Hum Genet (1993) 91 : 312-316 human ..

genet, cs �9 Springer-Verlag 1993

Support for founder effect for two lipoprotein lipase (LPL) gene mutations in French Canadians by analysis of GT microsatellites flanking the LPL gene

Stephen Wood, Michael Schertzer, Michael Hayden, Yuanhong Ma

Department of Medical Genetics, University of British Columbia, 6174 University Boulevard, Vancouver, British Columbia V6T l Z3, Canada

Received: 20 July 1992 / Revised: 25 September 1992

Abstract. Mutations in the human lipoprotein lipase (LPL) gene are one of the major causes of familial chylomi- cronemia. We have characterized two polymorphic GT microsatellites f lanking this gene. Two LPL mutations that are extremely frequent in French Canadians appear to be in complete l inkage disequi l ibr ium with specific LPL microsatellite haplotypes indicating a founder effect with- in this population.

Introduction

Lipoprotein lipase (LPL) hydrolyzes the triglyceride core of very low density lipoproteins (VLDL) and chylomi- crons making free fatty acids available for energy metabo- lism or further storage. Patients with LPL deficiency, which is inherited as an autosomal recessive trait, often present with eruptive xanthomas, chylomicronemia, and recurrent pancreatitis (Brunzell 1989). This familial chy- lomicronemia is estimated to occur with a frequency of about I per mil l ion in most populations but is 100- to 200- fold more frequent in parts of French Canada (Gagne et al. 1989).

The human LPL cDNA has been cloned and sequenced ( W i e n e t al. 1987). The LPL gene is located at 8p22 (Sparkes et al. 1987) and consists of 10 exons distributed over 30 kilobases (kb) DNA (Deeb and Peng 1989). Nu- merous mutations responsible for the recessive lipoprotein lipase deficiency phenotype have been characterized (Hay- den and Ma 1992) including two missense mutations at residue 207 and 188 that are common in French Canadi- ans. The Pro-->Leu mutation at residue 207 accounts for over 70% of the mutant alleles while the Gly--->Glu muta- tion at residue 188 accounts for most of the remaining mu- tant alleles in a group of 37 French-Canadian patients with LPL deficiency (Ma et al. 1991).

The hydrolysis of VLDL by LPL modulates plasma LDL and HDL levels (Nikkila et al. 1978). Possible asso-

Correspondence to: S.Wood

ciations between restriction f ragment length polymor- phism (RFLP) markers within the LPL gene and plasma triglyceride levels (Chamberlain et al. 1989) as well as the levels of both HDL cholesterol and total cholesterol (Heiz- mann et al. 1991) have been reported. To provide highly polymorphic markers for investigation of the ancestry of particular LPL mutations as well as possible association between particular LPL haplotypes and lipid profiles we have screened the LPL region for highly informative GT microsatellite polymorphisms (Weber 1990). We have iso- lated two GT simple sequence repeat (SSR) loci f lanking the LPL gene and characterized allelic variants. We have found that each of the two common French-Canadian LPL mutants are carried by a single SSR haplotype.

Materials and methods

The flow-sorted chromosome-8 cosmid library LA08NC01 (prepared at Los Alamos as part of the National Laboratory Gene Library Pro- ject supported by the U. S. Department of Energy) was screened for cosmids containing LPL sequences (Wood et al. 1992) using the LPL35 cDNA clone (Wion et al. 1987). Four positive cosmids were isolated 114C1 I, 165B9, 178F10, and 179G2. An additional cosmid 5" from the gene, 17B7, was isolated by cosmid walking (Fig. 1).

Cosmid DNAs were digested with EcoRI, Southern blotted, and fragments containing GT microsatellites identified by hybridization with poly (dG dT)-(dC-dA). These fragments were subcloned into pBluescript II and the GT microsatellite sequenced using a modified (Seto 1990) Sequenase (US Biochemical Corp.) protocol.

The Centre d'Etude du Polymorphisme Humain (CEPH) panel is a mixed population consisting of Utah Mormons, French, Venezuelan, and Amish individuals (Dausset et al. 1989). We also examined a to- tal of 33 French-Canadian probands with LPL deficiency who were unrelated to each other for at least three generations (Ma et al. 1991 ).

Allelic variation within GT tracts was detected by polymerase chain reaction (PCR) amplification of genomic DNA using flanking oligonucleotide primers. Amplification reactions of 30 cycles con- sisted of 1 min at 94~ 30s at 58~ and 2min at 72~ A 40-ng sam- ple of genornic DNA was used with 10pmol each primer in 25-1al re- actions. The 1 x reaction buffer contain 1.5mM MgCI2, 50mM Tris- Cl, pH8.3, 0.02% NP 40, 0.02% Tween. Each dNTP was 200gM fi- nal concentration. The CA strand primer was end labeled with 32p- 7- ATP and I pmol (0.5 ~aCi) was added to each reaction. Sizes of alleles were determined with reference to the cloned allele using an M13 se- quencing reaction as a secondary standard. Reactions were run out on 5% Long Ranger modified polyacrylamide denaturing gels (AT Bio

0 10 20 30 40 50 60 70 80

I I I I I I I I I

GT GT E EE E E E E , , , , , , ,

1 7 8 F 1 0

1 1 4 C l l

LPL Gene 17137

1 6 5 B 9

179G2

kb

313

Fig. 1. Map of the LPL region. Distances are shown in kb. The positions of EcoRI restriction sites and the characterized GT repeats are indi- cated. The extent of the LPL gene is indicated by a thick line and the extent of isolated cosmids by thin lines. Isolate numbers for cosmids are shown

Table 1. Polymerase chain reaction (PCR) primer sequences for sim- ple sequence repeats (SSRs) flanking the lipoprotein lipase (LPL) lo- cus

SSR site Primer a Primer sequence

LPL5GT LPL5-CA LPL5-GT

LPL3GT LPL3-CA LPL-GT

5"-TAGAGCACACTATCCAGGTGA-3" 5"-CAGTGGGTTATTTGTGGGATA-3"

5"-ATTCTGGTATGAATGTACATGTG-3" 5"-ATCTGCCTCTGCAGCTCTCA-3"

a The primers are named -CA or -GT according to the dominant com- position of the strand produced by that end-labeled primer

I

1 3 8 - - 1 3 6 - - 1 3 4 - - 132 - -

chemicals). Primers were synthesized using an Applied Biosystems 380B oligonucleotide synthesizer.

Linkage disequilibrium was assessed by calculating the ;(2 and as- sociated P values for haplotype contingency tables using CONTING, one of the linkage utility programmes of Ott (1991). The magnitude of significant disequilibrium was calculated using the standardized dise- quilibrium value D' of Lewontin (1964).

R e s u l t s

Detection, sequencing and amplification of GT microsatellites

Over 80kb of genomic D N A has been isola ted at and sur- rounding the LPL locus by screening the L A 0 8 N C 0 1 f l o w - s o r t e d c h r o m o s o m e - 8 c o s m i d l ib ra ry wi th the LPL35 c D N A clone fo l lowed by a cosmid walk. Three of the f ive i sola ted cosmids cover this region with min ima l over lap. Three GT microsate l l i tes were ident i f ied in these cosmids . Two of these SSRs, f lanking the LPL gene, were se lected for sequencing and ana lyzed for al lel ic variat ion. A single EcoRl f ragment different in size for each of the cosmids 17B7, 165B9, and 179G2 (Fig. 1) was posi t ive with the p o l y - G T probe. The 4 ,0-kb EcoRI f ragment f rom 179G2 was subcloned, and a 0 .8-kb PstI f ragment con- taining the SSR was sequenced (GenBank access ion no. M94222, 830bp) . This SSR, referred to as the L P L 5 G T site, contains a (GT)I 6 repeat. The largest EcoRI f ragment present in both 178F10 and 114C11 was also posi t ive with the p o l y - G T probe but the SSR was not isolated. The ter- minal 4 .0-kb EcoRI f ragment o f 114C11, conta in ing the vector EcoRI site, was isola ted and subcloned. The SSR was sequenced f rom a 0.5-kb SacI/PstI f ragment (Gen-

118 116

Fig.2. Genotyping of CEPH family 21 for LPL5GT and LPL3GT al- leles. DNA from the parents amplifies to give fragments of 118, 116 (father) and 118, 116 (mother) at the LPL5GT locus and fragments of 138, 132 (father) and 136, 134 (mother) at the LPL3GT locus (not shown). The genotypes of the six children are: 118, 116; 134, 132 (2103); 118, 116; 138, 136 (2104); 118, 118; 136, 132 (2105); I16, 116; 138, 134 (2106); 118, 118; 136, 132 (2107); and 118, 116; 134, 132 (2108). This segregation infers that the paternal haplotypes are 1 1 8 - 1 3 2 (6-6) and 116-138 (7-3) and the maternal haplotypes are 118-136 (6-4) and 116-134 (7-5). The additional secondary bands form a l-bp ladder for the LPL5GT system and a 2-bp ladder for the LPL3GT system

Bank access ion no. M94221, 206bp) . This SSR, referred to as the L P L 3 G T site, contains a (CA)22 repeat.

F lanking pr imers were selected, which p roduced a 116- bp product with the c loned L P L 5 G T site and a 128-bp product with the c loned L P L 3 G T site (Table 1).

Allelic variation at the LPL5GT and LPL3GT sites

The CEPH panel (Dausset et al. 1990) was typed for both these f lanking sites (Fig. 2). At the L P L 5 G T site 231 inde- pendent al le les vary ing f rom 106 to 134bp were obse rved whi le 255 independent al leles varying f rom 118 to 142bp were observed at the L P L 3 G T site (Table 2). He te rozy- gosi t ies were ca lcula ted at 48% and 83% and po lymorph ic

314

Table 2. Allele frequency and size of PCR product

Allele Frequency PCR product Repeats ~' number size (bp)

LPL5GT I 0.004 134 25 2 0.009 132 24 3 0.013 126 21 4 0.169 124 20 5 0.004 120 18 6 0.104 118 17 7 0.693 116 16 8 0.004 106 11

LPL3GT I 0.020 142 29 2 0.062 140 28 3 0.110 138 27 4 0.212 136 26 5 0.263 134 25 6 0.180 132 24 7 0.055 130 23 8 0.043 128 22 9 0.039 126 21

10 0.008 124 20 II 0.008 118 17

~' The number of repeats if the allelic differences are due solely to va- riation in number of the dinucleotide repeat. Reference genotypes for 133101 were LPL5GT 3;7 and LPL3GT 3;4 and for 133102 were LPL5GT 6;7 and LPL3GT 5;6

Table 3. Distribution of 222 LPL chromosome haplotypes among the Centre d'Edute du Polymorphisme Humain (CEPH) panel

LPL3GT LPL5GT Total

Table 4. Distribution of 46 French LPL chromosome haplotypes among CEPH panel

LPL3GT LPL5GT

I 2 3 4 5 6 7 8

Total

1 0 0 0 0 0 0 1 0 I

2 0 0 0 1 0 0 2 0 3 3 0 0 1 3 0 0 4 0 8 4 0 0 0 2 0 I 7 0 10 5 0 0 0 I 0 0 8 0 9 6 0 0 0 0 1 3 3 0 7 7 0 0 0 1 0 0 2 0 3 8 0 0 0 0 0 0 3 0 3 9 0 2 0 0 0 0 0 0 2

10 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0

Total 0 2 1 8 1 4 30 0 46

Table 5. SSR haplotype of 33 French Canadian patients with LPL de- ficiency

LPL mutant n LPL5GT LPL3GT Haplotype genotype alleles alleles

188/188 4 116;116 138;138 7 3;7-3 207/207 20 I 16; 116 130; 130 7-7;7-7 188/207 1 116;116 138;130 7-3;7-7 250/207 1 116; 116 134; 130 7-5;7-7 ~/207 1 116; I 16 136; 130 7 4;7-7 ~/250 1 116; 116 140; 134 7-2:7-5

Denotes an uncharacterized mutant allele

l 2 3 4 5 6 7 8

I 0 0 0 4 0 0 1 0 5

2 0 0 0 6 0 0 7 0 13 3 0 0 3 6 0 2 15 0 26 4 0 0 0 11 0 4 34 0 49 5 0 0 0 5 0 12 39 0 56 6 0 0 0 2 I 6 29 I 39 7 0 0 0 2 0 0 11 0 13 8 0 0 0 0 0 0 10 0 10 9 1 2 0 0 0 0 5 0 8

10 0 0 0 0 0 0 1 0 1 I 1 0 0 0 1 0 0 1 0 2

Total 1 2 3 37 I 24 153 1 222

i n f o r m a t i o n c o n t e n t ( P I C ) v a l u e s at 0 .44 a n d 0.81 for the L P L 5 G T a n d L P L 3 G T sites, r e spec t i ve ly .

Linkage disequilibrium between SSR alleles

A tota l o f 28 d i f f e r e n t h a p l o t y p e s w e r e f o u n d a m o n g the 222 i n d e p e n d e n t h a p l o t y p e s tha t w e r e o b s e r v e d in the C E P H p a n e l (Tab le 3), w h i l e a m o n g the F r e n c h s u b s e t o f

the p a n e l 18 d i f f e r e n t h a p l o t y p e s w e r e s e e n a m o n g 46 in-

d e p e n d e n t h a p l o t y p e s (Tab le 4). T h e c a l c u l a t e d h e t e r o z y - gos i ty for the h a p l o t y p e was 9 0 . 8 % w i t h a P IC v a l u e o f 0 .90. T h e f ive ra re a l l e les at the L P L 5 G T si te w e r e f o u n d in h a p l o t y p e s tha t w e r e o b s e r v e d on a to ta l of o n l y e igh t

c h r o m o s o m e s . A m o n g h a p l o t y p e s o b s e r v e d at l eas t f ive t i m e s w e r e t h r e e o f the e i g h t L P L 5 G T a l le les a n d e i g h t o f the e l e v e n L P L 3 G T al le les . A tes t fo r a l le l ic a s s o c i a t i o n , c a r r i e d ou t o n th i s h a p l o t y p e t ab l e (Tab le 3) a f t e r it h ad

b e e n c o l l a p s e d to a 3 x 3 t ab l e so tha t no en t ry c o n t a i n e d f e w e r t h a n f ive o b s e r v a t i o n s , g a v e a X 2 o f 22 .97 ( P <

0 .0005 ) . To e v a l u a t e the bas i s o f th i s d e t e c t e d d i s e q u i l i b - r i u m all 14 h a p l o t y p e s tha t w e r e f o u n d at leas t f ive t i m e s w e r e t e s t ed in 2 x 2 c o n t i n g e n c y t ab l e s u s i n g Yates co r r ec - t ion . H a p l o t y p e 4 -2 ( L P L 5 G T - L P L 3 G T ) w i t h a X 2 v a l u e o f 6 .54 ( P = 0 .01) a n d h a p l o t y p e 6-5 w i t h a X 2 v a l u e o f 7 .35 ( P < 0 . 0 1 ) w e r e the o n l y h a p l o t y p e s t ha t g a v e s i g n i f i c a n t a l le l ic a s soc ia t ion . B o t h these h a p l o t y p e s were f o u n d m o r e o f t e n t han e x p e c t e d . H a p l o t y p e 6-5 was f o u n d a m o n g the 161 c h a r a c t e r i z e d U t a h h a p l o t y p e s o n l y and g a v e a s tan- d a r d i z e d d i s e q u i l i b r i u m v a l u e D ' ( L e w o n t i n 1964) o f 0 .45 in th i s M o r m o n p o p u l a t i o n ; h a p l o t y p e 4 - 2 w a s f o u n d a m o n g U t a h , F r e n c h , a n d V e n e z u e l a n h a p l o t y p e s and g a v e a D ' o f 0.35.

315

Linkage disequilibrium of SSR haplotype with French-Canadian LPL mutations

We characterized 33 French-Canadian patients with LPL deficiency for their SSR haplotype at the LPL5GT and LPL3GT loci (Table 5). The majority of patients were ho- mozygous for the ProZ~ mutation, which accounts for over 70% of the mutant alleles in a group of 37 LPL deficient patients (Ma et al. 1991). All 20 of these 207 mu- tation homozygotes were also homozygous for the 7-7 haplotype. The four Gly188---)Glu mutation homozytotes (Monsalve et al. 1990) were all homozygous for the 7-3 haplotype, whereas patients with the compound 188/207 genotype were heterozygous for the 7-3 and 7-7 haplo- types. This indicates that both the 188 and 207 mutations are in complete linkage disequilibrium with the 7-3 and 7- 7 haplotypes, respectively, in this population The remain- ing three patients, who are genetic compounds, were all heterozygous. This data is compatible with the Asp25~ Asn mutation (Ma et al. 1992) being associated with the 7- 5 haplotype and suggests that the two uncharacterized mu- tations are likely to be different, since they are associated with different haplotypes.

Discussion

Several RFLPs have been described at the LPL locus. These are mostly two-allele systems with PIC values of less than 0.37. Furthermore, the more informative mark- ers, such as the HindIII (Heinzman et al. 1987) and PvuH (Li et al. 1988) RFLPs that have PIC values around 0.36, give rise to a haplotype that has reduced informativeness due to linkage disequilibrium (Heinzmann et al. 1991). A more informative tetranucleotide repeat in intron 6 with a PIC of 0.47 has been reported (Zuliani and Hobbs 1990). Even more informative are the two polymorphic GT dinu- cleotide repeats, reported here, that flank the LPL locus. The PIC values for the LPL5GT and LPL3GT sites are 0.44 and 0.81, respectively. The haplotype PIC value of 0.90 shows no reduction in informativeness, such as that seen for the HindIII and PvuII RFLPs due to linkage dise- quilibrium. Nevertheless analysis of allelic association showed that two haplotypes (4-2 and 6-5) were observed more often than expected. This linkage disequilibrium did not, however, interfere with the informativeness of the haplotype, which was at the expected value. The basis for the disequilibrium, detected for only 2 of the 14 test- ed haplotypes, is unknown but might reflect selection, population admixture, or recent mutation. Disequilibrium may exist for the other 12 haplotypes but not be detectable in this study because of sample size (Thomson et al. 1988).

The LPL3GT site (17-29 repeats) is more informative than the LPL5GT site (11-25 repeats), consistent with the correlation of repeat number with informativeness (Weber 1990). Nine alleles at the LPL3GT site and three alleles at the LPL5GT site occur with a frequency of 2% or greater. The distribution of LPL3GT alleles around the most fre- quent allele (25 repeats) is more Gaussian than the distrib- ution of LPL5GT alleles around the most common allele

(16 repeats). Mutational gain and loss of repeats (Kwiat- kowski et al. 1992) will lead to an equilibrium where the allele distribution is Gaussian. Thus it appears that the LPL3GT site is older than the LPL5GT site, since the for- mer appears closer to equilibrium.

The two common French-Canadian LPL missense mu- tations appear to be in complete disequilibrium with spe- cific LPL haplotypes (Table 5). The GlylSS---~Glu mutation is found on the 7-3 haplotype while the Pro2~ muta- tion is found on the 7-7 haplotype. The 7-3 haplotype has a frequency of 6.8% and the 7-7 haplotype, a frequency of 5% in the CEPH panel. These frequencies are close to those observed in the French subset of the CEPH panel. Complete disequilibrium for these two common LPL mu- tations is further evidence of a founder effect in this popu- lation. Since three sets of seventeenth century founders have been described for three different regions of Quebec (De Braekeleer et al. 1991), it appears that two of these sets introduced the same ProZ~ mutation and haplo- type to both Beauce and the Charlevoix/Saguenay-Lac St- Jean regions (Hayden et al. 1992). The Gly~SS-->Glu mis- sense mutation found in the Trois-Riviers/Montreal re- gion (Hayden et al. 1992) has been identified among dif- ferent populations. Genealogical reconstruction of French- Canadian heterozygote carriers led to the recognition of four founders for the GlylSS-->Glu missense mutation, all emigrants from the western part of France to Quebec in the seventeenth century. It will be of interest to determine haplotype associations for the Gly~S8-->Glu mutation in other populations.

Acknowledgements. This work was supported by grants from the Medical Research Council of Canada MT-10613 (S. W.) and MT- 11253 (M. R. H.) and the BC Heart Foundation (M. R. H.). Y. Ma is a postdoctoral fellow of the Medical Research Council of Canada and M. R. Hayden is an established investigator of the British Columbia Children's Hospital. We thank our colleagues in Quebec, Drs. C. Gagne, P. Lupien, J. Davignon, and G. Roederer, for access to DNA from their patients.

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