Two Chinese pedigrees for adenomatous polyposis coli: new mutations at codon 1309 and predisposition to phenotypic variations

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease caused by a mutation in the adenomatous polyposis coli (APC) gene. Some studies have attempted to correlate mutations at codon 1309 with classic FAP (≥100 colorectal polyps). We report two Chinese FAP pedigrees with new frameshift mutations at codon 1309, in which affected individuals manifest phenotypic variations. Comprehensive physical examinations were performed for all living individuals and the medical data of deceased patients were collected. Screening of the APC and human mutY homolog (MUTYH) genes for germline mutations was conducted by direct polymerase chain reaction (PCR) sequencing. In two pedigrees, a heterozygous deletion in exon 16 of the APC gene was present in all FAP patients but absent in the unaffected individuals. There were no changes to the MUTYH gene. The first pedigree, with a new frameshift mutation at c.3926_3930 del AAAAG (p. Glu1309Aspfs X4), exhibited obvious differences in the polyp number such that the proband manifested only three colorectal polyps, whereas another patients showed the symptoms of classic FAP. The second pedigree, also traced a new mutation at c.3922_3925 del AAAG (p. Glu1309Argfs X11). Although all of the patients presented with classic polyposis, one of them exhibited a delayed onset of colorectal cancer in his 50s. Two novel mutations at codon 1309 in two Chinese families suffering from FAP could enrich the germline mutation spectrum of the APC gene. Families of individuals might manifest different phenotypes, even with an identical codon 1309 mutation, unlike in previous studies.     

Correlation between the location of germ-line mutations in the APC gene and the number of colorectal polyps in familial adenomatous polyposis patients.

Recently we have isolated the adenomatous polyposis coli (APC) gene which causes familial adenomatous polyposis (FAP), and its germ-line mutations in a substantial number of FAP patients have been identified. On the basis of this information, we compared the location of germ-line mutations in the APC gene in 22 unrelated patients (12 of whom have been reported previously) with the number of colorectal polyps developed in FAP patients; 17 were sparse types and five were profuse types. All but one of the mutations were considered to cause truncation of the gene product by frame-shift due to deletion (14 cases) or nonsense mutation (seven cases). The location of the germ-line mutations seems to correlate with the two clinical types; germ-line mutations in five FAP patients with profuse polyps were observed between codon 1250 and codon 1464, whereas mutations in 17 FAP patients with fewer polyps were observed in the other regions of the APC gene. The result suggests that the number of colorectal polyps in FAP patients may be associated with a difference in the stability or biological function of the truncated APC protein.     

MUTYH-associated polyposis: 70 of 71 patients with biallelic mutations present with an attenuated or atypical phenotype

To determine the frequency, mutation spectrum and phenotype of the recently described autosomal recessive MUTYH-associated polyposis (MAP), we performed a systematic search for MUTYH (MYH) mutations by sequencing the complete coding region of the gene in 329 unselected APC mutation-negative index patients with the clinical diagnosis of familial adenomatous polyposis (FAP) or attenuated FAP (AFAP). Biallelic germline mutations in MUTYH were identified in 55 of the 329 unselected patients (17%) and in another 9 selected index cases. About one-fifth (20%) of the 64 unrelated MAP patients harboured none of the 2 hot-spot missense mutations Y165C and/or G382D. Including 7 affected relatives, almost all MAP patients presented with either an attenuated (80%) or with an atypical phenotype (18%). Fifty percentage of the MAP patients had colorectal cancer at diagnosis. Duodenal polyposis was found in 18%, thyroid and stomach cancer in 1 case, other extraintestinal manifestations associated with FAP were not observed. In 8 families, vertical segregation was suspected; in 2 of these families, biallelic mutations were identified in 2 generations. Monoallelic changes with predicted functional relevance were found in 0.9% of the 329 patients, which is in accordance with the carrier frequency in the general population. In conclusion, biallelic MUTYH mutations are the underlying genetic basis in a substantial fraction of patients with adenomatous polyposis. The phenotype of MAP is best characterised as attenuated or atypical, respectively. Colorectal surveillance starting at about 18 years of age is recommended for biallelic mutation carriers and siblings of MAP patients, who refuse predictive testing.     

Detection of APC gene deletion by double competitive polymerase chain reaction in patients with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominant familial cancer syndrome caused by germline mutations of the tumor suppressor adenomatous polyposis coli (APC) gene. Heterozygous apc mutations have been identified in the majority of classical FAP patients who develop more than 100 colorectal adenomas. However, classical FAP patients often fail to display germline APC mutations detectable by routine mutation analysis. These apparently mutation-negative cases may be caused by heterozygous large genomic deletions. In the present study, FAP patients who showed no APC germline mutation detectable by the protein truncation assay and direct sequencing of protein coding exons were screened for APC gene deletion by a gene dose assay based on double competitive polymerase chain reaction. Gene dosage measurements within exon 15 of the APC gene identified two patients with gene deletion and one with possible gene duplication among 41 apparently mutation-negative patients. The deleted sequences in the two patients were determined by fine gene dose mapping around the APC gene and nucleotide sequencing of the deletion breakpoints. They were approximately 435-kilobase pair (kb) and 737-kb regions including the whole APC gene and flanked by a 4-base pair repeat and LINE-1 repetitive sequences, respectively. The chimeric LINE-1 element created at the breakpoint in the latter case also contained a short sequence derived from another LINE-1 element, suggesting a complex unequal homologous recombination event. These findings indicate that this gene dose assay is a useful technique to detect large gene deletions of the APC gene and to determine their genomic breakpoints.     

Somatic Mutation of the APC Gene in Thyroid Carcinoma Associated with Familial Adenomatous Polyposis

We report the existence of both germline and somatic mutations of the APC gene in thyroid carcinomas from familial adenomatous polyposis (FAP) patients. One papillary thyroid carcinoma from a 20-year-old woman, with germline mutation of the APC gene (TCA to TGA at codon 1110), showed a somatic mutation of AAAAC deletion between codons 1060 and 1063. Another somatic mutation of CAG to TAG at codon 886 was also found in one of multiple thyroid carcinomas from a 26-year-old woman with attenuated FAP and germline mutation at codon 175 (C deletion). This is the first evidence that total absence of the normal function of the APC gene is involved in development of thyroid carcinomas in FAP.     

Somatic mutation of the APC gene in thyroid carcinoma associated with familial adenomatous polyposis.

We report the existence of both germline and somatic mutations of the APC gene in thyroid carcinomas from familial adenomatous polyposis (FAP) patients. One papillary thyroid carcinoma from a 210-year-old woman, with germline mutation of the APC gene (TCA to TGA at codon 1110), showed a somatic mutation of AAAAC deletion between codons 1060 and 1063. Another somatic mutation of CAG to TAG at codon 886 was also found in one of multiple thyroid carcinomas from a 26-year-old woman with attenuated FAP and germline mutation at codon 175 (C deletion). This is the first evidence that total absence of the normal function of the APC gene is involved in development of thyroid carcinomas in FAP.     

Difference in characteristics of APC mutations between colonic and extracolonic tumors of FAP patients: variations with phenotype

To clarify the differences in characteristics of adenomatous polyposis coli (APC) mutations between colorectal tumors from various phenotypes of familial adenomatous polyposis (FAP) and between colorectal and extracolonic tumors, we analyzed APC mutations in 86 colorectal tumors from FAP patients including profuse, sparse and attenuated types, 23 sporadic colorectal tumors and 40 FAP extracolonic tumors including duodenal, gastric and desmoid tumors. In all tumors, 1 allele of the truncated APC gene retained armadillo repeats, 15-amino-acid (aa) repeats and various numbers of 20-aa repeats, but lacked SAMP repeats, as a result of germline and somatic mutations. Regarding 20-aa repeats, the truncated APC gene retained 1 repeat due to allele loss in 96% (27/28) of colorectal tumors from profuse-type FAP, 69% (36/52) of sparse-type retained 2 repeats due to somatic mutation, and 100% (6/6) of attenuated-type retained 2 or 3 repeats due to the third or second hit. In sporadic colorectal tumors 74% (17/23) retained 1 or 2 repeats. The truncated APC gene retained 3 repeats in 88% (7/8) of FAP duodenal tumors, 100% (26/26) of gastric tumors retained 2 or 3 repeats and 83% (5/6) of desmoid tumors retained 2 repeats. These data suggest that the number of beta-catenin downregulating 20-aa repeats in truncated APC gene associated with colorectal tumorigenesis is different in profuse, sparse and attenuated types of FAP, and that the association with tumorigenesis is also different between colorectal and extracolonic tumors.     

Analysis of Small Fragment Deletions of the APC gene in Chinese Patients with Familial Adenomatous Polyposis,a Precancerous Condition

Background: : Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease mainly causedby mutations of the adenomatous polyposis coli (APC) gene with almost complete penetrance. These colorectalpolyps are precancerous lesions that will inevitable develop into colorectal cancer at the median age of 40-yearold if total proctocolectomy is not performed. So identification of APC germline mutations has great implicationsfor genetic counseling and management of FAP patients. In this study, we screened APC germline mutationsin Chinese FAP patients, in order to find novel mutations and the APC gene germline mutation characteristicsof Chinese FAP patients. Materials and Methods: The FAP patients were diagnosed by clinical manifestations,family histories, endoscope and biopsy. Then patients peripheral blood samples were collected, afterwards,genomic DNA was extracted. The mutation analysis of the APC gene was conducted by direct polymerasechain reaction (PCR) sequencing for micromutations and multiplex ligation-dependent probe amplification(MLPA) for large duplications and/or deletions. Results: We found 6 micromutations out of 14 FAP pedigrees,while there were no large duplications and/or deletions found. These germline mutations are c.5432C>T(p.Ser1811Leu), two c.3926_3930delAAAAG (p.Glu1309AspfsX4), c.3921_3924delAAAA (p.Ile1307MetfsX13),c3184_3187delCAAA(p.Gln1061AspfsX59) and c4127_4126delAT (p.Tyr1376LysfsX9), respectively, and alldeletion mutations resulted in a premature stop codon. At the same time, we found c.3921_3924delAAAA andtwo c.3926_3930delAAAAG are located in AAAAG short tandem repeats, c3184_3187delCAAA is located in theCAAA interrupted direct repeats, and c4127_4128 del AT is located in the 5’-CCTGAACA-3’ ,3’-ACAAGTCC-5palindromes (inverted repeats) of the APC gene. Furthermore, deletion mutations are mostly located at condon1309. Conclusions: Though there were no novel mutations found as the pathogenic gene of FAP in this study,we found nucleotide sequence containing short tandem repeats and palindromes (inverted repeats), especiallythe 5 bp base deletion at codon 1309, are mutations in high incidence area in APC gene.     

Biallelic inactivation of the APC gene is associated with hepatocellular carcinoma in familial adenomatous polyposis coli

BACKGROUND: Certain primary hepatic tumors have been associated with familial adenomatous polyposis (FAP), a condition caused by germline mutations of the adenomatous polyposis coli (APC) gene. However, a genetic association between FAP and hepatocellular carcinoma (HCC) has not been shown. This study tested the hypothesis that biallelic inactivation of the APC gene contributed to the development of HCC in a patient with FAP and a known germline mutation of the APC gene at codon 208, but no other risk factors for HCC. METHODS: Total RNA and genomic DNA were isolated from the tumor, and in vitro synthesized protein assay and DNA sequencing analysis were used to screen for a somatic mutation in the APC gene. RESULTS: A somatic one-base pair deletion at codon 568 was identified in the wild-type allele of the APC gene. CONCLUSIONS: To the authors' knowledge, this study provides the first evidence that biallelic inactivation of the APC gene may contribute to the development of HCC in patients with FAP.     

大肠肿瘤APC基因突变的研究

目的检测ＡＰＣ基因在国人散发性大肠肿瘤中的突变情况,并探讨ＡＰＣ基因突变与大肠肿瘤生物学行为的关系。方法采用ＰＣＲＳＳＣＰ、ＤＮＡ测序对２７例（３３份标本）大肠息肉,３６例大肠癌和相应正常粘膜及３例家族性腺瘤性息肉瘤（ＦＡＰ）标本中ＡＰＣ基因“突变密集区”（ｍｕｔａｔｉｏｎｃｌｕｓｔｅｒｒｅｇｉｏｎＭＣＲ）的突变进行了研究。结果大肠息肉和癌组织中,ＡＰＣ基因ＭＣＲ突变率分别为３０．３％（１０／３３）和３５．９％（１４／３９）,两者差异无显著性,而正常大肠粘膜、炎性息肉和增生性息肉均无突变。３例ＦＡＰ发现有２例突变,其中１例从正常粘膜到腺瘤、到癌变组织均有突变,另１例则在＞１．０ｃｍ的腺瘤和肝转移癌组织有突变,测序结果证实该例在１４２５号密码子存在相同点突变。ＡＰＣ突变与肿瘤的临床病理特征无明显的相关性。结论ＡＰＣ不仅在ＦＡＰ中存在突变,而且在散发性大肠肿瘤中亦存在突变;它的突变至少参与了部分大肠癌的发生发展过程,而且是这一过程中较早发生的事件。     

Multiple approach to the exploration of genotype-phenotype correlations in familial adenomatous polyposis.

PURPOSE: Familial adenomatous polyposis (FAP), caused by a mutation in the APC gene, is a colorectal cancer predisposition syndrome associated with several other clinical conditions. The severity of the FAP is related to the position of the inherited mutation in the APC gene. We analyzed a large series of FAP patients to identify associations among major clinical manifestations and to correlate the mutation site with specific disease manifestations. MATERIALS AND METHODS: APC mutations were identified in 953 FAP patients from 187 families. We used unconditional logistic regression models and a method involving generalized estimating equations to investigate the association between genotype and phenotype. We used multiple correspondence analysis to represent the interrelationships of a multiway contingency table of the considered variables. RESULTS: APC germline mutations were located between codons 156 and 2011 of the APC gene. Mutations spanning the region between codons 543 and 1309 were variable, but strongly associated with congenital hypertrophy of retinal pigment epithelium. Mutations between codons 1310 and 2011 were associated with a six-fold risk of desmoid tumors relative to the low-risk reference region (159 to 495). Mutations at codon 1309 were associated with early development of colorectal cancer. Mutations between codons 976 and 1067 were associated with a three- to four-fold increased risk of duodenal adenomas. The cumulative frequency of extracolonic manifestations was highest for mutations between codons 976 and 1067, followed by mutations between 1310 and 2011. CONCLUSION: Analysis of the relation between APC mutation site and phenotype identifies subgroups of FAP patients at high risk for major extracolonic disease, which is useful for surveillance and prevention.     

Prevalence of MYH germline mutations in Swiss APC mutation-negative polyposis patients

In 10-30% of patients with classical familial adenomatous polyposis (FAP) and up to 90% of those with attenuated (<100 colorectal adenomas; AFAP) polyposis, no pathogenic germline mutation in the adenomatous polyposis coli (APC) gene can be identified (APC mutation-negative). Recently, biallelic mutations in the base excision repair gene MYH have been shown to predispose to a multiple adenoma and carcinoma phenotype. This study aimed to (i) assess the MYH mutation carrier frequency among Swiss APC mutation-negative patients and (ii) identify phenotypic differences between MYH mutation carriers and APC/MYH mutation-negative polyposis patients. Seventy-nine unrelated APC mutation-negative Swiss patients with either classical (n=18) or attenuated (n=61) polyposis were screened for germline mutations in MYH by dHPLC and direct genomic DNA sequencing. Overall, 7 (8.9%) biallelic and 9 (11.4%) monoallelic MYH germline mutation carriers were identified. Among patients with a family history compatible with autosomal recessive inheritance (n=45), 1 (10.0%) out of 10 classical polyposis and 6 (17.1%) out of 35 attenuated polyposis patients carried biallelic MYH alterations, 2 of which represent novel gene variants (p.R171Q and p.R231H). Colorectal cancer was significantly (p<0.007) more frequent in biallelic mutation carriers (71.4%) compared with that of monoallelic and MYH mutation-negative polyposis patients (0 and 13.8%, respectively). On the basis of our findings and earlier reports, MYH mutation screening should be considered if all of the following criteria are fulfilled: (i) presence of classical or attenuated polyposis coli, (ii) absence of a pathogenic APC mutation, and (iii) a family history compatible with an autosomal recessive mode of inheritance.     

Inherited multitumoral syndromes including colorectal carcinoma

Inherited multitumoral syndromes including colorectal carcinoma are the followings: familial adenomatous polyposis (FAP), hereditary non-polyposis colon cancer (HNPCC) and Peutz-Jeghers syndrome (PJS). The studies of genotype-phenotype correlation have shown that various types of colonic polyposis are related with germline mutation of adenomatous polyposis coli (APC) gene, that is also responsible for a wide number of extracolonic manifestations. Concerning severity of polyposis, it has been suggested to perform ileorectal anastomosis in patients with intermediate FAP, whereas it has been suggested to remove the rectum from the beginning and to perform ileopouch anastomosis in patients with severe FAP (germline APC mutations between codons 1250 and 1464, i.e. mutation cluster region-MCR). Concerning extracolonic manifestations, original studies from our laboratory have shown that three peculiar, even if unusual extracolonic manifestations of FAP, were in the 5' portion of the gene, but almost always outside of MCR. In particular, in some patients or in some kindreds all these manifestations may be present together. It is suggested, from one hand, because of the wide variety of genotype-phenotype correlation word of caution before selecting surgical treatment simply on the basis of the germline mutations, on the other hand co-segregation of some extracolonic manifestations could facilitate intensive screening, early diagnosis and optimal time for treatment.     

The relationship between frequencies of extracolonic manifestations and the position of APC germline mutation in patients with familial adenomatous polyposis

BACKGROUND: Familial adenomatous polyposis (FAP) patients develop various extracolonic lesions; however, the relationship between germline mutation of the APC gene and extracolonic manifestations is mostly unknown. To examine the genotype-phenotype relationship, we compared the APC mutation and clinical data. METHODS: Germline mutations from codon 157 to 1465 of the APC gene were identified in 39 families of FAP and clinical data were collected from 80 patients of these families. Germline mutations were classified into two groups: mutations from exon 4 to 9 (codon 157 to 416, Group 1) and those from exon 10 to 15H (codon 564 to 1465, Group 2). The complication rates of extracolonic manifestations were compared between these two groups. RESULTS: Frequencies of duodenal polyps and gastric adenomas in Group 2 were higher than those in Group 1 (p < 0.0001 and p < 0.0004, respectively) and development of osteoma was more frequent in Group 2 (p = 0.01). The number of colorectal polyps and retinal pigments also correlated with the germline mutation, which was consistent with previous reports. However, such correlations were less obvious with regard to gastric fundic polyps, desmoid tumors, soft tissue tumors and colorectal cancer. CONCLUSION: There are two types with regard to extracolonic manifestations of FAP: one is more severely affected according to the position of germline mutation of the APC gene and the other is not affected.     

Different expressivity of two adjacent mutations of the APC gene

AIMS AND BACKGROUND: The phenotypic expression of different APC mutations in familial adenomatous polyposis (FAP) is variable: two to three variants of the disease have been defined based on the severity of colonic manifestations. Age of onset and number of polypectomies per person-year of post-surgical follow-up were compared in two FAP families with very close mutation sites in the APC gene, in order to ascertain mutation-specific variation of expressivity. FAMILIES AND APC MUTATIONS: Family A (5 patients) carried a newly characterized mutation, a four bp deletion at codon 843. Family B (5 patients) carried a previously identified mutation at codon 835. RESULTS: Mean age of onset was 49.7 years in family A and 30.5 years in family B; number of polypectomies per person-year of follow-up was 1.05 for family A and 10.1 for family B (P < 0.001). CONCLUSIONS: There is significant variation of expressivity (allelic heterogeneity) in FAP between two mutations separated by only eight codons, located at the 5' extremity of APC gene exon 15.     

Refining the relation between 'first hits' and 'second hits' at the APC locus: the 'loose fit' model and evidence for differences in somatic mutation spectra among patients

The site of the 'first hit' in the APC tumour suppressor gene determines the type of the 'second hit', both in familial adenomatous polyposis (FAP) and sporadic colorectal tumours. Mutations near codon 1300 are associated with loss of heterozygosity (LOH) of the wild-type allele; other tumours tend to have two protein-truncating mutations. In this study, we have confirmed and refined the LOH-associated region in colorectal FAP: allelic loss in adenomatous polyps tended to occur when the germline mutation lay in the region of the APC gene between the first and second beta-catenin degradation repeats (codons 1285-1378). LOH generally occurred by mitotic recombination, leaving two identical alleles, each encoding a protein with one remaining beta-catenin degradation repeat. For patients with germline mutations that truncated the protein before the first repeat (codon 1264), LOH was very rare and tumours generally acquired a somatic mutation which left two, or less often one, repeats remaining in the protein. In our sample set, patients with germline mutations after the second beta-catenin degradation repeat tended to have undetectable, presumably cryptic, somatic mutations in their polyps. Exceptions to these rules were, however, not uncommon. Although the site of the germline mutation was the strongest determinant of the somatic mutation in FAP tumours and most patients showed no clear tendency to acquire specific types of truncating 'second hit', a minority of patients did have unusual somatic mutation spectra in their polyps. Thus, some individuals may be predisposed to particular types of 'second hit' (for example, frameshift rather than nonsense changes). Overall, disease severity (polyp number) did not vary with individuals' spectrum of somatic APC mutations, providing no clear evidence for modifier genes that influence disease severity in this fashion. Our data are consistent with the hypothesis that there exists an optimal level of beta-catenin signalling in colorectal tumours and that the APC mutation spectrum principally reflects this fact. The association between 'first hits' and 'second hits' at APC is not, however, so strong as to suggest that tumorigenesis only occurs if the genotype is optimum; we suggest 'relaxed' terminology, the 'loose fit' model, to describe this situation.     

Mutation analysis of the MYH gene in an Australian series of colorectal polyposis patients with or without germline APC mutations

The MYH gene has recently been shown to be associated with a recessive form of colorectal adenomatous polyposis. Two common mutations in the MYH gene have been identified that lend themselves to rapid screening. We have examined a series of 302 individuals comprising 120 control subjects, 120 patients diagnosed with adenomatous polyposis but without germline mutations in the APC gene and 62 patients diagnosed with familial adenomatous polyposis all harbouring confirmed causative APC germline mutations. The results reveal that MYH accounts for 16 percent of polyposis patients without germline mutations in the APC gene and that it does not appear to be a modifier gene in FAP patients diagnosed with APC germline mutations.     

Somatic APC Inactivation Mechanisms in Sporadic Colorectal Cancer Cases in Hungary

The role of germline inactivation of the adenomatosis polyposis coli (APC) gene in hereditary colorectal cancer is well known, being the most important cause of familial adenomatosus polyposis (FAP) syndrome. Hereditary cases with germline mutations, however, account only for 5-10% of colorectal cancers. The somatic inactivation of this gene has also been observed in sporadic cases. In order to examine the inactivation mechanisms of the APC gene we screened 70 sporadic colorectal cancer cases (27 rectal, 43 intestinal) of different stages for promoter hypermethylation, allelic imbalance (AI) and somatic mutations. The presence of promoter hypermethylation was observed in 21 cases (30%). Fifteen of the examined tumors (21%) showed AI, and also 15 tumors (21%) carried at least one somatic mutation. Thirteen of the detected alterations were novel variations: seven frameshifts, four missense mutations and two polymorphisms. Biallelic inactivation was found in 15 patients (21%). These results suggest that the inactivation of the APC gene is very common in sporadic colorectal cancer, and the main inactivation mechanism of the APC gene is promoter hypermethylation. Allelic imbalance has the same frequency as mutations, and mutations in the APC gene are more common in the early stages and in tumors located in the rectum.     

云南省家族性腺瘤性息肉病APC基因胚系突变的研究

目的研究云南省家族性腺瘤性息肉病(FAP)APC基因胚系突变的特点。方法收集云南地区10个FAP家系,抽取10例先证者的外周静脉血,提取脱氧核糖核酸(DNA),应用聚合酶链反应(PCR)方法扩增APC基因,应用DNA自动测序仪进行测序。结果 10例FAP先证者中,1例检出APC基因致病突变,此突变存在于APC基因第15外显子上c.3587 C>A(S1196X);随后对检出突变的先证者家系中另外8名成员进行该突变位点筛查,其中7人有突变。结论云南地区FAP患者APC基因致病突变检出率明显低于国内外报道;未检出APC基因致病突变的FAP患者可能存在其他发病的因素。