A636P testing in Ashkenazi Jews

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1 Familial Cancer 3: , Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands. A636P testing in Ashkenazi Jews Jose G. Guillem 1, Harvey G. Moore 1, Crystal Palmer 2, Emily Glogowski 1,2, Rob Finch 2, Khedoudja Nafa 2, Arnold J. Markowitz 2, Kenneth Offit 2 and Nathan A. Ellis 2 Departments of 1 Surgery and 2 Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA Key words: Ashkenazi Jewish, A636P, hereditary nonpolyposis colorectal cancer, mismatch repair Abstract Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited colorectal cancer syndrome attributable to mutations in one of several DNA mismatch repair genes, most commonly MLH1 and MSH2. In certain populations, founder mutations account for a substantial portion of HNPCC. In this report we summarize the literature and our personal experience testing for a specific founder mutation in the Ashkenazi Jewish population, MSH2*1906G>C, also known as A636P. Although rare in the general population, the A636P mutation is detected in up to 7% of Ashkenazi Jewish patients with early age-of-onset colorectal cancer, and may account for up to one third of HNPCC in the Ashkenazi Jewish population. In addition, we summarize our initial experience with a prospective A636P testing protocol aimed at Ashkenazi Jewish patients at high or intermediate risk for harboring the A636P mutation. Introduction A hereditary predisposition to colorectal cancer is present in the syndromes familial adenomatous polyposis (FAP), which is caused by mutations of the APC gene, and hereditary nonpolyposis colorectal cancer (HNPCC), which is caused by mutations in the mismatch repair (MMR) genes, predominantly MSH2, MLH1, and MSH6 [1 5]. In some populations, a founder mutation can explain a significant fraction of diseasecausing mutations segregating in the population that cause a particular inherited disease. For example, several founder mutations have been reported for genes associated with HNPCC, including in Newfoundland an exon 5 MSH2 mutation (MSH2*IVS5+3A>T) [6], in Finland two unique MLH1 mutations, one a 3.5-kilobase genomic deletion comprising exon 16 and the other an exon 6 splice site mutation (MLH1*3.5kbdel exon 16 and MLH1*IVS5-1G>A) [7], in the Valais region of Switzerland an exon 19 MLH1 nonsense mutation (MLH1*2141G>A, T714X) [8], in the Southern region of China an exon 9 MSH2 frameshift mutation (MSH2* delAATG) [9], and a recently described 16 kb deletion of MSH2 in the United States [10]. In the Ashkenazi Jewish population, a mutation in the APC gene that changes a T base to an A base at nucleotide residue 3920 in the cdna, referred to as the I1307K mutation, has been identified as a founder mutation [11, 12]. The I1307K allele, which is present in approximately 6% of healthy Ashkenazi Jews, causes a fold increase in the risk of developing colorectal cancer [12]. Another founder mutation that was recently described in the Ashkenazi Jewish population involves the MSH2 gene. The MSH2*1906G>C mutation is a missense mutation that causes the substitution of a proline for an alanine at amino acid residue 636 (thus A636P) in the MSH2 protein. This allele is the rarest founder mutation identified to date, being present in less than 0.22% of healthy Ashkenazi Jews. The identification and characterization of founder mutations is not only of research interest but has clinical implications in that ethnic-specific analysis can be performed before a more widespread search for disease-causing mutations is initiated [13]. Because testing for founder mutations is simpler and less costly than testing for unspecified mutations in the entire gene sequence, the threshold for recommending testing is lower and many more patients can be tested easily. In this article, we review what is known about the A636P mutation from population-based and clinic-based casecontrol studies. The current information suggests that any Ashkenazi Jew who develops colorectal cancer at or before age 50 or with a family history of HNPCCrelated cancers should be tested for the A636P mutation. Correspondence to: Nathan A. Ellis, PhD, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. Tel: ; Fax: ; ellisn@mskcc.org

2 224 J. G. Guillem et al. Identification and characterization of the A636P founder mutation The A636P mutation was originally described in 1997 in a single family meeting Amsterdam criteria; however, there was incomplete evidence at the time that the mutation was disease-causing. Initial evidence supporting the pathogenicity of the A636P mutation included the lack of the mutation in a series of 100 Ashkenazi Jewish patients not known to have colorectal cancer, the absence of any other mutation in MSH2 or MLH1 in affected persons who carried the A636P mutation, and evidence that the mutant MSH2 protein was non-functional in a yeast mismatch repair assay [14]. It was shown subsequently that the addition of a single human chromosome containing the MSH2*1906G>C mutation did not correct the mismatch repair deficiency of mouse Msh2 )/) cells [15]. Although these data were strongly suggestive, further genetic studies were required to establish definitively that the A636P mutation was disease causing. A636P mutation and HNPCC Subsequent studies showed that the A636P mutation accounts for a significant portion of HNPCC in the Ashkenazi population. The mutation was detected in 15 unrelated Ashkenazi families who presented to genetics clinics with a family history consistent with hereditary colon cancer. Of these 15 families, 9 met Amsterdam criteria for HNPCC [13]. In a group of 109 kindreds systematically ascertained at Memorial Sloan-Kettering Cancer Center between 1995 and 2001, 37 were of Ashkenazi descent, including 12 that fulfilled Amsterdam criteria for HNPCC. The remaining 25 Ashkenazi patients met HNPCC-like criteria (a family history suspicious for HNPCC but not fulfilling Amsterdam Criteria) or Bethesda criteria. Overall, the A636P mutation was detected in 6 of the 37 families (16%). Of the 12 families that met the Amsterdam criteria, 6 had a mutation in either MSH2 or MLH1, including 4 with the A636P mutation. Thus, the A636P mutation accounted for two thirds of the HNPCC cases in which an MMR mutation was identified and for one third of the kindreds meeting Amsterdam criteria overall [13]. The colorectal cancers of A636P carriers were shown to exhibit microsatellite instability (MSI), and they also demonstrated highly reduced or absent expression of the MSH2 protein when assessed by immunohistochemical methods [13]. Moreover, in those families in which multiple affected persons were available for analysis, the mutation segregated with disease status. Altogether, these data indicated that the A636P mutation is diseasecausing. Molecular genetic analysis of DNA markers in the region surrounding the MSH2 gene in Ashkenazi Jews who carried the A636P mutation demonstrated the mutant chromosomes shared marker alleles in a physical region of over 10 million base pairs. These data indicated that the mutation arose in a single founder individual. Because the region of allele sharing is quite large relative to the size of regions surrounding other founder mutations, we speculate that the mutation arose relatively recently (approximately generations ago). The present day carriers of the A636P mutation do not know themselves to be related; however, with respect to this portion of chromosome 2 surrounding the MSH2 gene, carriers are in fact cousins. A636P mutation in Jewish colorectal cancer cases In a total of 1345 Ashkenazi Jews affected with colorectal cancer (including a series of 108 consecutive cases from the Memorial Sloan-Kettering Cancer Center), eight A636P mutation carriers were identified (0.6%) [13]. In comparison, the mutation was not detected in any of 566 and 1022 unaffected Ashkenazi Jewish patients from Israel and New York, respectively (0/1588, P ¼ ) [13]. Thus, although the A636P mutation is very rare in the Jewish population, it is significantly more frequent in colorectal cancer cases than in healthy controls. These data contribute further evidence to the conclusion that the A636P mutation is disease-causing. A636P and early age-of-onset colorectal cancer Patients with early age-of-onset colorectal cancer comprise a uniquely interesting and relatively unstudied cohort. Although this group accounts for almost 8000 cases of CRC annually in the United States (SEER Data), conflicting reports on stage at presentation and prognosis suggest that we do not fully understand the natural history of this subset of patients. Perhaps most importantly, patients with early age-of-onset CRC represent a heterogeneous group: nearly half will have no family history of CRC while one fifth will have a recognizable hereditary form of CRC, or strong familial clustering of CRC [1, 16, 17]. In so far as early age-of-onset CRC is a consistent feature in all current and past criteria for HNPCC (Warthin s Family Cancer Syndrome, Lynch Syndromes I and II, Amsterdam criteria I and II, Japanese criteria, and Bethesda Guidelines) [18 20], efforts have focused on identifying novel predisposition genes in this enriched population. Young patients with CRC have an increased likelihood of having MSI-positive tumors and for harboring a germline MMR gene mutation, as high as 42 58% in patients with CRC 35 years of age, compared to 1 12% in a non-age stratified population [21 23]. However, in a significant fraction of early age-ofonset colorectal cancer, a genetic basis has yet to be identified. The A636P mutation may account for a significant percentage of colorectal cancer in early age-of-onset Ashkenazi population. The A636P mutation was found to be present in 2.1% (3/140) of Ashkenazi patients diag-

3 A636P in Ashkenazi Jews 225 Table 1. Intermediate risk criteria for HNPCC. 1. CRC diagnosed at 50 years of age. 2. CRC diagnosed at any age with histology suggestive of HNPCC (tumor infiltrating lymphocytes, mucinous or signet-cell histology, medullary growth pattern). 3. Individual with multiple HNPCC-related primary tumors at any age. 4. Individual with an HNPCC-related cancer in a family with two or more HNPCC-related cancers (inclusive of the proband) with at least one of the cancers being CRC. CRC = colorectal cancer; HNPCC = hereditary nonpolyposis colorectal cancer; MSI = microsatellite instability. nosed with colorectal cancer at < 60 years [13]. In a more recent series from Memorial Sloan-Kettering Cancer Center, the A636P mutation was detected in 3 of 41 (7%) Ashkenazi patients who had a colorectal cancer diagnosed at or under age 40. The individuals affected were all male and were diagnosed at ages 40, 32, and 28 [24]. This incidence is significantly greater than the 8/1345 (0.6%) reported in Foulkes et al. [13] in Ashkenazi Jews not selected for age (P ¼ 0.004). These results suggest that for Ashkenazi Jews diagnosed with colon cancer at age 40 or earlier, initial testing for the MSH2*1906G>C mutation is clinically indicated. Methodologies used for A636P testing Multiple methods have been used to indicate the presence or absence of the A636P mutation. All methods depend on an amplification step utilizing the polymerase chain reaction (PCR) with oligonucleotides flanking exon 12 of the MSH2 gene. For clinical purposes, the most reliable test is direct DNA sequencing of genomic DNA isolated from a venous blood specimen. For high throughput screening purposes, laboratories have used denaturing high performance liquid chromatography (D-HPLC), single stranded conformation polymorphism analysis, restriction enzyme digestion, and allele-specific hybridization. Testing can also be reliably performed on DNA prepared from formalin-fixed, paraffin embedded tissue specimens. Single-amplicon A636P testing protocol for high-risk Ashkenazi patients Full sequencing of MSH2 and MLH1 to find a mutation is complex and expensive; screening only for a specific mutation in cases with a greater probability of harboring that mutation is both rapid and cost efficient. We have recently utilized a prospective testing strategy directed at the A636P mutation in Ashkenazi Jewish individuals to determine the rate of detection of the A636P mutation when the threshold for ordering testing has been lowered. From December 2002 through April 2004, 21 Ashkenazi Jewish individuals of high (meeting Amsterdam I or II criteria) or intermediate risk (Table 1) have undergone this prospective HNPCC testing protocol. Patients were offered single-amplicon testing for the A636P mutation in conjunction with MSI testing. If both tests were negative, no further testing was performed and low-risk screening was recommended. If the A636P test result was negative, but the individual was MSI positive, full sequencing of MSH2 and MLH1 would then be carried out. This latter scenario has not yet occurred. Full gene mutational analysis was ordered for an additional six persons during this period. In the 21 individuals who had singleamplicon testing, the A636P mutation was identified in 3 individuals. In the six individuals who had full-gene analysis, the A636P mutation was identified in one individual and two different disease-causing mutations were identified in two unrelated individuals. No mutations in MSH2 or MLH1 were detected in the remaining three individuals. In summary, the A636P founder mutation was identified in 4 of 27 families (15%) tested with this strategy in the last 17 months. A summary of the clinicopathologic characteristics of all A636P carriers identified at Memorial Sloan- Kettering Cancer Center between 1995 and 2004 is given in Table 2. Of note, 8 of 11 patients had a diagnosis of CRC or an HNPCC-related cancer at or before 50 years of age. Seven individuals had family histories that fulfilled Amsterdam I or II criteria. The observation of kindred 5085, in which only two CRCs after age 50 were diagnosed with no other HNPCCrelated cancers in the family, suggests that screening colorectal cancer patients with a single first- or seconddegree relative with colorectal cancer may identify A636P carriers. Limitations of focused screening for this founder mutation must be recognized. Because of genetic heterogeneity, specificity of a negative result is limited; it is possible that an individual without the A636P MSH2 founder allele will have another mutation of MSH2, MLH1, MSH6 or an as-yet-undiscovered gene. Lower penetrance colon cancer predisposition alleles, e.g. I1307K or blm Ash, may also be present. However, individuals from kindreds in which the A636P mutation is found may be offered testing and, if the mutation is detected, may undergo risk-reducing surveillance or surgical procedures. Conclusions The A636P mutation is rare in the general colorectal cancer population, and probably accounts for only

4 226 J. G. Guillem et al. Table 2. Clinicopathologic characteristics of Ashkenazi Jewish A636P carriers evaluated at MSKCC ( ). Reference Other HNPCCrelated cancers (exclud. CRC) Family history Of CRC Number of CRCs in family Other cancers (age at dx) First cancer (age at dx) Individual Kindred MSI A636P allele Other MMR mutation Present Present Not tested CRC (55) None 2 SDR 0 Present study Not tested Present Not tested CRC (70) None 3 ACI 0 Present study Not tested Present Not tested Uterine (50) Duodenum (71), 1 Bethesda 1 Present study CRC (75), Breast (83) Not tested Present None detected Ovarian (50) Pancreas (50) 6 ACII 2 Present study Present Present None detected CRC (26) None 3 ACI Present Present None detected Fallopian tube (44) None 4 Bethesda Present Present None detected CRC (46) None 3 ACI Present Present Not tested CRC (30) None 5 ACI Present Present Not tested TCC-U (41) CRC (45) 2 Bethesda Present Present Not tested CRC (40) None 3 ACI 1 Present study a Present Present None detected Uterine (36) CRC (51, 55) 5 ACI 1 13 Detected prior to the commencement of prospective A636P testing. MSI = microsatellite instability; MMR = mismatch repair; CRC = colorectal cancer; HNPCC = hereditary nonpolyposis colorectal cancer; SDR = second degree relative; ACI = Amsterdam Criteria I; ACII = Amsterdam Criteria II; TCC-U = transitional cell carcinoma of ureter. a % of colorectal cancer in the overall Ashkenazi Jewish patient population. However, the mutation is significantly more prevalent in the early age-of-onset Ashkenazi population, accounting for over seven percent of colorectal cancers in patients diagnosed at < 40 years of age. Even more striking is the percentage of Ashkenazi HNPCC kindreds attributable to the A636P mutation. The A636P mutation accounted for one third of families that met Amsterdam criteria and for two thirds of HNPCC cases in which an MMR gene mutation was identified. Taken together, Ashkenazi Jewish patients diagnosed with colorectal cancer at or before age 50 or with a family history of HNPCC-related cancer should be offered A636P testing. Acknowledgements We would like to acknowledge support from the Lymphoma Foundation, the Koodish Fellowship, the Tavel Reznik Foundation, the Cancer Research Foundation of America, the Frankel Fellowship Fund, the Grace Fippinger Foundation, the Bianco Family Fund and the Ferdinand Koch Fund. References 1. Guillem JG, Smith AJ, Puig-La Calle J, Ruo L. Gastrointestinal polyposis syndromes. Curr Probl Surg 1999; 36: Fishel R, Lescoe MK, Rao MR et al. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 1993; 75: Bronner CE, Baker SM, Morrison PT et al. Mutation in the DNA mismatch repair gene homologue hmlh1 is associated with hereditary non-polyposis colon cancer. Nature 1994; 368: Akiyama Y, Sato H, Yamada T et al. Germ-line mutation of the hmsh6/gtbp gene in an atypical hereditary nonpolyposis colorectal cancer kindred. Cancer Res 1997; 57: Miyaki M, Konishi M, Tanaka K et al. Germline mutation of MSH6 as the cause of hereditary nonpolyposis colorectal cancer. Nat Genet 1997; 17: Desai DC, Lockman JC, Chadwick RB et al. Recurrent germline mutation in MSH2 arises frequently de novo. J Med Genet 2000; 37: Moisio AL, Sistonen P, Weissenbach J et al. Age and origin of two common MLH1 mutations predisposing to hereditary colon cancer. Am J Hum Genet 1996; 59: Salovaara R, Loukola A, Kristo P et al. Population-based molecular detection of hereditary nonpolyposis colorectal cancer. J Clin Oncol 2000; 18: Chan TL, Wai Chan Y, Ho JW et al. MSH2 c delaatg is a founder mutation and an important cause of hereditary nonpolyposis colorectal cancer in the southern Chinese population. Am J Hum Genet 2004; 74: Lynch HT, Coronel SM, Okimoto R et al. A founder mutation of the MSH2 gene and hereditary nonpolyposis colorectal cancer in the United States. JAMA 2004; 291: Niell BL, Long JC, Rennert G, Gruber SB. Genetic anthropology of the colorectal cancer-susceptibility allele APC I1307K: evidence of genetic drift within the Ashkenazim. Am J Hum Genet 2003; 73:

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