Hum Hered 1991:41:188-194

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DNA Polymorphism and Linkage Disequilibrium within the Apolipoprotein C1I Locus on Human Chromosome 19 Alex E. MacKenzie, Heather L. MacLeod, Suzanne C. Leblond, Nancy Monteith, Donelda Lahey. Robert G. Korneluk Division of Genetics, Children's Hospital of Eastern Ontario, Ottawa, Ont., Canada

Key Words. Apolipoprotein Cl I • RFLPs • Linkage disequilibrium

Introduction Human apolipoprotein CI1 is a small 79-amino-acid-containing protein in­ volved in the activation of lipoprotein li­ pase and is encoded on the proximal long arm of chromosome 19 [1, 2]. The rare au­ tosomal recessive deficiency of apolipo­ protein CI I results in hypertriglyceri­ demia and a propensity to recurrent bouts of acute pancreatitis [3]. The clinical rele­ vance of the apolipoprotein CII gene (APOCII) also derives from its identifica­ tion as a closely linked marker for myo­ tonic dystrophy (DM), the most common

form of adult muscular dystrophy [4], Six restriction fragment length polymor­ phisms (RFLPs) in the APOCII region have been identified [5-8]. However, a de­ gree of linkage disequilibrium exists be­ tween some of these polymorphisms which reduces the potential information content of the APOCII RFLPs in DM link­ age analyses [6, 9]. In this report we ex­ amine the nature and degree of the dis­ equilibrium between 5 of these APOCII RFLPs in 213 non-DM haplotypes ob­ tained in the course of a DM linkage study. The physical ordering of these loci has been elucidated.

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Abstract. The gene for human apolipoprotein Cl I (APOCI1) is located on the proxi­ mal long arm of chromosome 19. It has been established as a closely linked marker for myotonic dystrophy (DM), the most common form of adult muscular dystrophy. In the present linkage study, we have analysed 6 APOCI1 RFLPs in 213 haplotypes: TaqI, 3.8/3.5 kb; Bgll, 12.0/9.0 kb; BanI, 2.5/1.6 kb; BamHI, 6.0/4.9 kb; Ncol, 14.5/11.5 kb, and Avail, 0.6/0.4 kb. The polymorphic enzyme sites were determined to be present at the following frequencies: Taql, 0.43; Bgll, 0.51; BanI, 0.25: BamHI, 0.99; Ncol, 0.51, and Avail, 0.52. Ordering of the polymorphic sites, 5' —3', has been determined to be (Ncol-Bgll)-Avall-Banl-Taql. Significant disequilibrium was seen between 5 of the APOCII RFLPs.

RFLPs and Linkage Disequilibrium within AP0CI1 Locus

Materials and Methods

189

Table I. Allelic frequencies (+ allele) of the APO­ CII RFLPs (n - 213)

Study Entries

DNA Analysis

DNA isolation, restriction enzyme digestion, probe labelling and hybridization conditions were all as previously described [9], Washed blots were ex­ posed to X-ray film (Kodak X-OMAT AR) at -70°C for 1-4 days. DuPont Lightning Plus screen intensifiers were used. A total of 6 APOCII RFLPs were screened: TaqI, 3.8/3.5 kb [5]; Bgll, 12/9 kb [6]: BanI, 2.5/I.6 kb: BamHI, 6.0/4.9 kb [10]; Ncol, 14.5/11.5 kb [11], and Avail, 0.6/0.4 kb [8]. The Bgll RFLP was detected with the genomic probe pSCI I, a gift of S.E. Hum­ phries [6]; the remaining RFLPs were detected with a full-length APOCII cDNA probe, kindly provided by F.E. Baralle [12], DNA Sequencing

DNA sequencing was performed on subcloned DNA by the dideoxy chain termination method [13] with either [,5S)a-dATP or [5:P]«-dATP (Amersham) and a sequencing system employing Klenow DNA polymerase (Promega). Direct sequencing was per­ formed as described by Korneluk et al. [14], Se­ quencing products were analyzed on 8 M urea-8% polyacrylamide gels. Statistical Analysis

The presence of linkage disequilibrium was deter­ mined by means of the standardized disequilibrium statistic 0 [15] as previously described [9]. Linkage disequilibrium was also quantified by calculating the parameter D as outlined by Thompson et al. [16].

Ncol

Bgll

Avail

TaqI

BanI

BamHI

0.51

0.51

0.52

0.43

0.25

0.99

Briefly, if D* = h -pq, Dm„ - p (l-q ), D„in ■= -p q , then D = either D*/DmM or D*/Dmm, which ever value is greater. The parameter h is the frequency of the haplotype comprised of the rare allele at both loci, and p and q are the frequencies of the rare al­ leles at both loci with p ¿ q ¿0.5.

Results A llelic Frequencies The ( + ) allelic frequencies for Ncol, Bgll, Avail and TaqI polymorphisms were all approximately 50% (table 1), in agreement with previously published re­ sults [6-8, 17, 18]. [The (+) symbol refers to the presence of a given restriction en­ zyme site, ( - ) to the absence of the poly­ morphic site.] The BanI and BamHI ( + ) allelic frequencies were 25 and 99% of the non-DM chromosomes, respectively, also in accordance with past studies [7, 18]. The allelic frequencies were virtually identical (i.e. no greater than 3% different) for the French Canadian and non-French Cana­ dian groups (data not shown). Linkage Disequilibrium between APOCII RFLPs Strong linkage disequilibrium was seen between 5 of the APOCII polymorphisms (Ncol, Bgll, Avail, TaqI, BanI); of the 32 haplotypes theoretically possible for these 5 RFLPs, only 9 were seen (table 2). Fur­ thermore, 90% of these were represent-

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Members of 52 DM families entered in our DM linkage study had APOCII RFLP identification per­ formed. Extensive haplotyping of DNA markers closely linked to APOCII permitted the unequivocal identification of the "non-DM" chromosomes for af­ fected individuals. APOCII haplotypes from these chromosomes as well as those of married-in family members were used in the data analysis. Care was taken not to score a given chromosome more than once. All individuals were Caucasian, the majority being of French Canadian (comprising over 50% of the study entrants), Scottish Canadian or Anglo-Ca­ nadian background. A total of 213 haplotypes from approximately 330 individuals were analysed.

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Table 2. Number of observed APOCII Ncol/ Bgll/Avall/BamHI/Taql/Banl RFLP haplotypes (n = 213) Haplotypes

Number

Nco Bgl Ava BamH Taq Ban

observed

expected3

+ + + —

106 52 34 2 II 1 4 2 1

12.2 2.6 7.8 0.1 10.4 9.2 11.3 8.5 3.5

+ + —

+ + + —

+ + + + + + + +

-

-

+ + + + + —

+ +

* Numbers of expected number of haplotypes us­ ing the allelic frequencies in table I and assuming lin­ kage equilibrium. y2> 1,000, p 1,000, d.f. = 31), indicating an exceedingly small likelihood of there being independence between the 5 polymorphisms. The extent of linkage disequilibrium between these 5 RFLPs was the same for both French Ca­ nadian and non-French Canadian study groups. The degree of linkage disequilib­ rium was even more marked between the Ncol, Bgll and Avail polymorphisms: 207 of the 213 observed N col/Bgll/Avall haplotypes were represented by 1 of 2 complementary haplotypes (+ + + , ------; table 2). The 0 parameter was 0.99 for the possible pairings of these 3 polymor­ phisms (table 3). Complete linkage diseq­ uilibrium in the coupling phase was ob­ served between the Ncol and Bgll poly­ morphisms (tables 2, 3). The high fre­ quency of the BamHI ( + ) allele (99%) ef­ fectively precluded meaningful statistical analysis with respect to this poly­ morphism.

191

sequences [19] and absent in the other [20], G AGTCCAGG(C/T)CCCCAGAC This proposed assignment has since been Fig. I. Twenty-nucleotide sequence of APOCII corroborated by in vitro genomic DNA region containing (C/T) polymorphic Avail site. amplification and direct DNA sequencing The polymorphism maps 80 bp 5' to APOCII exon 4. studies (fig. 1). These data yield an order Bgll-Avall-Taql, leaving the location of the Ncol polymorphic site unresolved. The Ncol RFLP is detected by both the APOCII cDNA probe and the genomic Thus, either the Ncol site lies such a small probe pSCl 1. Since the latter probe does distance downstream from the Bgll site not overlap APOCII and lies 5' to the that the resultant difference in fragment gene [6], the hybridizing Ncol restriction size is undetectable by Southern blotting fragment must extend upstream of APO­ or, a more likely situation, the Ncol site CII. Furthermore, in both published APO­ lies to the 5' side of the BII site. The ordering of (Ncol-Bgll)-AvalICII sequences, there were contiguous Ncol sites situated approximately 100 bp Taql is supported by the observed devia­ downstream from the 4th exon. It is clear tions from the 2 common N col/B gll/that neither of these could be the polymor­ Avall/Taql haplotypes, + + + - and phic Ncol site, given that the observed re­ ------ + . The 4 rare N col/B gll/A vall/ striction fragment lengths are 11.5 and TaqI haplotypes found on 19 chromo­ 14.5 kb. It was therefore deduced that the somes [+ + + + (n = 1 ),-------- (n = 12), Ncol polymorphic site lies 11.5 kb to the ----- F - (n = 4) and + + - + (n = 2); table 5' side of the intra-APOCII Ncol sites. 2] can be explained by single recombina­ This placed the Ncol site very close to the tion events occurring to 1 of the 2 natural polymorphic Bgll site, which lies 9 kb up­ haplotypes as follows: between the Avail stream of the 5' end of APOCII [6]. How­ and TaqI sites in the first 2 cases ever, the order of the 2 sites was unre­ (+ + -1— x ------- 1—- + + + + , -------- ) and solved. To clarify this issue, Ncol/Bgll between the Bgll and Avail sites in the double digests of DNA from 3 Ncol/Bgll second 2 cases (--------h x + - M----- * + + homozygous individuals were per­ — + - , + + - + ) . No other polymorphic formed and blot hybridization with pSCl 1 site ordering is compatible with single was carried out. It was reasoned that if the recombination events generating the rare polymorphic Ncol site was 5' to the poly­ haplotypes. The BanI site has been previously local­ morphic Bgll site then a 9-kb fragment such as that ordinarily seen with a Bgll (+) ized at the 3' end of the 4th APOCII exon genotype would be seen. If the Ncol site [21], We have obtained preliminary data lay 3' to the Bgll site, that is closer to from in vitro DNA amplification experi­ APOCII, then a smaller fragment would ments confirming this general localization be seen. A band of 9 kb, indistinguishable but placing it approximately 50 bp down­ from that normally seen following Bgll di­ stream from the 4th APOCII exon. The re­ gestion of an individual with a Bgll (+) spective polymorphic enzyme site posi­ genotype was visualized (data not shown). tions are shown in figure 2.

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RFLPsand Linkage Disequilibrium within APOCII Locus

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MacK.enzie/MacLeod/Leblond/Monteith/Lahey/Korneluk

(Neo I, Bgl I)

Ava II Ban I

_____ i___i_______

1

I H

Taq I ___ L_

2 3 4

1 kb I---------1

5 ’----- >3'

Discussion Allelic Frequencies The frequencies of the 6 APOCII poly­ morphic enzyme sites in both non-French Canadian and French Canadian chromo­ somes were all comparable to previously reported values. No significant differences in allelic frequencies were observed be­ tween the two linguistic groups. This is in agreement with the report of Williams et al. [17], who found no significant differ­ ence between the APOCII TaqI RFLP al­ lelic frequencies in such disparate groups as the Japanese and Africans. Linkage Disequilibrium between APOCII RFLPs Strong linkage disequilibrium exists be­ tween all 5 polymorphisms: 3 main N col/ B gll/A vall/B anl/Taql haplotypes are seen. As outlined below, the Ban I site is interposed between the Avail and TaqI polymorphic sites. Despite this placement, this polymorphism appears to be in weaker linkage disequilibrium with its flanking markers (0 = 0.60 for A vail/ BanI, 0 = 0.64 for Banl/TaqI) than these latter markers are with each other (0 = 0.88 for Avall/TaqI). However, the link­ age disequilibrium constant 0 is affected by polymorphic allelic frequencies; in or­

der to attain the theoretical maximum of |l|, 2 alleles have to have equal allelic fre­ quencies. This is true of Bgll/TaqI (p«*0.45-0.50) but not of Avall/Bgll or Avall/TaqI (Avail p = 0.25). When ana­ lysing the linkage disequilibrium with the allelic frequency-independent parameter D, the degree of linkage disequilibrium is constant between these 3 polymorphisms (table 3). However, there are some diffi­ culties with the latter means of statistical analysis; for example, if a mutation gen­ erates a new unique polymorphism in an individual, it would automatically be in 100% disequilibrium with all other poly­ morphisms as measured by D. Given the physical sequence of sites, it is possible that in the past there existed 2 main N col/B gll/A vall/B anl/T aql haplotypes: + + + — a n d ------+ + . At some point thereafter, a mutation oc­ curred resulting in the loss of the BanI pol­ ymorphic site (i.e .------ + + —--------- + ), leading to the current situation (N col/ B gll/A vall/B anl/Taql + + + — , 50%; ------ + + , 24% ;---------+, 17%; table 2). We feel that this was likely to have been a mutation as a recombinant event between the 2 common haplotypes (------+ + , + + + — ) would not generate a -------- + N col/B gll/A vall/B anl/T aql haplotype. This mutation may have happened once in

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Fig. 2. Disposition of polymorphic restriction enzyme sites within the APOC1I region. X = Polymorphic restriction enzyme sites: ■ = APOCI1 exons:---- = APOCII gene.

RFLPs and Linkage Disequilibrium within APOCI1 Locus

both the physical position and the intrapolymorphic linkage disequilibrium rela­ tionships for 5 polymorphisms in the APOCII region of chromosome 19.

Acknowledgements We would like to thank Drs S.E. Humphries and F.E. Baralle for supplying the APOCII probes. We are grateful to Dr. A.G.W. Hunter and our other clin­ ical colleagues for the arranging of the necessary blood sampling. We wish to thank T.L. Shenstone for his help in computer programming. This work was funded by a grant to R.G.K. from the Muscular Dys­ trophy Association of Canada. A.E.M. was a post­ doctoral fellow of the Canadian Heart Foundation when this work was conducted.

References 1 Huselbos T, Brunner H, Wieringa B, et al: Re­ gional assignment of C3, GPI, APOC2 and betaHCG and their linkage relationships with DM and I9cen. Human Gene Mapping 8. Cytogenet Cell Genet 1985:40:658. 2 Lusis AJ. Heinzmann C, Sparkes RS, et al: Re­ gional mapping on human chromosome 19: Apo­ lipoprotein E, apolipoprotein C2, low density lipoprotein receptor, peptidase D and glucose phosphate isomerase. Human Gene Mapping 8. Cytogenet Cell Genet 1985:40:683. 3 Nikillâ EA: Familial apolipoprotein C-II defi­ ciency; in Stanbury JB, Wyngaarden J, Fredrick­ son DS, Goldstein JL, Brown MS (eds): The Me­ tabolic Basis of Inherited Disease. New York, McGraw-Hill, 1983, pp 632-633. 4 Shaw DJ, Meredith AL, Sarfarazi M, et al: The apolipoprotein CI I gene: Subchromosomal local­ isation and linkage to the myotonic dystrophy lo­ cus. Hum Genet 1985;70:271-273. 5 Humphries SE, Jowett Nl, Williams, et al: A DNA polymorphism adjacent to the human apo­ lipoprotein CI I gene. Mol Biol Med 1983; 1: 463-471. 6 Wallis SC, Donald JA, Forrest LA, et al: The iso­ lation of a genomic clone containing the apoli-

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the distant past and dispersed thereafter or this may be a recurrent event due to an un­ stable Ban I site. The absence of disequilib­ rium between the Banl polymorphism and the DM locus in the French Canadian population in contrast with the other APOCI1 polymorphisms is compatible with the latter model [9], Such a Banl site loss could be assayed for in male sperm (if a meiotic event) or lymphoblasts (if a ran­ dom event) using PCR with a primer with a 3' end homologous to the DNA contain­ ing the lost Banl site. If there is a Banl site loss, even at a low frequency, then DNA amplification will be seen with such pri­ mers; if there is no such site loss, then no amplification will be seen. Linkage disequilibrium has been re­ ported previously between APOCII RFLPs [6, 7, 22-24], but only one study presented the data in a fashion which was amenable to the statistical analysis used here. In that study [6], a 0 of -0.76 is ob­ tained for the disequilibrium between the Bgll and TaqI polymorphisms. This is comparable to the value of -0.83 obtained in this report. We have shown that 5 of the 6 DNA polymorphisms detected in and around the APOCII locus are located in transcrip­ tionally inactive regions. This is in keep­ ing with Sephrenia et al.’s immuno-isoelectric focussing study of apolipoprotein CM polymorphism [25]. Of the 186 Ameri­ can Caucasians studied, only 1 individual showed an immunoblotting pattern devi­ ating from that otherwise seen. This pro­ tein polymorphism (allelic frequency 1/186) may represent the polymorphic BamHI site [(-) allelic frequency 2/213] which we have not mapped in this study. In conclusion, we have documented

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poprotein CII gene and the detection of linkage disequilibrium between two common DNA poly­ morphisms around the gene. Hum Genet 1984; 68:286-289. 7 Appleby VL, Coleman RT, Frossard PM: Link­ age disequilibrium at the human apolipoprotein CII gene locus. Am J Hum Genet 1986; 39(suppl):AI45. 8 Korneluk RG, MacLeod HL, Leblond SC, et al: Ava II RFLP at the human apolipoprotein CII (APOC2) gene locus. Nucleic Acids Res 1987:15: 6769. 9 MacKenzie AE, MacLeod HL, Hunter AGW, et al: Linkage analysis of the apolipoprotein C2 gene and myotonic dystrophy on human chromo­ some 19 reveals linkage disequilibrium in a French-Canadian population. Am J Hum Genet 1989;44:140-147. 10 Frossard PM, Coleman RT, Assmann G: Genetic polymorphisms at the apolipoprotein CII locus. Am J Hum Genet 1985;37(suppl):AI53. 11 Frossard PM, Coleman RT, Assmann G: Nco I RFLP at the human apolipoprotein CII locus. Nucleic Acids Res 1986:14:5120. 12 Sharpe CR, Sidoli A, Shelley CS, et al: Human apolipoproteins AI, All, CII and C lII: cDNA se­ quences and mRNA abundance. Nucleic Acids Res 1984;12:3917-3923. 13 Sanger F, Nicklen S, Coulson AR: DNA sequenc­ ing with chain-terminating inhibitors. Proc Natl AcadSci USA 1977;74:5463-5467. 14 Korneluk RG, Quan F, Gravel RA: Rapid and re­ liable sequencing of double stranded DNA. Gene 1985:40:317-323. 15 Hill WG. Robertson A: Linkage disequilibrium in finite populations. Theor Appl Genet 1968:38: 226-231. 16 Thompson EA, Deeb S, Walker D, et al: The de­ tection of linkage disequilibrium between closely linked markers: RFLPs at the AI-CIII apolipo­ protein genes. Am J Hum Genet 1988;42:113-124. 17 Williams LG, Jowett NI. Vella MA, et al: Allelic variation adjacent to the human insulin and apo­ lipoprotein CII genes in different ethnic groups. Hum Genet 1985;71:227-230.

18 Bird TD, Boehnke M, Schellenberg GD, et al: The use of apolipoprotein CII as a genetic marker for myotonic dystrophy. Arch Neurol 1987:44:273-275. 19 Fojo SS, Law SW, Brewer BJ Jr: The human preapolipoprotein CII gene: Complete nucleic acid sequence and genomic organization. FEBS Lett 1987;213:221-226. 20 Wei C-F, Tsao Y-K, Robbcrson DL, et al: The structure of the human apolipoprotein CII gene. J Biol Chem 1985:260:1521-1522. 21 Frossard PM, Coleman RT, Funke H, et al: Di­ morphic markers for the human apolipoprotein CII gene locus. Gene 1987;51:103-106. 22 Lunt PW, Meredith AL, Huson SM, et al: The ap­ plication of closely linked restriction fragment length polymorphism in counselling families with myotonic dystrophy. J Med Genet I987;24: 239. 23 MacLeod HL. Hunter AGW, Korneluk RG: A new RFLP in linkage disequilibrium at the hu­ man apolipoprotein CII gene locus and linkage relationship in French-Canadian families. Am J Hum Genet 1987;4l(suppI):A175. 24 Boileau C, Benlian P, Bonait'i C, Pastier D, Loux N, Coulon M, Ragab A, Cambien F, Ruidavets JB, Junien C: Positive linkage disequilibrium in the apolipoprotein gene cluster on chromosome 19. Cytogenet Cell Genet 1989:51:965. 25 Sephrenia B. Kamboh MI, Ferrell RE: Genetic studies of human apolipoproteins. III. Polymor­ phisms of apolipoprotein C-II. Human Hered 1988;38:136-143.

Alex MacKenzie. MD Division of Genetics Children's Hospital of Eastern Ontario Ottawa. Ont. KI H 8 LI (Canada)

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194

DNA polymorphism and linkage disequilibrium within the apolipoprotein CII locus on human chromosome 19.

The gene for human apolipoprotein CII (APOCII) is located on the proximal long arm of chromosome 19. It has been established as a closely linked marke...
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