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Heterozygosity for the BLM^Ash Mutation and Cancer Risk¹

Centre for Cancer Genetics, Samuel Lunenfeld Research Institute, Mount Sinai Hospital [S. P. C., N. D., A. S., R. H., S. G., R. G.], Department of Surgery [S. P. C., M. A., R. H., S. G., R. G.], Centre for Research in Women’s Health, Sunnybrook and Women’s College Health Sciences Centre [W. Z., J. A., S. A. N.], University of Toronto, Toronto, Ontario, M5G 1X5, Canada, and Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts [M. R.]

	ABSTRACT

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Bloom syndrome is an autosomal recessive disorder whose characteristics include an increased risk for many types of cancers. In contrast to the homozygous mutations of Bloom syndrome, heterozygous carriers of BLM mutations may be at increased risk for developing colorectal cancer. We have screened 2,333 Jewish individuals, including 497 individuals with colorectal cancer, 125 with adenomatous polyps, 767 with noncolorectal cancers and 944 controls for the truncating BLM^Ash founder mutation. The BLM^Ash mutation was carried by 0.80% of individuals with colorectal neoplasia, 0.87% of those with any type of cancer and 0.85% of controls. In addition to case-control data, we found no evidence to support a significant relationship between increased cancer risk and heterozygous BLM^Ash mutations with respect to age of cancer diagnosis, tumor multiplicity or family cancer history.

	Introduction

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Bloom syndrome is a rare autosomal recessive disorder characterized by immunodeficiency, growth retardation, male sterility, and a predisposition to many types of cancers (1) . The gene responsible for Bloom syndrome, BLM,³ encodes a homologue of recQ helicase (2) . Loss of helicase activity in the cells of individuals with Bloom syndrome leads to genomic instability, which is characterized by increased rates of somatic recombination, chromosomal breakage, and gene mutation (3, 4, 5) . Multiple types of cancer, including colorectal cancer and premalignant adenomatous polyps, have been observed in individuals with Bloom syndrome (1 , 6) . Approximately one-third of individuals with Bloom syndrome are of Jewish descent and carry the founder BLM^Ash mutation, a frameshift mutation in exon 10 (2281 delATC TGA insTAG ATT C; Ref. 7 ). In contrast to Bloom syndrome, which occurs in homozygous BLM mutation carriers, there are some data supporting the hypothesis that BLM heterozygotes are at increased risk of cancer. Increased rates of somatic recombination have been observed in cells from individuals who carry a single mutant BLM allele (BLM heterozygotes; Ref. 8 ), and transfection of wild-type BLM only partially corrects excessive sister chromatid exchange in BLM-deficient cells (9) . Recently, Gruber et al. (10) studied Jewish populations from Israel and New York and reported that heterozygosity for BLM^Ash is associated with a 2.3–2.8-fold increase in the risk of colorectal cancer. Despite the obligate carrier status of parents of individuals with Bloom syndrome, this increased risk of cancer does not appear to have been previously appreciated. Confirmation of an increased risk of colorectal cancer is of obvious clinical importance to the 1% of Ashkenazi Jewish individuals who carry the BLM^Ash mutation, as well as heterozygous BLM carriers from other ethnic groups. In this study, we have screened Jewish individuals with and without a personal history of cancer for the BLM^Ash mutation to assess the association of this allele with colorectal and other cancers.

	Materials and Methods

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Patient Populations.
Study subjects were recruited from a variety of sources, including patients referred for colonoscopy or for cancer treatment at one of several teaching hospitals in Toronto or through the genetic epidemiology repository at the Centre for Research in Women’s Health in Toronto. Control subjects for this study were Jewish individuals without a personal history of cancer who had either undergone colonoscopy without any neoplastic mass identified or had been referred for BRCA1 or BRCA2 germ-line testing (i.e., at risk testing) but were found to not harbor mutations in these genes. A portion of tissue analyzed for this study was obtained from a previously described cancer DNA repository linked to an anonymous clinical database (11) . The remaining DNA samples were used after individual informed consent was obtained. The use of DNA samples from human subjects for this study received institutional approval.

BLM^Ash Mutation Analysis.
DNA was extracted from either peripheral blood lymphocytes or from nonneoplastic paraffin-embedded tissues. The DNA samples were screened for the BLM^Ash mutation by either SSCP analysis of a 75-bp PCR product (forward primer: 5'-CTTTTATACTTAGATTCCAGC-3', reverse primer: 5'-TGGAGGTAAATATTTGTAGCT-3'; PCR and SSCP conditions available upon request) or digestion of a 310-bp PCR product (forward primer: 5'-GATATGTGACTAATAAAATA-3'; reverse primer: 5'-ATTCTTTGCACTACGTATAC-3'; PCR conditions available upon request) with the restriction enzyme Hpy188 I. Cases observed to be positive for the BLM^Ash mutation were confirmed by direct sequencing.

APC^I1307K Polymorphism Analysis.
Genomic DNA samples were screened for APC^I1307K by SSCP (forward primer: 5'-AGATTCTGCTAATACCCTGC-3', reverse primer: 5'-GAACTTCGCTCACAGGATC-3) as described previously (11) . Cases observed to be positive for the APC^I130K polymorphism were confirmed by direct sequencing.

Statistical Analysis.
ORs were calculated from proportions of BLM^Ash carriers among cases and controls and compared using Fisher’s exact test. Clinical features of BLM^Ash carriers were compared with noncarriers using Mann-Whitney nonparametric testing. PAR% was calculated as follows:

	Results

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Among 2333 Jewish individual tested, we identified 21 (0.90%) heterozygous BLM^Ash carriers (Fig. 1) . No individual was found to be homozygous for the BLM^Ash mutation. We observed no significant difference in the frequency of the BLM^Ash allele in Jewish individuals with either a personal history of cancer or controls without such a history (Table 1) . Our carrier rate of 0.85% in 944 controls nearly identically mirrored a carrier rate of 0.86% in 8260 Jewish individuals analyzed in multiple previous studies (12, 13, 14, 15) . In our series, the BLM^Ash mutation was present in only 0.80% of the 617 patients with colorectal neoplasms, a frequency not significantly different from that observed in 9204 controls from this series and others (OR = 0.94; 95% CI, 0.38–2.32; Refs. 12, 13, 14, 15 ). Similarly, the risk for any type of cancer did not appear to be increased in BLM^Ash carriers (OR = 1.01; 95% CI, 0.54–1.91). Interestingly, when our colorectal neoplasia data were combined with those previously published, the OR for BLM^Ash remained significant but fell to a modest 1.79 (95% CI, 1.17–2.74; P = 0.009).

View larger version (115K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. BLMAsh mutation detection. A, SSCP testing revealed a shifted band (Lane 2) that corresponded to a heterozygous BLMAsh mutation. B, Hpy188 I digestion of a 310-bp product from a BLMWT specimen yields three fragments, 173, 115, and 22 bp (the smallest fragment is not visualized on the gel). Heterozygous BLMAsh samples yield a 137-bp product in addition to the wild-type fragments (Lane 8). C, direct forward sequence analysis of a heterozygous BLMAsh sample demonstrates the frameshift delATC TGAinsTAG ATT C sequence compared with BLMWT.

At the population level, the significance of a specific genotype on disease prevalence may be appreciated by calculating the PAR%. Using our observed data for APC^I1307K (control prevalence = 5.3%; OR = 2.10), the PAR% for colorectal neoplasia for this allele was 5.5%. Our data could not be used to calculate a meaningful PAR% for BLM^Ash as our OR was < 1. However, when our data were combined with those previously published (control prevalence = 0.84%; OR = 1.79), the PAR% for BLM^Ash was estimated to be 0.60% (10 , 12, 13, 14, 15) .

	Discussion

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Bloom syndrome is associated with increased risk of cancer, including leukemia and lymphoma in younger patients and carcinomas of larynx, lung, esophagus, colon, breast, and cervix in adults (1) . It has recently been reported that heterozygous BLM^Ash mutations were carried by 1 of 54 (1.85%) Jewish individuals with colorectal cancer (10) . This heterozygous carrier frequency was predicted to confer a 2.3–2.8-fold increased risk for colorectal cancer. To confirm this increased risk of colorectal cancer risk and examine whether this risk association is observed with other cancer types, we screened 2333 Jewish individuals with and without a personal history of cancer for the BLM^Ash founder mutation. The frequency of BLM^Ash heterozygous mutations in our series of individuals with colorectal and other cancers was nearly identical to our own control population and those reported previously (12, 13, 14, 15) . In fact, the frequency of BLM^Ash heterozygosity in 622 Jewish individuals with a personal history of colorectal neoplasia was slightly lower than unaffected controls in our series. When our population is combined with near identical historical control study estimates (12, 13, 14, 15) , the power to rule out the unadjusted OR of 2.45 previously reported by Gruber et al. (10) was 63%. To rule out an OR of 1.3 (the lower 95% CI of the previous OR estimate) with 80% power (assuming = 0.05), a staggering 25,737 cases and 25,737 controls would have been required for analysis (10) .

Classically, cancer-causing germ-line mutations have been associated with an observable family cancer history, a younger age of a cancer diagnosis, and an increased rate of synchronous and metachronous neoplastic lesions. In the current series, carriers of the BLM^Ash mutation did not appear to have a higher likelihood of a family history of colorectal cancer, nor did they appear to have a younger age of diagnosis of colorectal cancer, or an increased number of colorectal neoplasms per patient compared with noncarriers. Similarly, Gruber et al. did not find a younger age of diagnosis or stronger colon cancer family history in BLM^Ash carriers with colorectal cancer, compared with noncarriers (S. Gruber, personal communication).

In addition to human case-control data, BLM heterozygosity has been implicated in colorectal tumorigenesis by observations that Apc^+/-Blm^+/- mice develop increased number of adenomas compared with littermate multiple intestinal neoplasia Apc^+/-Blm^+/+ mice (16 , 17) . However, spontaneous intestinal neoplasia was not observed in studies of two separate Blm^+/- mouse models (17 , 18) and was rare in a third (16) . Furthermore, the use of mouse modeling for BLM heterozygosity is questionable as homozygous BLM mutation causes the Bloom syndrome in humans but is embryonic lethal in the mouse (16, 17, 18) . Nonetheless, it may be hypothesized from previous mouse studies that rather than providing a direct risk, heterozygous BLM mutation may modify the risk of other cancer causing alleles. The APC^I1307K polymorphism has been observed in >10% of Ashkenazi Jews with colorectal neoplasia and is estimated to approximately double the risk of colorectal cancer (11 , 19) . If BLM^Ash modifies the risk of cancer in individuals with other genetic predispositions to colorectal cancer, we might postulate that the BLM^AshAPC^I1307K genotype would be overrepresented in individuals with colorectal cancer. However, similar to our other results, the BLM^AshAPC^I1307K genotype was not observed in 581 Jewish individuals with colorectal neoplasia.

On the basis of this case-control data, cancer phenotype, and cancer family history analyses, it did not appear in our series that heterozygosity for the BLM^Ash allele was a significant risk factor for colorectal cancer or cancer from any site in general. Furthermore, it appears that if in fact BLM^Ash confers increased risk for colorectal neoplasia, this risk is more modest than originally estimated. Given only a modest individual risk in addition to the low prevalence of this allele, the PAR% for BLM^Ash heterozygosity was just 0.60%. Thus, although our data cannot fully rule out subtle increases in the risk for colorectal or other cancers because of BLM^Ash, if present, these differences are not likely to appreciably alter individual patient care, nor are they likely to impact significantly on the Ashkenazi Jewish population.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ S. P. C. is a Research Fellow of the National Cancer Institute of Canada supported with funds provided by the Canadian Cancer Society. R. G. is a Charles H. Hollenberg Senior Fellow/Clinician Scientist supported with funds provided by Eli-Lilly Canada, Cancer Care Ontario, and the Canadian Institutes of Health Research.

² To whom requests for reprints should be addressed, at Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Suite 455, Toronto, Ontario, M5G 1X5 Canada. E-mail: rgryfe{at}mtsinai.on.ca

³ The abbreviations used are: BLM, Bloom gene; Ash, Ashkenazi Jewish; SSCP, single-strand conformation polymorphism; OR, odds ratio; CI, confidence interval, APC, adenomatous polyposis coli gene; PAR%, population attributable risk.

Received 11/13/02. Accepted 3/ 4/03.

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