treatment and end of the study. RESULTS: Overall, a statistically significant decrease in the number of adenomas was observed (120 +/- 112 to 28 +/- 64, P = 0.007). After cessation of sulindac treatment the number of adenomas increased to 48 +/- 44.5, but remained significantly lower than the values observed at baseline. In the ten patients with intact colons, adenomas decreased by sevenfold in the proximal colon (103 +/- 73 to 15.1 +/- 47.4, P = 0.011) and twofold in the distal colon (80 +/- 52 to 29.6 +/- 37.2, P = 0.005). The size of adenomas and the grade of dysplasia also decreased. No correlation could be seen between the APC mutation site and the response to treatment. CONCLUSION: These data indicate that sulindac reduces the number of adenomas in the entire colon and that the effect seems to be more pronounced in the proximal colon.

A polymorphism in the ATM gene modulates the penetrance of hereditary non-polyposis colorectal cancer.

Germ-line mutations in MLH1 and MSH2 genes predispose to hereditary non-polyposis colorectal cancer (HNPCC) syndrome, but they do not predict a specific phenotype of the disease. We speculated that the ataxia-telangiectasia mutated gene (ATM) was a candidate gene to modulate the phenotypic expression of HNPCC, as heterozygous individuals for germ-line ATM mutations have been considered at higher risk of developing epithelial malignancies. The frequency of the ATM D1853N polymorphism was evaluated in 167 individuals from 20 HNPCC families in which MLH1 or MSH2 germ-line mutations co-segregated with the disease. Among the 67 MLH1 or MSH2 mutation carriers, the ATM 1853N variant was associated with a significantly higher incidence of colorectal and other HNPCC-related cancers, when compared with individuals carrying the ATM 1853D variant [12/13 (92%) vs. 31/54 (57.5%); p = 0.02]. MLH1 and MSH2 mutation carriers who concomitantly carried the ATM 1853N variant, had an 8 times increased risk of developing colorectal and other HNPCC-related cancers (OR: 8.9; p = 0.02), when compared with MLH1 or MSH2 mutation carriers with the ATM 1853D variant. Our results suggest that the ATM D1853N polymorphism modulates the penetrance of MLH1 and MSH2 germ-line mutations.

Germline alterations in the cyclooxygenase-2 gene are not associated with the development of extracolonic manifestations in a large swiss familial adenomatous polyposis kindred.

Familial adenomatous polyposis (FAP) is an autosomal dominant condition leading to the development of multiple colorectal polyps and other features. Intrafamilial variation in phenotype is known to occur in FAP; despite carrying the same causing mutation in the APC gene, disease expression may considerably differ in affected individuals, likely due to the existence of modifier genes. Several lines of evidence suggest the cyclooxygenase-2 (COX-2) gene to be a candidate modifier in FAP. Since COX-2 appears to be expressed in tissues prone to be affected in FAP, it might influence the occurrence of extracolonic manifestations in this disorder. Herein, we investigated whether alterations in the COX-2 gene are involved in the development of extracolonic polyps and extragastrointestinal features. Mutational analysis using single-strand conformation polymorphism (SSCP) in 130 members of a FAP family displaying strong phenotype variation revealed 3 polymorphic sites within the coding region of the COX-2 gene. None of these allelic variants, however, segregated with a particular disease phenotype. In addition, expression analysis was performed in 31 family members with representative phenotypes. Neither of the two polymorphisms detected within the COX-2 promoter was associated with a given phenotype nor was there a significant difference in quality or quantity of COX-2 mRNA in lymphocytes as measured by reverse transcription- and real time quantitative reverse transcription PCR (RT-PCR and TaqMan). In conclusion, germline alterations in the COX-2 gene are unlikely to account for the development of extracolonic disease in FAP patients.

Genetics of hereditary colon cancer - a basis for prevention?

Hereditary colorectal cancer syndromes are among the best in vivo models to study colorectal carcinogenesis and the influence of putative modifiers of the cancer risk. The present knowledge regarding the wide range of colorectal cancer (CRC) susceptibilities and the histological and molecular changes they elicit is leading to a very dynamic and integrated concept of tumorigenesis in the colon and to new views about prevention and early treatment of cancer.

Cortisol producing adrenal adenoma--a new manifestation of Gardner's syndrome.

INTRODUCTION: Familial adenomatous polyposis (FAP) is an autosomal dominant disorder which typically presents with colorectal cancer in early adult life secondary to extensive adenomatous polyps of the colon. Gardner's syndrome is a variant of FAP in which desmoid tumors, osteomas and pigmented retinal lesions occur together with intestinal manifestations. The APC gene (adenomatous polyposis coli) at 5q21 is a tumor suppressor gene which is mutant in FAP. PATIENT: A 36 year old woman presented with a history of polyposis ventriculi, ovarian desmoid cysts, and disseminated desmoid tumors. Her familial history was unremarkable. On admission she complained weight gain, secondary amenorrhea, and episodes of hypertension followed by paroxysmal headache. RESULTS: Elevated urinary free cortisol (878 microg/24h), suppressed basal ACTH (< 5 pg/ml) and insuppressible serum cortisol after low dose dexamethasone (189 ng/ml) revealed adrenal Cushing's syndrome. Abdominal NMR showed an adrenal mass two centimeter in diameter with inhomogeneous contrast enhancement. Unilateral adrenalectomy was performed and an adrenal adenoma was diagnosed by histological criteria. For mutational detection DNA from peripheral blood leucocytes was extracted. A protein truncation test was performed, which revealed a termination mutation between codon 1099 and 1623 of the APC gene. Direct sequencing showed a point mutation in exon 15 of the APC gene at position 1542 (CAG --> TAG). This region is known to be altered in patients with extraintestinal manifestation of FAP. CONCLUSION: In patients with Gardner's syndrome adrenal tumors leading to hormonal excess should be considered. Whether mutations in the APC gene have implications in sporadic adrenal tumorigenesis needs to be proven.

N-acetyltransferase 2 influences cancer prevalence in hMLH1/hMSH2 mutation carriers.

Hereditary nonpolyposis colorectal cancer (HNPCC), an inherited cancer predisposition syndrome, has been associated with germline mutations in DNA mismatch repair (MMR) genes. Because a deficiency in MMR does not predict a specific cancer phenotype, modifying genes may account in part for the variation in disease expression. We determined the N-acetyltransferase 2 (NAT2) genotype in 26 unaffected and 52 cancer-affected hMLH1/hMSH2 mutation carriers coming from 21 Swiss HNPCC families. Slow acetylators were found to be significantly (P < 0.03) more prevalent in the group of affected mutation carriers. Our results suggest a protective effect of the NAT2 rapid acetylator phenotype, an observation that could have implications for genetic counseling and management of MMR gene mutation carriers.

[Early diagnosis of familial adenomatous polyposis based on multiple osteomas of the facial skull]. / Frühzeitige Diagnose einer familiären adenomatösen Polypose wegen multipler Osteome des Gesichtsschädels.

We describe a 21-year-old patient hospitalised because of a dislocated mandibular fracture and accidentally found to have multiple osteomas of the skull. A subsequent gastroenterological examination revealed the presence of multiple polyps in the large intestine, typical of familial adenomatous polyposis. A disease causing germline mutation in the adenomatous polyposis coli gene was identified by molecular genetic analysis. Although extracolonic features such as multiple osteomas, multiple epidermal cysts and desmoids are frequently found, most cases without a family history of familial adenomatous polyposis or colorectal cancer are only diagnosed because of colonic disease manifestations with colorectal cancer already present. This case report strikingly illustrates that in the absence of a family history the presence of extracolonic features allows presymptomatic diagnosis in familial adenomatous polyposis patients before colorectal cancer has developed.

Influence of selection criteria on mutation detection in patients with hereditary nonpolyposis colorectal cancer.

BACKGROUND: Hereditary nonpolyposis colorectal cancer (HNPCC) is linked genetically to mutations in DNA mismatch repair (MMR) genes. Because a deficiency in MMR does not predict a specific phenotype, the original selection criteria may be too restrictive in identifying additional families. The current study was performed to determine whether a relaxation of the Amsterdam criteria (AC) could be applied to identify more families associated with DNA MMR. METHODS: Twenty-eight unrelated Swiss families (15 complying with the AC and 13 fulfilling extended criteria [EC] to include other tumors of the HNPCC spectrum as well) were screened for mutations in the MMR genes hMSH2 and hMLH1, using single-stranded conformation polymorphism and direct DNA sequencing. Microsatellite instability (MSI) was determined in 14 families. A comparison was made between the phenotypic characteristics of the mutation positive and mutation negative families. RESULTS: Ten AC families (67%) harbored germline mutations in hMLH1 (6 kindreds) or hMSH2 (4 kindreds). In none of the EC kindreds could an unambiguous disease-causing mutation be identified. Seven of eight AC families were found to display MSI whereas all colorectal carcinomas (CRC) in eight EC kindreds were MSI stable. CRC patients from mutation positive families had an earlier age at diagnosis (44 years vs. 49 years) and appeared to have a better survival (11.1 years vs. 7.7 years). CONCLUSIONS: Extending the AC to include extracolonic tumors of the HNPCC spectrum results in a very low mutation detection rate for hMSH2 and hMLH1. The EC families appear to represent an alternative genetic entity not necessarily related to DNA MMR gene mutations because they do not display MSI.

[Identification and genetic counseling of people with HNPCC (hereditary nonpolyposis colorectal cancer): old and new research goals]. / Erfassung und genetische Beratung von Personen mit HNPCC ("hereditary non-polyposis colorectal cancer"): alte und neue Forschungsziele.

Hereditary non-polyposis colorectal cancer (HNPCC) is a relatively common autosomal dominantly inherited predisposition leading to a familial occurrence of cancer of the colon, rectum, endometrium and some other organs. Cancer mortality can be significantly reduced by appropriate intervention. The diagnosis of HNPCC is suspected on the basis of early onset and multiple foci of colorectal cancer (CRC), in many cases affecting the proximal part of the colon, and of endometrial cancer. It may be confirmed by molecular genetic analysis of the mismatch repair genes, especially hMLH1 and hMSH2. In spite of considerable progress in the understanding of hereditary colon cancer, many questions which are of basic importance for the identification and appropriate genetic counselling of gene carriers remain to be answered. HNPCC defined on clinical and genealogical grounds is by no means identical with the presence of mutated mismatch-repair genes. This impedes the identification of persons/families at increased cancer risk. Mutations of other, mainly as yet unidentified genes may lead to a similar phenotype. Not only heterogeneity of the predispositions underlying CRC, but also penetrance and expressivity of the identifiable mutations of the MMR-genes, have been explored only superficially. The process of carcinogenesis in the colon can follow different routes depending on the genetic background of the patients. Its investigation will open up new possibilities of cancer prevention. In addition, genetic counselling must be developed into a more "evidence"-based medical undertaking. These gaps in the understanding of hereditary CRC and in the care of persons at risk can only be overcome through structured collaboration between family doctors, medical specialists such as gastroenterologists, oncologists and surgeons, medical geneticists and basic researchers.

Phenotypic differences in familial adenomatous polyposis based on APC gene mutation status.

BACKGROUND: Familial adenomatous polyposis (FAP) is a clinically well defined hereditary disease caused by germline mutations within the adenomatous polyposis coli (APC) gene. Although several techniques are applied in the mutation analysis of FAP kindreds about 20-50% of cases remain unclear, with no APC mutation identified (APC negative). AIMS: To delineate phenotypic differences between APC positive and APC negative patients with respect to colonic and extracolonic disease in order to determine whether additional mechanisms are involved in the pathogenesis of FAP. METHODS: The entire coding region of the APC gene was analysed using single stranded conformation polymorphism and protein truncation tests in 50 Swiss FAP families with a total of 161 affected individuals. Differences in phenotypic manifestation were statistically evaluated by Student's t test, Fisher's exact test, and chi2 test. RESULTS: Thirty six families (72%) were APC positive. Statistically significant differences between APC positive and APC negative groups were found for the mean age at diagnosis of colonic polyposis (35.2 versus 45.3 years, respectively) and for the occurrence of stomach polyps (14 patients, all APC positive). Additionally, APC negative patients displayed lower polyp numbers at diagnosis and less extracolonic manifestations. CONCLUSIONS: FAP kindreds without detected APC gene mutations present with a notably milder disease phenotype compared with APC positive families, suggesting that different genetic factors might be involved.

Identification of a modifier gene locus on chromosome 1p35-36 in familial adenomatous polyposis.

Phenotypic variability based on nonallelic heterogeneity is a characteristic feature of the dominantly inherited disease, familial adenomatous polyposis (FAP). A modifying locus, called Mom-1, which strongly influences disease expression has been mapped in the mouse model of FAP to the region of murine chromosome 4, which has synteny to human chromosome 1p35-36. In the present study, this chromosomal region was investigated by using 14 microsatellite markers within a large FAP kindred in which patients harbor the same germ-line mutation but show markedly different disease characteristics. The linkage program MLINK was used to determine whether any correlation exists between these markers and the development of extracolonic symptoms in polyposis coli patients. Depending on the mode of inheritance of the affected locus, a maximum lod score was observed for markers D1S211 and D1S197, reaching 2.08 and 1.77, respectively. The observed values obtained within one large FAP family are supportive of a phenotype-modifying locus within this chromosomal region.

Association of extracolonic manifestations of familial adenomatous polyposis with acetylation phenotype in a large FAP kindred.

Familial adenomatous polyposis coli (FAP) has been shown to be associated with germline mutations of the adenomatous polyposis gene (APC) on chromosome 5. Extra-colonic manifestations also occur in FAP and include desmoid tumors, epidermoid cysts and osteomas. The combination of FAP with extracolonic symptoms is commonly referred to as Gardner's syndrome. It remains difficult, however, to predict which patients may have a propensity to develop extracolonic manifestations. The rapid acetylation phenotype is believed to be associated with an increased likelihood of sporadic colorectal cancer, whereas the slow acetylation phenotype is recognized as a predisposing factor for bladder cancer. The slow acetylation phenotype is caused by mutant alleles of the cytosolic enzyme N-acetyltransferase (NAT2). In this study, we determined the NAT2 genotype in members of one large FAP family and three smaller ones all of which had been shown to harbor the same germline APC gene mutation. We observed a significant correlation between slow acetylation genotypes and extracolonic manifestations of the disease. Rapid acetylation genotypes were not overrepresented in colorectal cancer cases in this family as compared to the frequency of this genotype in the normal Caucasian population.

Secretory phospholipase A2 does not appear to be associated with phenotypic variation in familial adenomatous polyposis.

Recent studies in mice have provided strong evidence for a modifier gene that is capable of effecting the expression of the mouse equivalent of familial adenomatous polyposis (FAP). A candidate gene has been proposed, namely secretory phospholipase A2 (sPLA2). Increased tumor number in mice was correlated with low levels of sPLA2 expression and the presence of truncating mutations within the sPLA2 gene. In an attempt to determine whether any genetic alterations in the sPLA2 gene were associated with the expression of FAP in man, we investigated the genetic structure of sPLA2 in 97 polyposis coli patients presenting with various disease phenotypes, and its expression in 8 FAP patients displaying markedly different disease characteristics. In the current study no inactivating mutations in the sPLA2 gene were identified, suggesting that human sPLA2 is not associated with phenotypic variation in FAP.