The authors evaluated the frequency of the carrier status of three ancestral colorectal neoplasm-associated mutations (APC:I1307K, BLMAsh, and MSH2*1906G>C) found in the Jewish population among a case series with documented colorectal neoplasms. They further compared family and personal histories plus environmental exposures of the carriers and noncarriers of the I1307K mutation and examined clinical differences with regard to the colorectal neoplasms and the specific molecular genetic changes in these lesions.
Analyses were performed on tissue from stored paraffin-embedded blocks for the three germline mutations plus the KRAS mutation and APC loss of heterozygosity (LOH) and APC gene sequencing.
Fifty-four of the 429 individuals (12.6%) were found to carry the APC:I1307K mutation, whereas 4 (0.9%) were found to be heterozygous for the BLMAsh mutation and 3 (0.7%) were carriers of the MSH21906G>C* mutation. Carriers of the I1307K mutation did not appear to differ from noncarriers with regard to the number of neoplasms, patient age at detection, or tumor location within the colon. There was no significant difference noted between I1307K carriers and noncarriers with regard to the percentage of patients with first-degree relatives with colorectal carcinoma. A significant risk for APC LOH was found in lesions from carriers who smoked cigarettes compared with nonsmokers. The I1307K mutation was found to be clearly associated with a somatic additional adenine insertion in the region of codons 1306–1309, but other mutations in the region of codons 1277–1348 were found to be no more prevalent in carriers than in noncarriers.
In Jewish individuals previously diagnosed with a colorectal neoplasm, MSH2*1906G>C is uncommon but has been associated with carcinoma occurring at a young age. The BLMAsh mutation is uncommon and appears to be of little effect. The I1307K mutation is common among Jews who have had colorectal neoplasms, but overall it was found to have little effect clinically in the current study group. There may be a gene–environment interaction between the I1307K mutation and cigarette use. Cancer 2005. © 2005 American Cancer Society.
Germline genetic changes are known to be associated with an increased risk of colorectal neoplasms.1 The genetics underlying the familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal carcinoma (HNPCC) syndromes have been elucidated and shown to be the result of mutations in the APC gene and genes involved in the mismatch repair pathway, respectively. However, evidence of further susceptibility genes remains to be identified.
The Ashkenazi population has been found to carry ancestral mutations for predisposition to a number of diseases including those associated with colorectal neoplasms. For example, one such genetic change, the APC:I1307K mutation, was first detected among Ashkenazi Jews. This variant is a T to A transversion at nucleotide 3920 of the APC gene that converts the sequence AAATAAAA to (A)8, resulting in a substitution of lysine for isoleucine at codon 1307 (I1307K). This change does not appear to affect the protein function; however, the (A)8 tract is unstable and may increase the rate of somatic mutations within the (A)8 region. Some studies have suggested this mutation confers an increased risk for the development of colorectal neoplasms. For example, Laken et al. estimated that this mutation nearly doubled the risk of colorectal neoplasia,2 although more recent (and larger) studies have suggested that the effect may have been overestimated by the earlier studies. Overall, the estimated increased risk of colorectal carcinoma has reportedly varied between 30–90%. However, to our knowledge, studies of adenomas have been limited in size, with no clear conclusion regarding an increase in the number of adenomas among carriers.3-5
Recently, an additional mutation contributing to colorectal carcinoma was identified within the Ashkenazi Jewish population. The MSH2*1906G>C mutation is a nucleotide substitution of cytosine for guanine at position 1906, resulting in a substitution of proline for alanine at codon 636 in the MSH2 protein. This mutation is uncommon and is estimated to explain 2–3% of colorectal carcinomas occurring in Ashkenazi patients with an age at diagnosis of < 60 years, although among mutation carriers the risk for colorectal carcinoma is high.6
Finally, Bloom syndrome is a rare autosomal recessive condition characterized by immunodeficiency, growth retardation, male sterility, and a predisposition toward many types of malignancy. Approximately one-third of individuals with Bloom syndrome are of Jewish descent and they carry the founder BLMash mutation, which is a frameshift mutation in exon 10 involving a deletion of ATC TGA at 2281 and an insertion of TAG ATT C. Recent reports have suggested that heterozygosity for BLMash is associated with a 2.3–2.8-fold increased risk for colorectal carcinoma; 11 of 334 (3.3%) patients with colorectal carcinoma among Ashkenazim living in New York were found to carry the mutation.7 However, others have questioned such a correlation.8
We identified patients of Jewish ancestry who were seen at two endoscopy suites in New Jersey who had had a colorectal neoplasm. Many were undergoing follow-up surveillance colonoscopy or an initial screening procedure. The current study was limited to individuals with a documented neoplastic lesion, on the assumption that such individuals would be more likely to have an underlying genetic predisposition. Individuals found to have an adenoma or carcinoma constitute the major target group for colonoscopic surveillance and represent a large percentage of the people undergoing colonoscopy. We evaluated the frequency of carrier status of the three mutations described above. We examined the characteristics of the cases, their family histories, and their lesions for differences between carriers and noncarriers with regard to the mutations, recognizing that the numbers of carriers of the BLMAsh and MSH2*1906G>C mutations were likely to be few. We also considered the exogenous influences of cigarette use and the daily use of vitamins, estrogens, and nonsteroidal antiinflammatory medications, which have been shown to affect the incidence of colorectal neoplasms.
MATERIALS AND METHODS
The institutional review board approved the current study. Informed consent was obtained in writing from all participants. Patients undergoing a colonoscopy at two large endoscopy suites between January 1, 2000 and March 30, 2003 were screened for eligibility. Permission to contact patients was obtained from the colonoscopists. Patient participation was dependent on verification of Jewish background, patient consent, and retrieval of stored paraffin-embedded colonic neoplastic tissue for analysis. Information was obtained by telephone interview regarding family history, personal medical history, and drug and cigarette use. Prior endoscopies were verified by reference to medical charts and pathology computerized records. We verified information on all patients spanning intervals up to 40 years. All information was entered into an independent computer by the interviewer. The pathology department associated with the endoscopy suites has retained all paraffin blocks since 1952 in a central warehouse. Right-sided lesions were defined as those removed from the cecum, as well as ascending and transverse colonic segments. Left-sided lesions were those removed from the descending, sigmoid, and rectal segments.
DNA was extracted from paraffin-embedded tissue using the QIAamp Tissue Kit (Qiagen, Valencia, CA). If necessary, DNA was extracted from peripheral leukocytes using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Purified DNA was reconstituted in 10 mM of Tris (pH of 8.0) buffer and placed in storage at 4 °C. A Beckman DU-20 spectrophotometer (Beckman Coulter, Inc., Fullerton, CA) was used to quantify the DNA. Assays for germline mutations were performed on DNA extracted from normal tissue in paraffin-embedded blocks. On six occasions, no normal tissue was present in the paraffin blocks and blood samples were obtained.
Mutation Analysis Of APC I1307K
The I1307K region was sequenced to screen for mutations. This method is preferred because of the number of other possible mutations that can occur in this region of the APC gene. Purified DNA was amplified using the primer set 5′-CCA ATA TGT TTT TCA AGA TGT AGT TC-3′ (sense) and 5′-AA TTC AAC AGC TTT GTG CCT-3′ (antisense). This primer set flanks the codon 1307 region of exon 15 of the APC gene and produces a 262 base product that effectively screens codons 1277–1348. Post polymerase chain reaction (PCR) product was purified for sequencing using the QIAquick PCR Purification Kit (Qiagen) and an aliquot was run on an agarose minigel to determine quality and estimate quantity.
PCR-based DyeDeoxy Terminator sequencing was performed using an ABI Prism 377 DNA Sequencing System® (Applied Biosystems, Foster City, CA). The sequencing reaction was performed with the BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems) and 5 pmol of either of the sense or antisense primer. Sequence reaction products were cleaned using AutoSeq G-50 spin columns (Amershan Pharmacia, Piscataway, NJ) and separated on a 5% Long Ranger acrylamide gel (Cambrex Bio Science Rockland, Inc., Rockland, ME).
Mutation Analysis of MSH2*1906G>C and BLMAsh
Single-strand conformational polymorphism (SSCP) analysis was used to screen for the MSH2*1906G>C and BLMAsh 2281delATC TGA insTAG ATT C mutations. The primers used for the MSH2*1906G>C reaction were: 5′-AGC ACC TGT TCC ATA TGT ACG A-3′ (sense) and 5′-AAT GCA ATT TCA TCT TGA ACT TC-3′ (antisense).6 The primers used for the BLMAsh reaction were 5′-CTA CTT TTA TAC TTA GAT TCC AGC-3′ (sense) and 5′-TGG AGG TAA ATA TTT GTA GCT-3′ (antisense).8 Within each primer set the sense and antisense primers are 5′-labled with different fluorescent tags.
The reaction and gel conditions for the MSH2*1906G>C and BLMAsh SSCP assays were the same. PCR reactions were performed in 30-μL volumes using Applied Biosystems reagents (Roche Molecular Systems, Inc., Branchburg, NJ). Six picomoles of each primer and a 1.5 mM of MgCl2 concentration were used in the PCR reactions. Reactions were run on a PE 9700 thermocycler (PE Applied Biosystems, Foster City, CA) under the following conditions: 6 minutes denaturation at 94 °C followed by 36 cycles of a 30-second denaturation at 94 °C, 25-second annealing at 55 °C, and a 50-second elongation at 72 °C, with a final 30-minute extension at 72 °C.
After amplification, the PCR products were mixed with gel-loading solution and heated to 95 °C for 5 minutes followed by flash cooling in ice water. PCR products were separated on a 0.6X MDE acrylamide gel (Cambrex Bio Science Rockland, Inc.) and run for 7 hours at 15 watts of constant power and 25 °C constant temperature. The samples were analyzed on an ABI Prism 377 DNA Sequencer with GeneScan collection software (PE Applied Biosystems). Normal samples were run simultaneously with neoplastic specimens to identify the wild-type SSCP banding pattern. Samples with mutations show additional bands with different electrophoretic mobilities. Each sample with a mutant SSCP band was sequenced to verify the point mutation. The mutation sites were reamplified with the respective untagged primers, purified, and sequenced as described in the mutation analysis for the I1307K section.
Loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene was determined through the PCR amplification of a CA repeat marker within the D5S346 locus of the DP1 gene using the fluorescent-labeled primer set: 5′– ACT CAC TCT AGT GAT AAA TCG GG-3′ (sense) and 5′– AGC AGA TAA GAC AAG TAT TAC TAG TT-3 (antisense).9 Reactions were run on a PE 9700 thermal cycler (PE Applied Biosystems) under the following conditions: 6 minutes denaturation at 94 °C, 36 cycles of a 30-second denaturation at 94 °C, 30-second annealing at 55 °C, and a 1-minute elongation at 72 °C, with a final 30-minute extension at 72 °C.
SSCP analysis was employed to screen for mutations within the KRAS oncogene. The codon 12/13 region of exon 1 in the KRAS oncogene was amplified using the primer set 5′-CCT GCT GAA AAT GAC TGA AT-3′ (sense) and 5′-TGT TGG ATC ATA TTC GTC CA-3′ (antisense).9 Samples showing KRAS mutation bands by SSCP were sequenced to verify the point mutation. PCR-based DyeDeoxy Terminator sequencing was performed using an ABI Prism 377 DNA Sequencing System (Perkin Elmer, Foster City, CA).
Lesions were evaluated for microsatellite instability using a panel of three microsatellite markers: the mononucleotide repeat BAT-26 and the dinucleotide repeats D5S346 and D18S58.10, 11 PCR primers for each marker were used to amplify DNA from normal and abnormal tissue. The amplified products were assayed side by side on an electrophoresis gel. Microsatellite instability for a given primer set was defined as the appearance of one or more new PCR products that were either larger or smaller than that produced by the DNA derived from normal tissue.
The frequencies of MSH2*1906G>C, BLMAsh, and APC:I1307K carriers were compared for patients with adenomas only and those with any colorectal neoplasm using the Fisher exact test. The characteristics of the patients and of the neoplasms were compared for those patients who were carriers versus those who were noncarriers of the I1307K mutation. Continuous variables were compared using the Student t test, and categoric variables were compared with contingency table analysis. Logistic regression was used to assess the odds ratio (OR) for APC LOH in the lesions of the patients by I1307K status, controlling for multiplicity of lesions and for most advanced histology. The general estimating equations (GEE) methods were used to compare the neoplasm characteristics of histology, location, APC LOH, and KRAS mutation, taking into account the correlation within a patient with multiple lesions over time.12 All analyses were performed using Stata statistical software (StataCorp LP, College Station, TX).
During the study interval, 38,674 individuals were seen at the 2 endoscopy suites, 10,176 of whom (26.3%) were Jewish. A total of 1056 patients (10.4% of the Jewish patients) had stored tissue blocks from either previous or current colonoscopies for which the retrieval rate from the warehouse was 98%. A total of 429 patients (40.6%) agreed to participate. Those not participating included 172 patients people who declined to participate after hearing about the study (16.3%), 375 patients who failed to return an initial telephone call (35.5%), and 80 patients who did not return the requested consent form despite having indicated a willingness to participate (7.5%).
There were 429 participants, all of whom identified themselves as Ashkenazim, which is consistent with the demographics of the location of the endoscopy clinics. Approximately 16% of the patients were undergoing their first colonoscopic screening, whereas 72% were undergoing a follow-up surveillance colonoscopy. Approximately 11% of the patients were being evaluated for specific symptoms and for 1% the indication was not clearly stated. There were 269 male patients (62.7%) and 160 female patients (37.3%) for a male:female ratio of 1.7. Of the 429 study participants, 417 (97.2%) were 100% Ashkenazi, whereas 11 participants (2.6%) were 50% Ashkenazi and 1 patient (0.2%) was 25% Ashkenazi. The majority of patients (355 [82.8%]) had a history of 1 or more adenomas only whereas 74 patients (17.2%) had a colorectal carcinoma (with or without an adenoma). Among those with a carcinoma, 69 patients had 1 carcinoma, 4 patients had 2 carcinomas, and 1 patient had 3 carcinomas. Among the 80 carcinomas, 24 were in situ carcinomas in a residual adenoma, and 56 were described pathologically as carcinomas with no evidence of residual adenoma; 12 carcinomas were rectal carcinomas and 68 were colonic carcinomas. There were 780 adenomas, 666 of which were classified histologically as tubular, 95 as tubulovillous, and 19 as villous. One lesion proved to be a hyperplastic polyp, and two lesions that were tubular/hyperplastic polyps were considered as tubular for the purposes of the analysis. In total, the patients reported 1512 colonoscopies; specific results were unknown for only 75 of the colonoscopies (5.0%).
Three patients were found to carry the MSH2*1906G>C mutation, all of whom were heterozygous, for a prevalence of 0.7%. All three were males with cecal carcinoma detected at ages 55 years, 46 years, and 41 years, respectively (average age, 47 years) (Table 1). Two had no family history of colorectal carcinoma. The father of the third patient had had colorectal carcinoma (diagnosed at age 59 years), as did this proband's paternal uncle (diagnosed at age 56 years) and the uncle's son (diagnosed at age 43 years). None of the 309 individuals with only adenomas carried this mutation, resulting in a significant difference in carrier rates between those patients with adenomas and those with malignancy (P = 0.005) (Table 1).
|Genetic statusa||Frequency among 355 adenoma-only patients (%)||Average age at first adenoma (years)||Frequency among 74 carcinoma patients (%)||Average age at cancer diagnosis (yrs)||P value for equality of carrier frequency between adenomas and carcinomab|
|MSH2* I906G >C carrier||0 (0.0%)||—||3 (4.1%)||47.0||0.005|
|BLMAsh carrier||4 (1.1%)||69.8||0 (0.0%)||—||1.00|
|APC:I1307K carrierc||42 (11.8%)||62.3||12 (16.2%)||66.2||0.34|
|No mutation detected||309 (87.0%)||63.0||59 (79.7%)||65.3||0.10|
- a No patient was found to have more than one mutation.
- b The P value was = 0.016 for comparison of MSH2* I906G >C versus BLMAsh or APC:I1307K.
- c Includes two patients who were homozygous.
All three carcinomas demonstrated microsatellite instability with all three markers. One individual also had two tubulovillous adenomas simultaneously with the carcinoma that were microsatellite stable by the three markers used; one of these two tubulovillous adenomas had a KRAS mutation (GGT to GAT, codon 12) and one did not. Neither lesion demonstrated APC LOH. The second individual had no adenomas. The third individual had three simultaneous right colon adenomas, two of which were tubulovillous and one of which was tubular. One tubulovillous adenoma was analyzed and demonstrated microsatellite instability by BAT-26, but not by the other two markers; it did not demonstrate any KRAS mutation nor APC LOH. In view of the limited number of mutation carriers, no further analysis of this group was conducted.
Four patients (3 females and 1 male) were found to be heterozygous carriers of the Ashkenazi Bloom mutation, for a prevalence of 0.9%. All had only adenomas; three patients had two tubular adenomas each and the fourth had three tubular adenomas. The average age at the time of detection of the first adenoma was 66.2 years (Table 1). Just one tubular adenoma demonstrated a KRAS mutation and none demonstrated APC LOH. In view of the limited number of mutation carriers, no further analysis of this group was conducted.
APC:I1307K Mutation: Clinical Information
Fifty-four of the 429 participants (12.6%) were found to have the APC:I1307K mutation, all of whom carried wild-type for BLMAsh and MSH2*1906G>C. Two individuals were homozygous and 52 were heterozygous. The size of this subgroup permitted further analyses, and comparisons were made with those individuals who were noncarriers for all three mutations. There was no statistically significant difference noted between those patients with and those without the I1307K mutation with regard to gender, incidence of colorectal carcinoma or other malignancies, and the average number of adenomas detected per colonoscopy (Table 2).
|Subgroup||Total no.||I1307K positive No. (%)||I1307K negative No. (%)||P value||ORsb (95% CI)|
|All participants||422||54 (12.8)||368 (87.2)|
|Male||265||35 (13.2)||230 (86.8)||0.74||1.11 (0.61–2.01)|
|Female||157||19 (12.1)||138 (87.9)||1.0|
|Average age at time of first neoplasm diagnosis||62.1 yrs||62.9 yrs||0.62|
|Average no. of adenomas/colonoscopies per patient||0.82||0.77||0.61|
|Personal history of colorectal carcinoma|
|Yes||71||12 (16.9)||59 (83.1)||0.25||1.50 (0.74–3.01)|
|No||351||42 (12.0)||309 (88.0)||1.0|
|Personal history of cancer other than colorectal carcinoma|
|Yes||58||10 (17.2)||48 (82.8)||0.28||1.52 (0.72–3.21)|
|No||364||44 (12.1)||320 (87.9)||1.0|
|In at least one lesion||105||18 (17.1)||87 (82.9)||0.11||1.66 (0.89–3.09)|
|All lesions normal||307||34 (11.1)||273 (88.9)||1.0|
|In at least one lesion||96||11 (11.5)||85 (88.5)||0.65||0.85 (0.42–1.72)|
|All lesions normal||325||43 (13.2)||282 (86.8)||1.0|
- ORs: odds ratios; 95% CI: 95% confidence interval; LOH: loss of heterozygosity.
- a Seven patients with either the BLMAsh or hMSH2* mutations were excluded.
- b An odds ratio of = 1.0 indicates the referent group for comparison.
- c For nine patients, the APC markers used for the study of loss of heterozygosity were uninformative or had a technical failure whereas for one patient no material was available.
- d No material was available for one patient.
Based on patient history as well as medical and pathology records, we ascertained that patients had undergone 1 or more colonoscopies with the following frequencies: 1 colonoscopy, 13.7%; 2 colonoscopies, 23.5%; 3 colonoscopies, 22.8%; 4 colonoscopies, 19.0%; 5 colonoscopies, 9.5%; 6 colonoscopies, 5.4%; 7 colonoscopies, 3.1%; 8 colonoscopies, 1.9%; 9 colonoscopies, 0.2%; and ≥ 10 colonoscopies, 0.95%. The average time interval between the colonoscopies did not appear to differ between the carriers and noncarriers. For example, the average time interval between the first and fourth colonoscopies was 7.7 years for the carriers and 8.9 years for the noncarriers (P = 0.27). The mean number of colonoscopies for those patients with the APC:I1307K mutation was 3.7, and the average number of colonoscopies for those without the mutation was 3.3 (P = 0.16). The mean number of neoplasms detected on each successive colonoscopy also did not differ between the carriers and the noncarriers for their first and second colonoscopies, but there were more lesions detected on the third and fourth colonoscopies among the carriers (38 carriers and 25 carriers underwent a third or fourth colonoscopy, respectively). For carriers, the mean numbers of neoplasms were first colonoscopy, 1.4 neoplasms; second colonoscopy, 0.7 neoplasms; third colonoscopy, 0.9 neoplasms; and fourth colonoscopy, 0.8 neoplasms. For noncarriers, the mean numbers and P values were: first colonoscopy, 1.2 neoplasms (P = 0.25); second colonoscopy, 0.7 neoplasms (P = 1.00); third colonoscopy, 0.5 neoplasms (P = 0.02); and fourth colonoscopy, 0.4 neoplasms (P = 0.01). It is possible that this finding indicates a greater tendency for I1307K carriers to develop new neoplasms.
The age at which a patient was first detected with a neoplastic lesion did not appear to differ between those with and those without the I1307K mutation (Table 2), nor when considered separately for each gender (females, 63.0 years and 64.6 years, respectively [P = 0.56] and males, 61.7 years and 61.9 years, respectively [P = 0.88]). Carriers and noncarriers of the I1307K mutation were found to be similar with regard to age at the time of colorectal carcinoma diagnosis (66.2 years for the carriers and 65.3 years for the noncarriers [P = 0.81]) (Table 1). Furthermore, there was no significant difference noted with regard to the patients' age at the time of the diagnosis of the first adenoma, for those with or without the I1307K mutation (Table 1).
APC:I1307K: Family History Data
There was no significant difference noted between those patients with and those without the I1307K mutation with regard to family history of a first-degree relative with colorectal carcinoma, other tumors, or an adenoma. Furthermore, there was no difference noted between those patients with and those without the I1307K mutation and at least two relatives diagnosed with colorectal carcinoma and/or an adenoma (Table 3).
|Characteristic||Total no.||I1307K positive No. (%)||I1307K negative No. (%)||P value|
|First-degree relative with CRC|
|Yes||139||16 (11.5)||123 (88.5)|
|No||283||38 (13.4)||245 (86.6)||0.58|
|First-degree relative with other cancer|
|Yes||177||26 (14.7)||151 (85.3)|
|No||245||28 (11.4)||217 (88.6)||0.32|
|First-degree relative with adenomas|
|Yes||88||10 (11.4)||78 (88.6)|
|No||334||44 (13.2)||290 (86.8)||0.65|
|Two or more relatives with CRC and/or adenomas|
|Yes||88||9 (10.2)||79 (89.8)|
|No||334||45 (13.5)||289 (86.5)||0.42|
- CRC: colorectal carcinoma.
APC:I1307K: Patient Genotyping
All patients carrying the I1307K mutation also carried a particular allele of the APC marker DP1 (D5S346) that we denoted as A10.13 For the group of 374 noncarriers of the I1307K mutation, the frequency of the A10 allele was only 17.9%. Genotyping for alleles of the APC marker D5S1385 (MC-11) also was performed for a limited number of patients. We identified seven alleles for this locus in our population. Seventeen carriers of the I1307K mutation were typed for D5S1385 15 of whom (88.2%) carried an allele we designated the A4 allele, whereas the remaining 2 (11.8%) carried the designated A6 allele. By contrast, of 32 noncarriers who were genotyped, 5 (15.6%) were found to have the A4 allele.
Analysis of Lesions
The carrier status was not found to differ based on the histologic type of the lesion when considering correlations within each patient because of multiplicity (Table 4). Multivariate analysis demonstrated no additional effect on histologic type based on age at first lesion, gender, or number of lesions (data not shown). The average number of adenomas per patient per colonoscopy performed was similar (0.82 for those with the I1307K mutation and 0.77 for those without; P = 0.61) (Table 2). There were 131 lesions from the patients with an I1307K mutation for which the exact location was known, and 2 lesions of an unknown site. Of these 131 lesions, 73 (55.7%) were from the left side of the colon and 58 (44.3%) were from the right side of the colon. A total of 708 lesions were from those individuals without the I1307K mutation; 358 (50.6%) were from the left side and 350 (49.4%) were from the right side. There was no statistical difference noted between the two groups with regard to the percentage of lesions obtained from each side (P = 0.28) (Table 4) The distribution of adenomas based on colorectal segment was similar for the 2 groups (carriers and noncarriers): cecum, 11.4% and 12.8%, respectively; ascending, 14.5% and 16.4%, respectively; transverse, 18.3% and 20.2%, respectively; descending, 16.8% and 11.7%, respectively; sigmoid, 27.5% and 29.7%, respectively; and rectum, 11.4% and 9.2%, respectively.
|Characteristic||Total no.b||I1307K positive||I1307K negative||P value|
|Total no. of lesions||No.||(%)||No.||(%)|
|Carcinoma (including in situ)||71||12||(18.3)||58||(81.7)|
|Tubulovillous and villous||111||17||(15.3)||94||(84.7)|
- LOH: loss of heterozygosity.
- a Seven patients with MSH2* I906G >C or BLMAsh mutations were excluded.
- b Information was not available for all lesions.
The likelihood of a patient having at least one lesion with LOH of the APC gene was marginally higher in those with I1307K compared with those without (17.1% vs. 11.1%; P = 0.11) (Table 2). There was no significant difference noted in the prevalence of APC LOH based on histology for carriers and noncarriers; among those patients with the I1307K germline mutation, APC LOH was detected in 5 of 12 carcinomas (41.7%), 3 of 16 villous and tubulovillous adenomas (18.8%), and 18 of 96 tubular adenomas (18.8%). The comparable data for those without the I1307K mutation were 18 of 56 carcinomas (32.1%), 24 of 89 tubulovillous and villous adenomas (27%), and 62 of 538 tubular adenomas (11.5%). For all lesions, there was no difference noted with regard to APC LOH prevalence between carriers and noncarriers when taking into account correlations within each patient for whom several lesions were studied (P = 0.19) (Table 4). The 24 informative lesions with APC LOH from I1307K carriers revealed 4 lesions had lost the APC allele containing the I1307K mutation whereas 20 lost the wild-type APC allele (P < 0.02 for equal rates of LOH among wild-type and mutation-bearing chromosomes).
APC LOH was not associated with the number of adenomas, number of colonoscopies, or patient age at the time of the first colonoscopy (data not shown). We investigated the interaction between the I1307K mutation and several separate exogenous influences on the presence of APC LOH. Specifically, we found no significant effect from multivitamin use, nonsteroidal antiinflammatory drug use, or the use of hormone replacement therapy (data not shown). However, there was a significantly increased risk for APC LOH among cigarette smokers who were I1307K carriers (OR, 3.27; P = 0.03 [95% confidence interval (95% CI), 1.12–10.53]). There was no excess risk noted for a lesion with APC LOH in I1307K carriers who did not smoke (OR, 0.50; P = 0.20 [95% CI, 0.24–1.33]).
APC Somatic Mutations
We performed sequencing of the I1307K region (codons 1277–1348) for 120 lesions from 54 patients who were carriers of the I1307K germline mutation. Forty-nine of the 120 lesions (40.8%) contained a somatic mutation in this region, 40 of which were an insertion of adenine in the 1306–1309 region and 9 that represented other genetic changes (Table 5). One carrier of the I1307K mutation was found to have an additional germline mutation in codon 1317 of G to C because this change was present in the normal tissue as well as the neoplastic tissue. The 9 nt3921insA mutations (5 point mutations and 4 ins/del mutations) represent only 7.5% of all 120 lesions and all were protein truncating (Table 5.) For the noncarriers, a random sample of 297 lesions were similarly sequenced and 35 lesions (11.8%) were found to contain a mutation in the sequenced region (Table 6). The frequency of nt3921insA somatic mutations was not found to be statistically different between the carriers and noncarriers (OR, 0.61; P = 0.20 [95% CI, 0.28–1.30]).
|Adenoma (36)||1306–1308||A insertion|
|Carcinoma (4)||1306–1308||A insertion|
|Carcinoma||1309||Point mutation G → T|
|Carcinoma||1311||1 base pair insertion|
|Carcinoma||1356||Point mutation C → G|
|Tubular adenoma||1287||1 base pair T insertion|
|Tubular adenoma||1300||1 base pair deletion|
|Tubular adenoma||1310||2 base pair deletion|
|Tubular adenoma||1284||Point mutation G → T|
|Tubular adenoma||1317||Point mutation G → T|
|Tubulovillous adenoma||1328||Point mutation C → T|
|Carcinoma (2)||1305||1 bp ins 1306|
|1306||G → T|
|Tubulovillous and villous (7)||1305||1 bp del|
|1311||1 bp del|
|1309–11||5 bp del|
|1297||7 bp del |
|1306||G → T |
|Tubular (26)||1282||1 bp del|
|1287||11 bp del|
|1297||2 bp del|
|1297||7 bp del|
|1297||11 bp del|
|1300||1 bp del|
|1300||11 bp del|
|1309||2 bp del|
|1309||5 bp del |
|1310||2 bp del|
|1311||8 bp del|
|1319||1 bp del |
|1324||1 bp del|
|1324||17 bp del|
|1291||C → T|
|1306||G → T |
|1317||G → C |
- bp: = base pair; ins: insertion; del: deletion.
Of the 40 lesions from I1307K carriers with a 3921insA, 12 lesions (30%) demonstrated APC LOH and all had loss of the allele that did not carry the I1307K mutation. Of the 9 lesions from carriers with a mutation other than the 3921insA, 3 (33%) had APC LOH, 2 of which (68%) lost the allele that carried the I1307K mutation whereas 1 lost the allele not carrying the I1307K mutation. For the 35 lesions from I1307K noncarriers with a demonstrated somatic mutation, 12 also had LOH of the APC gene, 10 of which lost the allele that did not carry the I1307K mutation and 2 of which lost the allele with the I1307K mutation.
There was no significant difference noted between carriers and noncarriers with regard to their lesions containing at least one KRAS mutation when considering correlations within each patient for multiplicity of lesions (P = 0.65) (Table 2). Among the carriers, a KRAS mutation was detected in 3 of 13 carcinomas (23.1%), 8 of 17 tubulovillous and villous adenomas (47.1%), and 2 of 100 tubular adenomas (2%). The comparable data for those patients without the I1307K mutation were 29 of 58 carcinomas (50%), 46 of 94 tubulovillous and villous adenomas (48.9%), and 51 of 553 tubular adenomas (9.2%).
No significant effect from the use of multivitamins, nonsteroidal antiinflammatory drugs, or hormone replacement therapy was found with regard to the interaction between the I1307K mutation and the presence of a KRAS mutation (data not shown). Cigarette use was associated with a decreased risk of KRAS mutations in the lesions of smokers who were I1307K carriers, but this did not achieve statistical significance (OR, 0.20; P = 0.16 [95% CI, 0.02–1.87]). KRAS mutation was not found to be associated with the number of adenomas, the number of colonoscopies the patient had undergone, or the patient's age at the time of the first colonoscopy (data not shown).
The majority of colorectal lesions detected by colonoscopy among the Jewish population of New Jersey are tubular, and not advanced villous adenomas or carcinomas. All the individuals we studied had an adenoma(s) and/or carcinoma and therefore may have some undetected inherited predisposition. Nonetheless, among this population are individuals carrying both common and uncommon mutations of varying penetrance and clinical significance that currently can be identified. The MSH2*1906G>C mutation and the BLMash mutation were both present in approximately 1% of the current study population. The data from the current study support the view that MSH2*1906G>C is highly penetrant, with all three individuals found to have colon carcinomas diagnosed at a young age, although only one patient had a close family history of colorectal carcinoma. Our data indicate that carriers of MSH2*1906G>C are more likely to have carcinoma than carriers of the I1307K or BLMAsh mutations. The four individuals identified as carriers of the BLMash mutation had only tubular adenomas. A previous report described one individual with BLMAsh syndrome and multiple colonic adenomas.14 However, Cleary et al. found no difference in the incidence of this mutation between 125 patients with adenomas and controls, even though the study was definitely underpowered to detect such an effect.8
To our knowledge, several articles published to date have evaluated the I1307K germline genetic mutation since the original report was published in 1997.2 These studies have shown this mutation to be present in approximately 6% of all Ashkenazi Jews, as well as others with whom a common ancestor was most likely shared, such as Sephardic Jews and Yemenites. The incidence of the I1307K mutation in patients with colorectal carcinoma or adenoma has been reported to be approximately 10–15%.3, 4, 15 We found a similar rate of 12.5% for a population in which all the patients had colorectal neoplasms. In previous studies, all carriers were shown to share a common haplotype that was indicative of a founder effect.16 Age estimates indicate that I1307K existed at about the time of the beginning of the Jewish diaspora, approximately 2200–2950 years ago.17 The mutation is rarely found in populations unrelated to Jews.18
Carriers of the I1307K mutation have been reported to have an increased risk of colorectal carcinoma, with an OR of 1.9,19 or an increased risk of combined colorectal carcinoma and adenomas, with a relative risk of 1.72.4 We did not study a population without colorectal neoplasm and therefore do not know the incidence of the mutation in our general population of individuals who were disease free.
There have been reported differences regarding the clinical features of colorectal neoplasms between carriers and noncarriers of the I1307K mutation. Some authors have reported no difference in phenotypic features3, 5, 20 or family history.3 Others have shown specific differences, such as increased numbers of adenomas and carcinomas in carriers,4 neoplasms occurring at a younger age in carriers,21 and carcinoma more likely to be located in the distal colon.22 We did not detect any differences between carriers and noncarriers with regard to the number of neoplasms, patient age at the time of detection, or location within the colon. In the current study, the estimated effect of a previous history of colorectal carcinoma was found to be slightly increased for carrier of the I1307K mutation (OR, 1.50; 95%CI, 0.74–3.01) with almost precisely the same estimates for a personal history of noncolorectal carcinoma but with a broad 95% CI, and therefore our results are consistent with no effect.
Relatives of carriers of the I1307K mutation have been reported to be more likely to have a previous diagnosis of colorectal carcinoma (OR, 1.3) as well as any type of tumor (OR, 1.2).19 Others have not reported any difference in family history.3 We found no evidence in the current study of an increased risk with a positive family history, although we could not exclude an effect as great as a 1.6-fold increased risk of a positive family history of colorectal carcinoma being associated with being an I1307K carrier.
In one study, the lifestyle parameters of alcohol intake, low physical activity, vitamin and antioxidant intake, and cigarette use did not appear to have a statistically significant effect on the interaction of risk for colorectal carcinoma and the carrier state.20 However, there was a (nonsignificant) suggestion of an interaction between being an I1307K carrier and smoking in terms of the risk of colorectal neoplasm. Our findings were similar with regard to the use of vitamins, nonsteroidal antiinflammatory drugs, and hormone replacement therapy. However, we found a significantly increased risk for APC LOH in those I1307K carriers who smoked. APC LOH is postulated to be an early molecular change in the carcinogenesis of colorectal carcinoma. The combined effects of the I1307K mutation and cigarette smoking may represent an example of gene-environment interaction that, over time, is critical for the initiation of the neoplastic process. We found no significant effect on the risk for KRAS mutations in carriers of the I1307K mutation who smoked.
The I1307K mutation has been shown to create an unstable tract of eight adenines, resulting in a small hypermutable region of the APC gene. Several additional genetic changes on the affected allele have been identified, particularly: 1) an A insertion in the 1306-9 region; 2) a G deletion at 1314; 3) a G to T change at codon 13092 as well as several other point mutations18; and 4) wild-type allele LOH.23 We also found these changes, as well as other genetic changes within the immediate vicinity of codon 1307.
One study found that, in a small group of patients with colorectal carcinomas, there was no difference noted between carriers and noncarriers with regard to p53 immunostaining; loss of B-catenin, E-cadherin, and deleted colorectal cancer (DCC) expression; or KRAS mutation frequencies.24 We found no difference between carriers and noncarriers with regard to their lesions containing at least one KRAS mutation.
We believe the results of the current study confirm that MSH2*1906G>C is uncommon but highly penetrant and results in carcinoma occurring at an early age. The BLMAsh mutation is uncommon and appears to have little to no apparent significant clinical effect. The APC:I1307K mutation is common among Ashkenazi patients with colorectal neoplasms. The combination of the findings of the current study and the literature are consistent with a modest increase in the risk of colorectal neoplasia. Furthermore, the similarity noted in the characteristics of the neoplasms in mutation carriers and noncarriers suggests that the clinical effect of this mutation is limited. However, the data suggest that this mutation may predispose patients to the more rapid development of new lesions. There also may be a gene-environment interaction between the I1307K mutation and cigarette use. Molecular analyses indicate the I1307K mutation is clearly associated with an additional adenine insertion in the region of codons 1306–1309, but other mutations within the region between codons 1277–1348 do not appear to be any more prevalent in carriers than in noncarriers. Despite the limited overall clinical impact of the I1307K, BLMAsh, and MSH2*1906G>C* mutations, it is nevertheless important that efforts continue to identify possible susceptibility germline mutations within the general population because other germline mutations may prove to be of widespread somatic importance.
The authors wish to thank Dr. Errol Berman for his review of histologic slides and Dr. George Chong of the Sir Mortimer B. Davis Jewish General Hospital (Montreal, Quebec, Canada) for assistance with the MSH2*1906G>C assay.
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