I-Thalassemia Genes in French-Canadians

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 nonsense 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-thalas 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 haplotype, in the Portneuf population. Genealogical reconstructions 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 genotype. 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 between-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). Unbiased ~~~EXON I 1,EXON 2 -im IVS2 IPOL estimates of b are positive. However, where one or both I samples are small in size and genetically similar, the WYs1,110 unbiased a can be negative because of adjustment for sampling error. In this case, the estimate of a is by con- 5' SGLOBIN PRIMER 5'TCCTAMCCAGTGCCAGAAG 3.1 vention put equal to zero (e.g., see Ewens 1979) or the 3' BGLOBIN PRIMER 5'TCATTCGTCTGTTTCCCATTCtAAAC 3' genetic distance is assumed to be zero (Nei 1987). Al- gorithms for genetic analysis were programmed in the 039 S language (Becker and Chambers 1984) as imple- NORMAL PROBE 5'CCTTHQACCCAGAGGIC 3* mented in S-Plus (Statistical Sciences, Inc. 1988). NITANT PROBE 5'ACAACCICTAGGCCAAGG 3' The data were the frequencies of 5' haplotypes (Hc Hd Hd Hc Hc sequence of polymorphic sites) of nor- IVS1 10 mal pA chromosomes for Europeans from the follow- NORMAL PROBE 5'AAAATAGACCAAIAGGCAG 3 ing sources: French-Canadians from Portneuf County MUlANT PROBE 5*'CGCCTATIJAGICTAI 11 3 (present study), Britons (Wainscoat et al. 1986), Ger- Figure 3 The 770-bp region of the 0-globin Igene, in which mans (Oehme et al. 1985), Italians (Wainscoat et al. the A+ IVS-1,ntllO and nonsense codon (C39) mlutations occur, 1986; Long et al., in press), Greeks (Athanassiadou et used here for amplification and mutation analysis. Er al. et in amplified product were determined by the 3-globin priidpointsaofthe 1987; Long al., press), Cypriots (Wainscoat The allele-specific oligonucleotides used here for ana elysis ofnormal et al. 1986), and Turks (Antonarakis et al. 1982; Oehme and mutant alleles (altered base pair underlined) alre also shown. et al. 1985). Samples of the same ethnic group were f3Thalassemia Genes in French-Canadians 129 pooled, giving the following numbers of chromosomes Mutation Analysis for the eight samples: British, N = 37; Portneuf, N 1-Thalassemia genes in the Portneufpopulation were = 33; German, N = 16; Italian, N = 84; Cypriot, associated with Mediterranean I or Mediterranean II N = 82; Greek, N = 81; and Turkish, N = 20. haplotypes exclusively; these haplotypes are designated in table 1. The mutation on the former was an RNA Results processing mutation (O + IVS-1,ntllO). It was identified in four families (fig. 4) in whom haplotype analysis was Haplotypes complete and by PCR analysis also in a fifth family (fam- We found two haplotypes (Mediterranean I and II) ily M; fig. 4) in whom haplotype analysis could not on 0-thalassemia chromosomes, both common in Med- be completed. The mutation on Mediterranean haplo- iterranean populations (Kazazian et al. 1984; Cao et type II encoded the nonsense codon 39 (fig. 4). It al. 1989). There were eight haplotypes on the 13A Port- occurred in only one of the six families studied here. neuf chromosomes; at least seven of them have been identified in Mediterranean populations (see table 1). Genealogical Reconstruction We constructed a sample of 13A chromosomes for Portneuf County (fig. 1), population -40,000, was Portneuf (number of chromosomes = 33) from indi- settled by a small number of families in the 17th cen- viduals who were known not to be directly related. The tury. Population expansion since then has been achieved distribution of the haplotypes for Portneuf is given in primarily by endogamous marriage and natural in- table 1. The distribution of the 5' haplotypes (Hc Hd crease. There has been no apparent recent immigration Hd Hc Hc polymorphic sites) was +--(N = 14), to Portneuf County from regions where ,B-thalassemia -+-++ (N = 13),and -++-+ (N = 6). ThePort- genes are prevalent. neuf sample of 1iA chromosomes consists of the three Genealogical reconstruction was undertaken to trace globally most common 5' haplotypes (Wainscoat et al. the ancestral origins of the oldest carriers confirmed 1986; Long et al., in press). by DNA analysis in each of the six families. Prelimi-

Table I Haplotype Frequencies on pA Chromosomes in Portneuf County, Mediterraneans, and Europeans HAPLoTYPEa PORTNEUFb GREEK MEDITERRANEAN GERMAN Hc Hd Hd Hc Hc Av Hda Bm N Frequency N Frequency N Frequency N Frequency I ...... + - - + [+] + 5 .16 20 .48 42 .34 4 .25 II...... - + + - + + [+1 + 4 .13 5 .12 8 .07 0 .00 III...... - + - + + + [+] - 5 .16 0 .00 8 .07 1 .06 IV ...... - + - + + - [-] + 2 .06 1 .02 4 .03 1 .06 V ...... + - - + [+ - 6 .19 8 .19 22 .18 2 .13 VI ...... - + + - - - [- + 0 .00 3 .07 0 .00 0 .00 VII ...... + [-] + 2 .06 3 .07 12 .10 1 .06 IX ...... - + - + + + [+] + 7 .22 2 .05 15 .12 3 .19 G ...... - + - + + + [-I + 1 .03 C d c d c d H ...... - + + - + - + 0 .00 0 .00 ...C d 1 .06

...... + + - _ + + 0 .00 0 .00 ...c d 2 .13

...... - + + _ 0 .00 0 .00 ...c d 1 .06 Other 0 .00 0 .00 11 .09 0 .00 Total 33' 42 122 15 SOURCES.-Portneuf County, Quebec (present study); southwestern Greece (Athanassiadou et al. 1987); Mediterranean aggregate (Kazazian et al. 1984); German families (Oehme et al. 1985). a For haplotype designations, see Orkin and Kazazian (1984). Data on the 3' HindIII site are available for the Portneuf County families. b Haplotypes from Portneuf individuals who were not closely related. All 3-globin gene region haplotypes were Hinfl + in the Portneuf sample. c Haplotypes not specified. d No data. ' Includes one chromosome which was classified only for the 5' haplotype, + 130 Kaplan et al.

lies; seven are prevalent in persons living today in Med- Family IVS 1, 110 C39 Genotype iterranean regions, and one is unusual (see table 1). The Normj MutMNoml Mut 10-thalassemia alleles in Portneuf segregated on two of I 4~I W-~f::,; I-M w these haplotypes, Mediterranean I and II, suggesting .s:. Pa AP 39 that at least two different k-thalassemia mutations could be segregating in the Portneuf population. Indeed, we T found two mutations, and they were those found most frequently in Mediterranean populations today (Cao D et al. 1989), namely, A+ IVS-1,nt110 (G-IA) and 1W nonsense codon 39 (C-UT). Up to now these mutations Pe had not been identified in French-Canadians. Evidence of eight different haplotypes in this small L sample of the regional population suggests that the founders of Portneuf were either few but already very M diverse genetically or more numerous and diverse in A_ origin than presumed. C As far as we are aware, the present study provides the first published report ofhaplotypes on the 3-globin Figure 4 Dot-blot analysis with allele-specific oligonucleotide gene cluster on both 0-thalassemia and ,BA chromo- probes of amplified genomic DNA shows that the A+ IVS-1,ntllO somes from a population of French origin. With re- mutation (five families) and the I° nonsense codon 39 mutation (one to the between Portneuf and family) segregate in a French-Canadian population where 13-thal- spect genetic distances the assemia minor occurs. other European populations, Nei's (1987) standard genetic distance not adjusted for sampling error is smallest between Portneufand the British sample. How- nary analyses showed that the majority of ancestors ever, unbiased estimates ofNei's minimum genetic dis- in the six thalassemia families came to PortneufCounty tance and the estimates of the coancestry coefficient 4-5 generations ago from Batiscan and La Perade, two of Reynolds et al. (1983) are negative for the pairwise small villages situated westward on the north bank of comparisons between Portneuf, British, and German the St. Lawrence River. Reconstruction back to France samples. By convention a negative estimate is taken to identified both a set of nine founder couples common be a genetic distance of zero. Thus, as a consequence to four of the five families with the A+ IVS-1,ntllO mu- ofgenetic similarity ofthe distributions ofthe 5' haplo- tation and a set of four founder couples common to types and because of small sizes ofcurrently published all five. The latter originated from four different regions samples, these three populations appear to belong to of France: Angoumois, Normandie, Saintonge, and the same European cluster. Data from many loci are Languedoc. The first three are in northwest France; Lan- required for reliable estimates of genetic distance, par- guedoc is in southern France and includes the Mediter- ticularly when genetic distance is small (Nei 1987). ranean littoral. Furthermore, Sokal (1988) has shown significant The family segregating the nonsense 39 mutation correlations between genetic and geographic distances shared only one founder couple with the set of nine in Europe. In addition, after adjustment for differenti- common to four of the five with the A+ IVS-1,ntllO ation by geographic region, there remained evidence mutation. This founder came from Perche, a western for genetic differences between language families. Long region of France. We have not been able to trace the et al. (in press) have developed an approach for choos- origin of a couple whose daughter was married in Port- ing a phylogenetic reconstruction of populations that neuf County in 1844. is based on a scheme for the evolution of the 5' haplotypes by mutation and recombination. Their method attempts to take into account both common and rare Discussion haplotypes. It would be interesting to apply this method We describe here six families living today in Portneuf to both 5' and 3' haplotypes of the P-globin gene clus- county, a region extending north of the St. Lawrence ter (e.g., see fig. 2 and table 1) were more data available. and west of Quebec City. We identified several RFLP Our findings are likely to encourage more research haplotypes at the P-globin gene cluster in these fami- on the origins and genetic structure of French-Cana- fBThalassemia Genes in French-Canadians 131 dians. We anticipate that some settlers of Portneuf Cao A, Gossens M, Pirastu M (1989) I-thalassemia muta- County either came from Languedoc directly or, ifthey tions in Mediterranean populations. Br J Haematol 71: came from elsewhere in France, had ancestors from Lan- 309-312 guedoc. The thalassemia mutations found in Portneuf Chakravarti A, Buetow KH, Antonarakis SE, Waber PG, are two most prevalent thalasse- Boehm CD, Kazazian HH Jr (1984) Non-uniform recom- French-Canadians the bination within the human 3-globin gene cluster. Proc Natl mia mutations found today in residents of the Lan- Acad Sci USA 79:137-141 guedoc region of France (Cao et al. 1989). Desjardins L, Rousseau C, Duplain JH, Valet JP (1978) La Delineation of the 13-thalassemia genes in French- thalassemie chez des Quebecois francophones. Can Med Canadians has implications for genetic and reproduc- Assoc J 119:709-713 tive counseling to avoid consequences of homozygos- Ewens WJ (1979) Mathematical population genetics. Springer, ity (Scriver et al. 1984). It is also relevant to medical Berlin, Heidelberg, New York practice; the "microcytic anemia" associated with ex- Haldane JBS (1949) The rate of mutation of human genes. pression ofthese thalassemia genes in carriers does not Proc VIII Int Congress Genet Hered (Suppl) 35:367 need treatment, and knowledge of carrier status can Jette R (1983) Dictionnaire genealogique des familles du Que- unnecessary iron medication. bec: des Origines a 1730. Presses de l'Universite de Mon- prevent treal, Montreal. Kaplan F, Akerman B, Bardanis M, Scriver CR (1988) The Acknowledgments 0-globin gene, a 13-thalassemia allele and associated haplotypes in French-Canadians. Am J Hum Genet 43:A216 The authors acknowledge the kindness of Haig H. Kazazian Kazazian Jr HH, Boehm CD (1988) Molecular basis and Jr., Corinne Boehm, and Carol Dowling in helpful discus- prenatal diagnosis of 1-thalassemia. Blood 72:1107-1115 sions and for their guidance in mutation analysis. We are in- Kazazian HH Jr, Orkin SH, Markham AF, Chapman CR, debted to Beverly Akerman and Nur Akalin for their assistance Youssoufian H, Waber PG (1984) Quantification ofthe dose and to Gerard Bouchard for advice about the genealogical association between DNA haplotypes and specific 1-thal- reconstruction. This work was supported in part by the MRC assemia mutations in Mediterraneans. Nature 310:152-154 Genetics Group, NSERC, Les Fonds de la Recherches en Sante Long JC, Chakravarti A, Boehm CD, Antonarakis SE, Kaza- du Quebec, the Interuniversity Population Research Center zian HH Jr. A phylogeny of human ,B-globin haplotypes (SOREP), and the Quebec Network of Genetic Medicine. and its implications for recent human evolution. Am J Phys Computing facilities were provided by the Howard Hughes Anthropol (in press) Medical Institute genome resources project and McGill Univer- Nei M (1987) Molecular evolutionary genetics. Columbia sity. K.M. is supported by Le Programme dActions Struc- University Press, New York. turantes du Ministere de l'Enseignement Superieur et de la Oehme R, Kohne E, Horst J (1985) DNA-polymorphic pat- Science du Quebec. We thank Jeffrey Long for a preprint of terns linked to 3-globin genes in German families affected the paper by J. C. Long, A. Chakravarti, C. D. Boehm, S. with hemoglobinopathies and 3-thalassemias: a compari- Antonarakis, and H. H. Kazazian, Jr. Our own work was son to other ethnic groups. Hum Genet 71:219-222 presented in part at the 1988 annual meeting of The Ameri- Orkin SH, Kazazian HH Jr (1984) The mutation and poly- can Society of Human Genetics (Kaplan et al. 1988). This morphism ofthe human ,B-globin gene and its surrounding is publication 89022 from the McGill University-Montreal DNA. Annu Rev Genet 18:131-171 Children's Hospital Research Institute. Orkin SH, Kazazian HHJr, Antonarakis SE, GoffSC, Boehm CD, SextonJP, Waber PG, et al (1982) Linkage of 3-thalas- References semia mutations and 0-globin gene polymorphims in the human 0-globin gene cluster. Nature 296:627-631 Antonarakis SE, Boehm CD, Giardina PJV, Kazazian HH Jr Pontbriand B (1978) Mariages du Comte de Portneuf (1982) Nonrandom association of polymorphic restriction (1679-1900). Sillery, Quebec sites in the P-globin gene cluster. Proc Natl Acad Sci USA Prevost C, Laframboise R, Bardanis M, Clow C, Lancaster 79:137-141 G, Desjardins L, Cantin F, et al (1988) La Gene de la ,B-thal- Antonarakis SE, Kazazian HHJr, Orkin SH (1985) DNA poly- assemie au Canada fransais: relance dans le comte de Port- morphism and molecular pathology of the human globin neuf. Union Med Can 118:141-144 gene clusters. Hum Genet 69:1-14 Reynolds J, Weir BS, Cockerman CC (1983) Estimation of Athanassiadou A, Zarkadis I, Papahadjopoulou A, Maniatis the coancestry coefficient: basis for a short-term genetic GM (1987) DNA haplotype heterogeneity of 1-thalassemia distance. Genetics 105:767-779 in Greece: feasibility of prenatal diagnosis. Br J Haematol Rioux E, Delage JH (1964) Thalassemie au Quebec. Union 66:379-383 Med Assoc J 119:709-713 Becker RA, ChambersJM (1984) An interactive environment Saiki RK, Sharf S, Faloona F, Mullis KB, Horn GT, Erlich for data analysis and graphics. Wadsworth, Belmont, CA HA, Arnheim N (1985) Enzymatic amplification of 13-globin 132 Kaplan et al.

genomic sequences and restriction site analysis for diagno- Wainscoat JS, Hill AVS, Boyce AL, Flint J, Hernandez M, sis of sickle cell anemia. Science 230:1350-1354 Thein SL, Old JM, et al (1986) Evolutionary relationships Scriver CR, Bardanis M, Cartier L, Clow CL, Lancaster GA, of human populations from an analysis of nuclear DNA Ostrowsky JT (1984) 0-Thalassemia disease prevention: polymorphisms. Nature 319:491-493 genetic medicine applied. AmJ Hum Genet 36:1024-1038 Weatherall DJ, Clegg JB, Higgs DR, Woods WG (1989) The Sokal RR (1988) Genetic, geographic, and linguistic distances Hemoglobinopathies. In: Scriver CR, Beaudet AL, Sly WS, in Europe. Proc Natl Acad Sci USA 85:1722-1726 Valle D (eds) The metabolic basis of inherited disease, 6th Statistical Sciences, Inc (1988) S-Plus for Sun workstations. ed. McGraw Hill, New York, pp 2281-2339 Statistical Sciences, Seattle Zannis-Hadjopoulos M, Gold RJM, Maag UR, Metrakos Tanguay C (1871) Dictionnaire genealologique des familles JD, Scriver CR (1977) Improved detection of,-thalassemia Canadiennes. Eusebe Senecal, Quebec carriers by a two-test method. Hum Genet 38:315-324