Am. J. Hum. Genet. 46:126-132, 1990 I-Thalassemia Genes in French-Canadians: Haplotype and Mutation Analysis of Portneuf Chromosomes Feige Kaplan,*I §, Georgia Kokotsis, * Marc DeBraekeleer, # Kenneth Morgan,1I and Charles R. Scriver* t §III DeBelle Laboratory for Biochemical Genetics, McGill University-Montreal Children's Hospital Research Institute; Departments of tBiology, *Epidemiology and Biostatistics, and SPediatrics, and IlCentre for Human Genetics, McGill University, Montreal; and #Population Research Group (SOREP), Universite du Qudbec A Chicoutimi, Chicoutimi, Quebec Summary 1-Thalassemia minor occurs at _1% frequency in French-Canadians-in families residing in Portneuf County (population ~40,000) of Quebec province. We found eight different RFLP haplotypes at the 3-globin gene cluster in 37 normal persons and in 12 j-thalassemia heterozygotes from six families. 13-Thalassemia genes in these families associated with two haplotypes only: Mediterranean I and Mediterranean II. There were two different P-thalassemia mutations segregating in the Portneuf population: an RNA processing mutation (P3+IVS-1,nt11O) on haplotype I (five families) and a point mutation leading to chain termination (300 non- sense codon 39) on haplotype II (one family). The distribution of 5' haplotypes on normal P3A Portneuf chromosomes compared with other European populations was most similar to that in British subjects (data for French subjects have not yet been reported). Genealogical reconstructions traced the ancestry of carrier couples to settlers emigrating from several different regions of France to New France in the 17th century. These findings indicate genetic diversity of a greater degree among French-Canadians than recognized heretofore. Introduction a few specific 0-thalassemia genes occur at high fre- The 13-thalassemias are a heterogeneous group of dis- quencies in regional populations, together with numer- orders affecting hemoglobin synthesis. They are caused ous rarer alleles (Cao et al. 1989), and particular regions by a variety of mutations (over 50 identified to date) tend to have their own prevalent mutations. which impair expression of the 0-globin gene (Kaza- The 60-kb region containing the 3-globin gene cluster zian and Boehm 1988; Weatherall et al. 1989), and, on chromosome 11 (region 11p15.5), where ,B-thal- as a group, they constitute the most common Mendelian assemia mutations occur, shows extensive polymor- disorder in the world; about 3% ofthe world's popula- phism in nucleotide sequence. At least 17 sites rec- tion probably has a 13-thalassemia gene. fVThalassemia ognized by restriction endonucleases are polymorphic heterozygotes, relative to normal homozygotes, were (Antonarakis et al. 1985), and there are additional poly- putatively more resistant to malarial parasite infection morphisms involving intragenic sequences not recog- (Haldane 1949), and selection was probably the prin- nized by restriction enzymes (Kazazian et al. 1984). The cipal determinant of gene frequency in regions of en- polymorphisms define two regions of linkage disequi- demic malaria in the past. In Mediterranean regions librium, referred to as the 5' and 3' RFLP haplotypes; they flank a region of randomization 5' to the P-globin gene liable to recombination (Antonarakis et al. 1982, Received July 5, 1989. 1985; Chakravarti et al. 1984). Within regional popu- Address for correspondence and reprints: Charles R. Scriver, M.D., lations, a particular 1-thalassemia gene tends to associ- DeBelle Laboratory, Montreal Children's Hospital, 2300 Tupper Street, ate with a RFLP et al. Montreal, Quebec H3H 1P3, Canada. particular haplotype (Orkin C) 1990 by The American Society of Human Genetics. All rights reserved. 1982). However, there can be a high degree of hetero- 0002-9297/90/4601-0016$02.00 geneity in these associations both within and between 126 f-Thalassemia Genes in French-Canadians 127 population and found two mutations (I + IVS-1,nt11O and 1W nonsense codon 39), each on a different haplo- type, in the Portneuf population. Genealogical recon- structions indicate that both mutations could have been brought to New France by settlers from France in the 17th century. Subjects and Methods Subjects We performed haplotype and mutation analyses in five families and performed mutation analysis only in one additional family. All individuals were in the Port- neuf pedigrees reported by Desjardins et al. (1978). ,B-Thalassemia heterozygotes were classified by eryth- rocyte phenotype (MCV < 80 fl, HbA2 >3%) (Zannis- Hadjopoulos et al. 1977) and were confirmed by ge- notype. Figure I Geographic localization ofPortneuf County in Que- DNA Haplotype Analysis bec Province. Portneuf County (population about 40,000) is on the north shore of the St. Lawrence river west of Quebec City. Genomic DNA (3-5 ig) from blood leukocytes was digested to completion with restriction endonucleases (HincII, HindIII, Hinfl, AvaII, and BamHI) under con- populations (Antonarakis et al. 1985; Athanassiadou ditions recommended by the supplier. Digested DNA et al. 1987), and both mutations reported here have was electrophoresed on agarose gels (0.8%-1.2%), been reported on multiple haplotypes in Mediterranean transferred to Nitroplus 2000 (Fisher), fixed, hybrid- populations (Cao et al. 1989). ized with [32P]dCTP-labeled probes, and washed be- 1-Thalassemia minor has been reported in immi- fore autoradiography by standard procedures. We ana- grants from Italy and Greece now living in Montreal lyzed nine polymorphic sites (fig. 2) and used six DNA (Scriver et al. 1984) and also in French-Canadians in probes provided by H. H. Kazazian, Jr.: (1) a 1.3-kb Quebec province (Rioux and Delage 1964; Desjardins BamHI-EcoRI fragment of the cloned s-globin gene; et al. 1978). In the latter case, the trait segregates in (2) a 0.93-kb BamHI-EcoRI fragment containing the a small region called Portneuf County (fig. 1), where IVS-2 of the cloned y-globin gene; (3) a 1.7-kb BglII- the frequency of heterozygosity is on the order of 1% XbaI fragment from the cloned Wi-globin gene; (4) a (Prevost et al. 1988). We performed haplotype and mu- 1.9-kb BamHI fragment containing 5' flanking sequence tation analyses to determine the association between ofthe 3-globin gene; (5) a 0.92-kb BamHI-EcoRI frag- RFLP haplotype and 0-thalassemia mutation in this ment containing the IVS-2 ofthe cloned 3-globin gene; 60 50 40 30 20. tO * 0* I Ikb 2kb s GM Ar 5 E a 3 3' I II It II Hc Hd Hd Hc Hc Hf Av Hd Bm 5' Haplotype 3' Haplotype Figure 2 Location of nine polymorphic restriction sites linked to the 13-globin gene used here to describe RFLP haplotypes: Hc = HincII; Hd = HindIII; Hf = Hinfl; Av = AvaII;r--Bm = BamHI. The S' and 3' regions in the haplotypes, referred to in text, are indicated. 128 Kaplan et al. (6) a 0.9-kb BglII-EcoRI fragment ofcloned DNA 10 kb baked at 801C for 1 h. Filters were hybridized in 5 x 3' to the 0-globin gene. All probes were lab)eled by ran- SSPE, 0.5% SDS, 5 x Denhardt solution containing dom primer extension with [32P]dCTP aLccording to 5 x 106 cpm hybridization buffer/ml for 1 h, rinsed the supplier's instructions. in 2 x SSPE, 0.1% SDS at 250C, washed for 10 min at the Tm of the ASO, and exposed to X-ray film for Mutation Analysis 30 min at -800C. Hybridization/wash temperatures Genomc DNA (0.5-1 rg) was amplified by the ther- were as follows: 1W 39 normal, 580C/60'C; 1W 39 mocycling method of Saiki et al. (1985) thrrough 30 cy- mutant, 561C/581C; 0+IVS-1,ntllO (normal), 500C/ cles by using the polymerase chain reacition (PCR), 520C; 1+ IVS-1,ntllO (mutant), 480C/500C. Thermus aquatus heat-stable Taq DNA polymerase (Perkin Elmer), and PCR primers that flaink a region Genealogical Reconstruction of the 0-globin gene where most "Mediteriranean" mu- Pedigrees were reconstructed over 3-4 generations, tations are clustered (fig. 3). The cycling rreaction was and the oldest member carrying a 3-thalassemia muta- performed in a programmable Perkin Elmer DNA Ther- tion was identified in each. Genealogies were then recon- mal Cycler set to heat samples to 941C for 30 s (to structed using several published marriage registries of denature DNA), cool them to 550C for 30 s (to anneal various regions of Quebec that allowed reconstruction primers), and heat them to 720C for 2 miin (to extend to the beginning ofthe 19th century. The pedigrees were annealed primers). After the last cycle, soamples were completed using genealogical dictionaries (Tanguay incubated for an additional 10 min to ensuire complete 1971; Pontbriand 1978; Jette 1983) to recognize the an- extension. Buffer and dNTPs were suppliced by Perkin cestors in Europe. Elmer. For direct mutation analysis, amplified D)NA was dot Population Genetic Analysis blotted onto nylon filters and hybridized with allele- For genetic distance analysis we computed Nei's mini- specific oligonucleotide probes (ASO), for the 1W non- mum and standard genetic distances. In addition, we sense codon 39, and i + IVS-1,ntllO mutations (fig. 3), used the procedure of Reynolds et al. (1983) to esti- labeled at the 5' end with [32P]ATP. Amj?lified DNA mate the coancestry coefficient, 0, by weighted analysis was mixed with 100 id 0.4 N NaOH and 2'5 mM EDTA of variance for pairs of populations. The estimate of (to denature DNA), applied to nylon memb)rane (Gene- the coancestry coefficient for a given locus is 0 = tran 45; Plasco), fixed to nylon by UV irradiation, and a/(a+b), where a is the unbiased estimate of the be- tween-populations component of variance and b is the estimate ofthe within-population component. The es- 39 timate of genetic distance is D = -ln(1- 0).