Characterization of a novel founder MSH6 mutation causing Lynch syndrome in the French Canadian population

Authors E. Castellsagué1,2,3, J. Liu4, A. Volenik5, S. Giroux6, R. Gagné7, B. Maranda8, A. Roussel-Jobin6, J. Latreille9, R. Laframboise10, L. Palma11, L. Kasprzak11, VA. Marcus12, M. Breguet13, S. Nolet14, Z. ElHaffaf14, K. Australie9, A. Gologan4, O. Aleynikova4, K. Oros-Klein15, C. Greenwood15,16, AM. MesMasson17, D. Provencher17, M. Tischkowitz18, G. Chong4, F. Rousseau6,19, WD. Foulkes1,2,11,20 Affiliations 1

Department of Human Genetics, McGill University, Montreal, QC, Canada; 2Department of Medical

Genetics, The Lady Davis Institute, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada; 3Translational Research Laboratory, Catalan Institute of Oncology, Bellvitge Institute for Biomedical Research, L’Hospitalet de Llobregat, Barcelona, Spain; 4Department of Pathology, Jewish General Hospital,Montreal, QC, Canada; 5Cancer Prevention Centre, Jewish General Hospital, Montreal, QC, Canada; 6Unité de recherche en génétique humaine et moléculaire, Axe Santé des Populations et Pratiques Optimales en Santé, Centre de recherche du CHU de Québec, Canada; 7

Department of Medical Genetics, CHU of Quebec and Laval University, QC, Canada; 8Service of

Genetics, Université de Sherbrooke, QC Canada; 9Faculté de médecine et des sciences de la santé, Université de Sherbrooke, CSSS Champlain Charles Le Moyne, QC, Canada; 10Génétique Médicale, CHU Laval, QC, Canada;

11

Department of Medical Genetics, Research Institute of the McGill

University Health Centre, Montreal, QC, Canada; Montreal, QC, Canada;

Department of Pathology, McGill University,

13

Medecine Genique, CHU de Montreal, QC, Canada;

Department, CHU de Montreal, QC, Canada; Montreal, QC, Canada;

12

16

14

Medical Genetics

15

Lady Davis Institute, Jewish General Hospital,

Departments of Oncology, Epidemiology Biostatistics and Occupational

Health, and Human Genetics, McGill University, Montreal, QC;

17

Centre de recherche du Centre

Hospitalier de l'Université de Montréal/Institut du Cancer de Montréal, QC, Canada; 18Department of Medical Genetics, University of Cambridge, Cambridge, UK; 19 Département de biologie moléculaire, biochimie médicale et pathologie, Université Laval;

20

Program in Cancer Genetics, Department of

Oncology and Human Genetics, McGill University, Montreal, QC, Canada

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Correspondence Dr. Ester Castellsagué, PhD Department of Human Genetics, McGill University Lady Davis Institute, Jewish General Hospital 3755 Cote Ste Catherine Road, Montreal, H3T 1E2 Email: [email protected] Tel: 1 (514) 340-8222 x3100 Fax : 1 (514) 340-8600 Conflict of interest statement The authors declare no conflicts of interest Acknowledgements EC is a recipient of a Marie Curie International Outgoing Fellowship (PIOF-GA-2012-327193), cosponsored with the Catalan Institute of Oncology-Bellvitge Institute for Biomedical Research, Barcelona, Spain. FR holds an MSSS/FQRS/CHUdeQuébec Research Chair in Technology Assessment and Evidence-based Laboratory Medicine. This project was also funded partly by the APOGÉE-Net/CanGèneTest Research and Knowledge Network in Genetic health Services, funded by the Canadian Institutes for Health Research, (grant number ETG-92250) (to FR and WDF). Additional funding came from the Ministère de l’enseignement supérieur de la recherche de la science et de la technologie (grant number PSR-SIIRI-846) (to WDF). ABSTRACT We identified an MSH6 mutation (c.10C>T, p.Gln4*) causing Lynch syndrome (LS) in 11 French Canadian (FC) families from the Canadian province of Quebec. We aimed to investigate the molecular and clinical implications of this mutation among FC carriers and to assess its putative founder origin. We studied 11 probands and 27 family members. Additionally 6,433 newborns, 187 colorectal cancer (CRC) cases, 381 endometrial cancer (EC) cases and 179 additional controls, all of them from Quebec, were used. Found in ~1/400 newborns, the mutation is one of the most common LS mutations described. We have found that this mutation confers a greater risk for EC than for CRC, both in the 11 studied families and in the unselected cases: EC (OR=7.5, pG and c.1614_1615delinsAG) have been published so far (14). In Finland, two atypical LS families with predominance of endometrial carcinoma appeared to carry the same mutation (c.2983G>T) and haplotype analysis pointed to a common ancestral origin. The mutation was not found in 268 healthy controls and 245 CRC cases of Finnish origin (16). Cederquist and colleagues described two founder Swedish MSH6 mutations (c.1346T>C and c.2931C>G) occurring in two large pedigrees of late 17th and 18th centuries showing a late age of onset but a high lifetime risk of LS tumours, with a significant predominance of EC (13). Finally, two Ashkenazi Jewish (AJ) MSH6 founder mutations (c.3984_3987dup and c.3959_3962del) were evaluated in large series of controls (n=3310), CRC (n=2685) and EC cases (n=337), and

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appeared to harbour higher risks to develop endometrial than colorectal malignancies. The mutations were calculated to arise around 585 CE and 685 CE, respectively (15). Here we describe a novel MSH6 founder mutation (c.10C>T; p.Gln4*) predisposing to LS in the FC population of Quebec. We have investigated 11 carrier families for genealogy, segregation of the mutation with LS tumours, MSI, IHC and loss of heterozygosity (LOH). We have assessed the frequency of this mutation in a large cohort of FC controls and in CRC and EC cases. Furthermore, we sought to assess its founder effect and calculate the age of the first MSH6 founder mutation described in the FC population of Quebec. MATERIALS AND METHODS Patients and samples This study includes 11 LS families fulfilling Amsterdam or Bethesda criteria from several hospitals in the Canadian province of Quebec that tested positive for a truncating mutation in the MSH6 gene (c.10C>T). Where available, germline DNA and tumour samples were obtained from 11 probands and 27 additional family members. For the haplotype analysis, germline DNA from 179 FC controls recruited at the Jewish General Hospital (JGH) was used. In order to calculate the risk of the mutation to develop LS-related tumours we screened two different sets of unrelated FC samples: germline DNA from 381 EC cases (Notre-Dame Hospital); and FFPE DNA from normal tissue of 187 CRC patients (Centre Hospitalier Universitaire de Québec). To set the allelic frequency, germline DNA from 6,433 FC anonymous-unlinked newborns was genotyped. Informed consent was obtained from participants and all analyses were approved by the appropriate ethics committee. Molecular testing strategy Familial samples were analysed in a routine manner in the molecular diagnostic units of the Pathology Departments of the JGH or Hotel-Dieu Hospital: germline DNA was tested for MSH6 mutations by High Resolution Melting (HRM) curve analysis and/or Sanger sequencing; IHC staining for MLH1, MSH2, MSH6 and PMS2 proteins, MSI analyses and LOH of the mutation were done where tumoural material was available. FC controls and EC cases were genotyped by HRM. CRC cases were genotyped using LightCycler®2.0 technology. Germline DNA from the newborn controls were pooled (in 8-plex) and screened as described previously (17) by allele-specific PCR (AS-PCR). Samples presenting abnormal patterns by HRM, LightCycler or AS-PCR were validated by Sanger sequencing. All primers, probes and specific conditions are available upon request. Haplotype analysis Thirty-eight DNA samples from members of the 11 studied families were haplotyped using 14 microsatellite markers spanning 12.3Mb upstream and 9.5Mb downstream the mutation (Table 3; primers and conditions available upon request). One-hundred and seventy-nine FC controls were

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haplotyped using 10 of the microsatellite markers (Table 3). To deduce the mutation-associated haplotype, intra-familial segregation analysis was performed and alleles were phased when possible. Statistical analysis Estimation of the mutation age was performed using DMLE+ (18, 19), a Bayesian method for fine mapping of a mutation by using the linkage disequilibrium patterns.

We assumed growth rates

between 1.38 and 1.48-fold increase per generation. These values were derived by comparing the 1851 population of 892,061 with the 1961 FC population (5,259,211) and assuming between 4.4 and 5.5 generations during these 110 years (20 or 25 years per generation). Furthermore, we assumed that we sampled 24/30,809 of the mutations among FC. The numerator is based on the 23 individuals carrying the mutation (one was homozygous) and the denominator is based on the estimated allele frequency and the 2011 Quebec population. The association between MSH6 mutation and risk to develop CRC, EC and OC was estimated by calculating odds ratio (OR) according to Altman (Table 2) (20). RESULTS Characterization of carrier families All families presented with either Bethesda or Amsterdam clinical criteria for LS (Table 1, Supplementary Table 1). Notably, only two of the families fulfilled Amsterdam criteria, but all affected carriers were diagnosed with at least one tumour that has been proposed to belong to the LS spectrum. The mutation co-segregated with cancer in 15/23 carriers, with an average age at diagnosis of 44.2 years (range, 10-59). The average age of the 8 unaffected carriers was 38.2 years (range, 3-62) (Table 1).There was an excess of mutation carriers among the 18 affected family members (15/18 [83%]) compared with those among the 20 unaffected individuals (8/20 [40%]) (Supplementary Table 1). All evaluable tumours showed loss of MSH6 protein and MSI (Table1). No LOH at the mutation position was identified in any of the evaluated tumours. We found that 7/8 (88%) non-affected carriers were men whereas 11/15 (73%) affected carriers were women. Related to that, and concerning the spectrum of tumours, 7/15 (47%) affected carriers presented at least one CRC. In 8/11 (73%) affected carrier females an EC was diagnosed (Table1). Risk of CRC and EC To assess the risk conferred by this mutation on CRC and EC development, we assessed the frequency of the mutation in a control population of 6,433 FC anonymous newborns, 187 CRC cases and 381 EC cases. Sixteen heterozygote carriers were found among the control group (16/6,433), giving an allelic frequency of the mutant allele among the FC population of 0.001242 (Table 2). Regarding CRC, we found only one carrier (1/187) giving a non-significant increased risk to develop CRC of 2.2 in association with the mutation (OR=2.2; 95%CI: 0.28-16.35; p-value: 0.46) (Table 2). Among the

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EC cases, 7 carriers were detected (7/381) - the mutation significantly increases the risk to develop an EC (OR=7.5; 95%CI: 3.07-18.36; p-value: < 0.0001) (Table 2). Haplotype analysis and estimation of mutation age The haplotype analysis strongly suggests that the mutations could have occurred as a unique event in a single founder individual. A common haplotype spanning a maximum of 3.42Mb and a minimum of 1.61Mb was determined to be shared among carrier individuals (Table 3). The minimum haplotype without the mutation was found in only 1 of the 179 FC controls analysed. The existence of a clear shared haplotype in carrier individuals permitted estimation of the age of the mutation.

Using DMLE+, and considering 1.42-fold per generation the growth rate of the FC

population of Quebec, the mutation is estimated to have occurred 23.3 generations ago with a 95% credible interval of 19-30 generations. Altering the growth rates makes only small alterations to this estimate (Supplementary Figure 1). Assuming 22 years per generation (corresponding to this growth rate), we would assume the mutation appeared for the first time 513 years ago (credible interval 430– 656 years). Therefore, the c.10C>T MSH6 mutation could easily have arrived with the first Quebec founders. Consistent with this hypothesis, the mutation has been recently been reported in France in one case (www.umd.be). DISCUSSION In the present study, we characterized a novel MSH6 founder mutation (c.10C>T, p.Gln4*) found in 11 LS families from the province of Quebec. We aimed to assess its clinical implications in the 11 affected families as well as in unselected CRC and EC cases from Quebec. We also confirmed its founder effect by finding that a common haplotype was present in all carriers among the 11 families. Additionally, we were able to calculate that the mutation appeared 430–656 years ago, probably coming with the first Quebec founders. All clinicopathological evidence pointed to the unequivocal pathogenicity of this mutation. In the carrier families we found an excess of mutation carriers among affected family members compared to unaffected (83% vs 40%, respectively). Significantly, a homozygous carrier of the mutation (FCMSH6-7.1) developed early CRC at age 10, which is consistent with constitutional MMR deficiency (CMMR-D) (21). Other typical features of CMMR-D such as haematological or brain malignancies (21) were not detected in this patient. All available tumours from carrier individuals showed MSI and IHC loss of MSH6 protein (two of them showed additional loss of MSH2, but tested negative for mutations in this gene). Although LOH in the mutation position was not found in any of the studied tumours, the gene was probably inactivated by point mutations or deletions elsewhere in MSH6. We found that 65% of carriers were affected by a LS cancer (15/23), with a similar average age at diagnosis than the age at interview of the 8 unaffected carriers, thus consistent with the incomplete

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penetrance characteristic of LS (22). Accordingly, we found 16 carriers of the mutation in a control population of 6,433 newborns in Quebec (~1/400). As far as we are aware, such a high prevalence of a MMR gene mutation has only been reported in another founder mutation in PMS2 (~1/399). However, no risk estimates to LS-associated cancers from unselected cases were provided in that study (23). Among the 11 studied families, a substantial increase in the presence of cancer was noticed among females compared to males (73% vs 27%), probably reflecting the fact that EC is the major cancer phenotype observed in female carriers of this mutation (8/11 affected carrier females developed an EC). This phenotype is a typical presenting feature of a MSH6 mutation in women, who are more commonly diagnosed with EC than CRC (5, 8-10). Consistent with this, by assessing the presence of the mutation in 381 and 187 unselected EC and CRC cases from Quebec, we found that the mutation confers a major risk to develop EC than CRC (OR=7.5, p100 polyps and 2 synchronous CRC at age 22) may be dominant over the LS phenotype, we have not excluded this family from our study because MSI and loss of MSH6 staining were found in a CRC from one of the two carriers, indicating that MSH6 probably played a major role in the tumourigenesis process. We present here the ninth described MSH6 founder mutation, which, based on available data, is the most frequent founder mutation found in this gene and at the population level, it is probably one of the most prevalent MMR mutations ever reported in LS. The high prevalence among FC LS families of this mutation (~50% of all MSH6 mutations in our database, data not shown) indicates that screening for c.10C>T should be the first step in the molecular diagnostic testing of MSH6-deficient FC tumours. We hope that the detailed clinical description of this mutation provided here will also facilitate the counselling of at-risk carriers. REFERENCES

1. Pineda M, Gonzalez S, Lazaro C et al. Detection of genetic alterations in hereditary colorectal cancer screening. Mutat Res 2010: 693: 19-31. 2. Win AK, Lindor NM, Jenkins MA. Risk of breast cancer in Lynch syndrome: a systematic review. Breast Cancer Res 2013: 15: R27. 3. Foulkes WD. Inherited susceptibility to common cancers. N Engl J Med 2008: 359: 2143-2153.

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4. Palomaki GE, McClain MR, Melillo S et al. EGAPP supplementary evidence review: DNA testing strategies aimed at reducing morbidity and mortality from Lynch syndrome. Genet Med 2009: 11: 42-65. 5. Hendriks YM, Wagner A, Morreau H et al. Cancer risk in hereditary nonpolyposis colorectal cancer due to MSH6 mutations: impact on counseling and surveillance. Gastroenterology 2004: 127: 17-25. 6. Plaschke J, Engel C, Kruger S et al. Lower incidence of colorectal cancer and later age of disease onset in 27 families with pathogenic MSH6 germline mutations compared with families with MLH1 or MSH2 mutations: the German Hereditary Nonpolyposis Colorectal Cancer Consortium. J Clin Oncol 2004: 22: 4486-4494. 7. Wagner A, Hendriks Y, Meijers-Heijboer EJ et al. Atypical HNPCC owing to MSH6 germline mutations: analysis of a large Dutch pedigree. J Med Genet 2001: 38: 318-322. 8. Baglietto L, Lindor NM, Dowty JG et al. Risks of Lynch syndrome cancers for MSH6 mutation carriers. J Natl Cancer Inst 2010: 102: 193-201. 9. Wijnen J, de Leeuw W, Vasen H et al. Familial endometrial cancer in female carriers of MSH6 germline mutations. Nat Genet 1999: 23: 142-144. 10. Ramsoekh D, Wagner A, van Leerdam ME et al. Cancer risk in MLH1, MSH2 and MSH6 mutation carriers; different risk profiles may influence clinical management. Hered Cancer Clin Pract 2009: 7: 17. 11. Ponti G, Castellsagué E, Ruini C et al. Mismatch Repair Genes Founder mutations and cancer susceptibility in Lynch syndrome. Clin Genet Under review. 12. Berends MJ, Wu Y, Sijmons RH et al. Molecular and clinical characteristics of MSH6 variants: an analysis of 25 index carriers of a germline variant. Am J Hum Genet 2002: 70: 26-37. 13. Cederquist K, Emanuelsson M, Wiklund F et al. Two Swedish founder MSH6 mutations, one nonsense and one missense, conferring high cumulative risk of Lynch syndrome. Clin Genet 2005: 68: 533-541. 14. Ramsoekh D, Wagner A, van Leerdam ME et al. A high incidence of MSH6 mutations in Amsterdam criteria II-negative families tested in a diagnostic setting. Gut 2008: 57: 1539-1544. 15. Raskin L, Schwenter F, Freytsis M et al. Characterization of two Ashkenazi Jewish founder mutations in MSH6 gene causing Lynch syndrome. Clin Genet 2011: 79: 512-522. 16. Vahteristo P, Ojala S, Tamminen A et al. No MSH6 germline mutations in breast cancer families with colorectal and/or endometrial cancer. J Med Genet 2005: 42: e22. 17. Giroux S, Dube-Linteau A, Cardinal G et al. Assessment of the prevalence of the 985A>G MCAD mutation in the French-Canadian population using allele-specific PCR. Clin Genet 2007: 71: 569-575. 18. Rannala B, Reeve JP. High-resolution multipoint linkage-disequilibrium mapping in the context of a human genome sequence. Am J Hum Genet 2001: 69: 159-178. 19. Reeve JP, Rannala B. DMLE+: Bayesian linkage disequilibrium gene mapping. Bioinformatics 2002: 18: 894-895. 20. Altman DG. Practical statistics for medical research. London: Chapman and Hall 1991. 21. Wimmer K, Kratz CP. Constitutional mismatch repair-deficiency syndrome. Haematologica 2010: 95: 699-701. 22. Stoffel E, Mukherjee B, Raymond VM et al. Calculation of risk of colorectal and endometrial cancer among patients with Lynch syndrome. Gastroenterology 2009: 137: 16211627. 23. Clendenning M, Senter L, Hampel H et al. A frame-shift mutation of PMS2 is a widespread cause of Lynch syndrome. J Med Genet 2008: 45: 340-345.

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Table 1: Clinicopathological features of c.10C>T MSH6 carriers Family ID

Hospital

FC-MSH6-1

HCLM

Clinical Criteria Individual ID Relation Gender Bethesda

FC-MSH6-1.1 Proband

F M F F F

FC-MSH6-2 CHUL-CHUQ

Bethesda

FC-MSH6-3 CHUL-CHUQ

Bethesda

FC-MSH6-1.2 Son FC-MSH6-2.1 Proband FC-MSH6-2.2 Sister FC-MSH6-3.1 Proband

Amsterdam Bethesda

FC-MSH6-3.3 Nephew FC-MSH6-4.1 Proband FC-MSH6-5.1 Proband

M F M

FC-MSH6-5.2 Sister FC-MSH6-5.3 Brother FC-MSH6-6.1 Proband FC-MSH6-7.1 Proband FC-MSH6-7.2 Mother FC-MSH6-8.1 Sister FC-MSH6-8.2 Sister

F M F M F F F

FC-MSH6-8.3 Nephew FC-MSH6-9.1 Proband

M F

FC-MSH6-9.2 Son FC-MSH6-9.3 Son Bethesda /FAP FC-MSH6-10.1 Proband

M M M

FC-MSH6-4 FC-MSH6-5

MGH MGH

FC-MSH6-6 HCLM FC-MSH6-7 CHUL-CHUQ

Amsterdam Bethesda

FC-MSH6-8

HD-CHUM

Bethesda

FC-MSH6-9

HD-CHUM

Bethesda

FC-MSH6-10

HD-CHUM

FC-MSH6-10.2

FC-MSH6-11 CHUL-CHUQ

Bethesda

Father

FC-MSH6-10.3 Son FC-MSH6-11.1 Proband

M M F

Status Carrier

Tumor

CRC CRC Carrier Unaffected Carrier EC Carrier BC Carrier Cervix EC Carrier Unaffected Carrier EC Carrier CRC SE Carrier EC Carrier Unaffected Carrier EC Homoz-Carrier CRC Carrier Unaffected Carrier EC Carrier CRC EC TC Carrier Unaffected Carrier EC OC BC CRC Adr Carrier Unaffected Carrier Unaffected Carrierc CRCd CRCd Carrier SCe NHL Carrierc Unaffected Carrier CRC

IHC MSI LOH MLH1 MSH2 MSH6 PMS2 53 + + + MSI 55 33 50 + + + MSI No LOH 34 37 43 + + + NE No LOH 45 55 + + + MSI No LOH 59 + + + MSI No LOH 59 + + + 50 + + + MSI No LOH 62 40 + + + MSI No LOH 10 + + -a + NE NE 55 55 57 + -b + MSI 65 67 43 44 + -b + MSI No LOH 44 51 54 54 30 35 22 + + + MSI No LOH 22 54 54 3 43 + + + MSI No LOH

Age

HCLM, Hôpital Charles-LeMoyne; CHUL, Centre Hospitalier Université Laval; CHUQ, Centre Hospitalier Universitaire de Québec; MGH, Montreal General Hospital; HD-CHUM, Hôtel-Dieu-Centre Hospitalier de l’Université de Montréal; FAP, Familial adenomatous polyposis; CRC, Colorectal cancer; EC, Endometrial cancer; BC, Breast cancer; CC, cervical cancer; SE, Sebaceous epithelioma; TC, Thyroid cancer; OC, Ovarian cancer; Adr, Adrenal tumor; SC, Stomach cancer; NHL, Non-hodgkin lymphoma; NE, Not evaluable (PCR failed to amplify the DNA); MSI, microsatellite instability; IHC, immunohistochemical analysis in tumor tissue (+, expression; -, lost expression); LOH, loss of heterozygosity. aDue to homozygosity for the mutation in the germline, the tissue showed lack of staining both in neoplastic and non-neoplastic cells bNo germline MSH2 mutations found. cCarrier of an additional de novo APC mutation (c.3927_3931del, p.Glu1309Aspfs*4). dSynchronous CRC. eNot confirmed by pathology report, confirmed by family history.

Table 2: Risk of CRC and EC conferred by c.10C>T MSH6 mutation Cancer CRC EC

Cases 1/187 (0.53%) 7/381 (1.84%)

Controls 16/6433 (0.25%) 16/6433 (0.25%)

OR* 2.2 7.5

95%CI 0.28-16.35 3.07-18.36

p-value 0.4572 T MSH6 mutation. Microsatellite

D2S2374

D2S2186

D2S177

D2S2272

D2S2306

D2S2174

D2S391

AT(n)

Mutation

CA(n)

D2S2739

D2S123

D2S2369

D2S2251

D2S1364

NC_000002.11

35709843

37072938

38026891

40896211

42604659

45178668

46411503

47866183

48010382

48131631

49622186

51288437

53224312

54209829

57582313

Family FC-MSH6-1

284

225

290

209

200

294

169

155

Yes

166

218

225

270

276

132/140

FC-MSH6-2

284

225

290

209

200

294

169

155

Yes

166

218

225

264

266

132

FC-MSH6-3

262/286

225

286/288

205

206

296

163/167

163

Yes

166

218

225

264

270

136

FC-MSH6-4

278/286

199/225

288/290

207/209

200/206

292/294

163

155/171

Yes

166/174

202/218

225

262/270

266/268

140

FC-MSH6-5

286

203

290

203

200

294

169

155

Yes

166

218

225

264

270

136

FC-MSH6-6

276/280

203/225

280/282

209/211

200/206

294/296

163/167

151/155

Yes

166

202/218

227

266/272

274/278

132

FC-MSH6-7a

262

223

292

205

204/206

302

169

155

Yes

166

218

225

264

284

136

FC-MSH6-7b

286

223

274

207

204/206

296

169

155

Yes

166

218

225

264

270

136

FC-MSH6-8

288

225

290

205

196

155

Yes

166

218

225

264

294

136

FC-MSH6-9

298

225

288

209

206

296 292/294

167 161/169

155

Yes

166

218

225

264

284

132

FC-MSH6-10

286

227

294

211

196

292

169

155

Yes

166

218

225

264

270

136

FC-MSH6-11

280/286

199/201

290/292

205/211

196/206

294/296

161/169

155

Yes

166/172

214/218

225/241

264/270

270/284

136/144

Max 3.42 Mb Min 1.61 Mb

The markers tested in a panel of French Canadian controls are shown in a box. The "/" symbol indicates that the phase of the disease haplotype cannot be established. Grey shading indicates nonrecombinant haplotypes. The sizes of the minimum and maximum conserved haplotypes are shown at the bottom. Disease-associated alleles within the conserved haplotypes appear in bold. a and b represent the two phases for the homozygote carrier in family FC-MSH6-7.