Kay Neale

Germline mutations in BMPR1A/ALK3 cause a subset of cases of juvenile polyposis syndrome and of cowden and bannayan-riley-ruvalcaba syndromes

Juvenile polyposis syndrome (JPS) is an inherited hamartomatous-polyposis syndrome with a risk for colon cancer. JPS is a clinical diagnosis by exclusion, and, before susceptibility genes were identified, JPS could easily be confused with other inherited hamartoma syndromes, such as Bannayan-Riley-Ruvalcaba syndrome (BRRS) and Cowden syndrome (CS). Germline mutations of MADH4 (SMAD4) have been described in a variable number of probands with JPS. A series of familial and isolated European probands without MADH4 mutations were analyzed for germline mutations in BMPR1A, a member of the transforming growth-factor beta-receptor superfamily, upstream from the SMAD pathway. Overall, 10 (38%) probands were found to have germline BMPR1A mutations, 8 of which resulted in truncated receptors and 2 of which resulted in missense alterations (C124R and C376Y). Almost all available component tumors from mutation-positive cases showed loss of heterozygosity (LOH) in the BMPR1A region, whereas those from mutation-negative cases did not. One proband with CS/CS-like phenotype was also found to have a germline BMPR1A missense mutation (A338D). Thus, germline BMPR1A mutations cause a significant proportion of cases of JPS and might define a small subset of cases of CS/BRRS with specific colonic phenotype.

Further observations on LKB1/STK11 status and cancer risk in Peutz-Jeghers syndrome

Germline mutations in the LKB1/STK11 tumour suppressor gene cause Peutz–Jeghers syndrome (PJS), a rare dominant disorder. In addition to typical hamartomatous gastrointestinal polyps and pigmented perioral lesions, PJS is associated with an increased risk of tumours at multiple sites. Follow-up information on carriers is limited and genetic heterogeneity makes counselling and management in PJS difficult. Here we report the analysis of the LKB1/STK11 locus in a series of 33 PJS families, and estimation of cancer risks in carriers and noncarriers. Germline mutations of LKB1/STK11 were identified in 52% of cases. This observation reinforces the hypothesis of a second PJS locus. In carriers of LKB1/STK11 mutations, the risk of cancer was markedly elevated. The risk of developing any cancer in carriers by age 65 years was 47% (95% CI: 27–73%) with elevated risks of both gastrointestinal and breast cancer. PJS with germline mutations in LKB1/STK11 are at a very high relative and absolute risk of multiple gastrointestinal and nongastrointestinal cancers. To obtain precise estimates of risk associated with PJS requires further studies of genotype–phenotype especially with respect to LKB1/STK11 negative cases, as this group is likely to be heterogeneous.

Whole-gene APC deletions cause classical familial adenomatous polyposis, but not attenuated polyposis or “multiple” colorectal adenomas

Familial adenomatous polyposis (FAP) is a dominantly inherited colorectal tumor predisposition that results from germ-line mutations in the APC gene (chromosome 5q21). FAP shows substantial phenotypic variability: classical polyposis patients develop more than 100 colorectal adenomas, whereas those with attenuated polyposis (AAPC) have fewer than 100 adenomas. A further group of individuals, so-called “multiple” adenoma patients, have a phenotype like AAPC, with 3–99 polyps throughout the colorectum, but mostly have no demonstrable germ-line APC mutation. Routine mutation detection techniques fail to detect a pathogenic APC germ-line mutation in approximately 30% of patients with classical polyposis and 90% of those with AAPC/multiple adenomas. We have developed a real-time quantitative multiplex PCR assay to detect APC exon 14 deletions. When this technique was applied to a set of 60 classical polyposis and 143 AAPC/multiple adenoma patients with no apparent APC germ-line mutation, deletions were found exclusively in individuals with classical polyposis (7 of 60, 12%). Fine-mapping of the region suggested that the majority (6 of 7) of these deletions encompassed the entire APC locus, confirming that haploinsufficiency can result in a classical polyposis phenotype. Screening for germ-line deletions in APC mutation-negative individuals with classical polyposis seems warranted.

Inherited Susceptibility to Colorectal Adenomas and Carcinomas: Evidence for a New Predisposition Gene on 15q14-q22

BACKGROUND & AIMS The aim of this study was to evaluate the role of known colorectal adenoma and carcinoma susceptibility genes and to locate a novel susceptibility gene in an Ashkenazi family (SM1311) with dominantly inherited predisposition to colorectal adenomas and carcinomas. METHODS Clinicopathologic and family history data were collected. Genetic linkage and mutational analyses were used to investigate the genetic basis of the familys disease. RESULTS Affected members of SM1311 develop multiple tubular, villous, tubulovillous, and/or serrated colorectal adenomas throughout the large bowel, and some develop colon carcinoma. There are no extracolonic features clearly associated with disease in SM1311. We have shown that the familys phenotype does not result from APC mutations (including the I1307K variant) or from genetic changes in the other known genes that predispose to colon cancer. Using genetic linkage analysis, supplemented by allele loss in tumors, we have provided evidence for a new colorectal cancer susceptibility gene, CRAC1 (colorectal adenoma and carcinoma), mapping to chromosome 15q14-q22. CONCLUSIONS We provide evidence for a novel colorectal adenoma and carcinoma susceptibility gene on chromosome 15q14-q22. Further studies are needed to confirm this localization and to evaluate the contribution of CRAC1 to this disease.

Analysis of genetic and phenotypic heterogeneity in juvenile polyposis

BACKGROUND Juvenile polyposis syndrome (JPS) is characterised by gastrointestinal (GI) hamartomatous polyposis and an increased risk of GI malignancy. Juvenile polyps also occur in the Cowden (CS), Bannayan-Ruvalcaba-Riley (BRRS) and Gorlin (GS) syndromes. Diagnosing JPS can be problematic because it relies on exclusion of CS, BRRS, and GS. Germline mutations in the PTCH, PTENand DPC4 (SMAD4)genes can cause GS, CS/BRRS, and JPS, respectively. AIMS To examine the contribution of mutations in PTCH,PTEN, and DPC4(SMAD4) to JPS. METHODS Forty seven individuals from 15 families and nine apparently sporadic cases with JPS were screened for germline mutations inDPC4, PTEN, andPTCH. RESULTS No patient had a mutation in PTEN orPTCH. Five different germline mutations were detected in DPC4; three of these were deletions, one a single base substitution creating a stop codon, and one a missense change. None of these patients had distinguishing clinical features. CONCLUSIONS Mutations in PTEN and PTCHare unlikely to cause juvenile polyposis in the absence of clinical features indicative of CS, BRRS, or GS. A proportion of JPS patients harbour DPC4 mutations (21% in this study) but there remains uncharacterised genetic heterogeneity in JPS.

Analysis of chromosomal instability in human colorectal adenomas with two mutational hits at APC

In vitro data show that the adenomatous polyposis coli (APC) protein associates with the mitotic spindle and that mouse embryonic stem cells with biallelic Apc mutations are karyotypically unstable. These findings led to suggestions that APC acts in chromosomal segregation and that APC inactivation leads to chromosomal instability (CIN). An alternative hypothesis based on allelic loss studies in colorectal adenomas proposes that CIN precedes and contributes to genetic changes at APC. We determined whether colorectal adenomas with two mutations at APC show features consistent with these models by studying 55 lesions (average size 5 mm; range 1–13 mm) from patients with familial adenomatous polyposis. A variety of methods was used depending on available material, including flow cytometry, comparative genomic hybridization, and loss of heterozygosity (LOH) analysis. Selected adenomas were assessed for proliferative activity by Ki-67 immunocytochemistry. Seventeen of 20 (85%) tumors were diploid, two were near-diploid, and one was hypotetraploid. Just one (near-diploid) tumor showed increased proliferative activity. LOH was found occasionally on chromosome 15q (2 of 49 tumors), but not on chromosome 18q (0 of 48). In 20 adenomas, LOH at APC was associated with loss at 5q but not 5p markers, with the former encompassing a minimum of 20 Mb. However, three of these lesions analyzed by comparative genomic hybridization displayed normal profiles, suggesting, together with other data, that the mechanism of LOH at APC is probably somatic recombination. Our results therefore do not support the hypothesis that CIN precedes APC mutations in tumorigenesis. Regarding the model in which APC mutations lead directly to CIN, if APC mutations do have this effect in vivo, it must be subtle. Alternatively, CIN associated with APC mutations might be essentially an in vitro phenomenon.

A 10-year review of surgery for desmoid disease associated with familial adenomatous polyposis

Desmoid tumours affect 10–25 per cent of patients with familial adenomatous polyposis and represent a major cause of morbidity and mortality. Surgery for intra‐abdominal desmoids has traditionally been used as a last resort or to manage obstructive complications. The aim was to review 10 years of desmoid surgery in patients with familial adenomatous polyposis from a single centre.

Peutz-Jeghers disease: most, but not all, families are compatible with linkage to 19p13.3.

A locus for Peutz-Jeghers syndrome (PJS) was recently mapped to chromosome 19p13.3. Each of 12 families studied was compatible with linkage to the marker D19S886. We have analysed 20 further families and found that the majority of these are consistent with a PJS gene on 19p13.3. Three families were, however, unlinked to 19p13.3 and none of the available PJS polyps from these families showed allele loss at D19S886. There were no obvious clinicopathological or ethnic differences between the 19p13.3 linked and unlinked families. There appears, therefore, to be a major PJS locus on chromosome 19p13.3 and the possibility exists of a minor locus (or loci) elsewhere.

Disease severity and genetic pathways in attenuated familial adenomatous polyposis vary greatly but depend on the site of the germline mutation

Background: Attenuated familial adenomatous polyposis (AFAP) is associated with germline mutations in the 5′, 3′, and exon 9 of the adenomatous polyposis coli (APC) gene. These mutations probably encode a limited amount of functional APC protein. Methods and results: We found that colonic polyp number varied greatly among AFAP patients but members of the same family tended to have more similar disease severity. 5′ Mutants generally had more polyps than other patients. We analysed somatic APC mutations/loss of heterozygosity (LOH) in 235 tumours from 35 patients (16 families) with a variety of AFAP associated germline mutations. In common with two previous studies of individual kindreds, we found biallelic changes (“third hits”) in some polyps. We found that the “third hit” probably initiated tumorigenesis. Somatic mutation spectra were similar in 5′ and 3′ mutant patients, often resembling classical FAP. In exon 9 mutants, in contrast, “third hits” were more common. Most “third hits” left three 20 amino acid repeats (20AARs) on the germline mutant APC allele, with LOH (or proximal somatic mutation) of the wild-type allele; but some polyps had loss of the germline mutant with mutation leaving one 20AAR on the wild-type allele. Conclusions: We propose that mutations, such as nt4661insA, that leave three 20AARs are preferentially selected in cis with some AFAP mutations because the residual protein function is near optimal for tumorigenesis. Not all AFAP polyps appear to need “three hits” however. AFAP is phenotypically and genetically heterogeneous. In addition to effects of different germline mutations, modifier genes may be acting on the AFAP phenotype, perhaps influencing the quantity of functional protein produced by the germline mutant allele.

Explaining variation in familial adenomatous polyposis: relationship between genotype and phenotype and evidence for modifier genes

Background: Familial adenomatous polyposis (FAP) is characterised by variable phenotypic expression. Part of this is attributable to a relationship between APC genotype and phenotype but there remains significant intrafamilial variation. In the Min mouse model of FAP, differences in the severity of gastrointestinal polyposis result from the action of modifier genes. Aims: To determine whether phenotypic variation in human FAP has an inherited component consistent with the action of modifier genes. Method: We systematically examined polyp numbers in colectomy specimens from patients with classical FAP. Variation both between and within families was analysed. Formal modelling of the segregation of disease severity in families was performed Results: There was strong evidence for a relationship between site of mutation and the number of colorectal polyps, with germline mutations in the “cluster region” causing the most severe disease and those with mutations between codons 1020 and 1169 having the mildest disease. In addition to this genotype-phenotype relationship, we found evidence for non-APC linked genetic modifiers of disease expression. First degree relatives had more similar polyp counts than more distant relatives. Formal modelling of the segregation of disease severity in families revealed further evidence for the action of modifier genes, with a best fit to a mixed model of inheritance. Conclusion: Our data provide good evidence to support the hypothesis that modifier genes influence the severity of FAP in humans.