Hum Genet (1990) 85:305-310
9 Springer-Verlag 1990
Linkage relationships of the apolipoprotein C1 gene and a cytochrome P450 gene (CYP2A) to myotonic dystrophy Kathryn V. Walsh, Helen G. Harley, J. David Brook, Shelley A. Rundle, Mansoor Sarfarazi, Peter S. Harper, and Duncan J. Shaw Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK Received September 30, 1989 / Revised January 9, 1990
Summary. We have studied the genetic linkage of two markers, the apolipoprotein C1 ( A P O C 1 ) gene and a cytochrome P450 (CYP2A) gene, in relation to the gene for myotonic dystrophy (DM). A peak lod score of 9.29 at 2 cM was observed for A P O C 1 - D M , with a lod score of 8.55 at 4 cM for C Y P 2 A - D M . These two markers also show close linkage to each other (0max= 0.05, Zmax = 9.09). F r o m examination of the genotypes of the recombinant individuals, C Y P 2 A appears to map proximal to D M because in one recombinant individual C Y P 2 A , A P O C 2 and C K M M had all recombined with DM. Evidence from another C Y P 2 A - D M recombinant individual places C Y P 2 A proximal to A P O C 2 and CKMM. Localisation of C Y P 2 A on a panel of somatic cell hybrids also suggests that it is proximal to DM and A P O C 2 / C U E gene cluster.
1986) and a phenobarbital-inducible cytochrome P450 gene, C Y P 2 A (formerly C Y P I : Phillips et al. 1985a; Davis et al. 1986; Hulsebos et al. 1986). We have also confirmed the localisation of these genes to 19q by use of a panel of somatic cell hybrids.
Materials and methods cDNA probes The cDNA probes for APOC1 (Knott et al. 1984) and CYP2A (pHP45011]; Phillips et al. 1985b) detect the following restriction fragment length polymorphisms (RFLPs): APOC1, DraI (Frossard et al. 1987a), BglI (Frossard et al. 1987b); CYP2A, SstI (Wainwright et al. 1985); see Fig. 1.
Family material and linkage analysis
Introduction Myotonic dystrophy (DM) is a relatively c o m m o n autosomal dominant disorder characterised by myotonia with progressive muscular weakness and wasting, plus a variety of other symptoms (Harper 1989). The underlying biochemical defect is still unknown. However, the D M gene has been localised to the long arm of chromosome 19, region 19q13.2-q13.3, by virtue of its linkage to several chromosome 19 markers (Shaw et al. 1985, 1986; Friedrich et al. 1987; B r o o k et al. 1987a; Stallings et al. 1988). The search for closely linked D N A markers is the first stage in the isolation of a disease gene by the "reverse genetics" approach; these markers are also of practical benefit in presymptomatic diagnosis. We have investigated the genetic linkage to D M of two genes previously assigned to the proximal long arm of chromosome 19: the apolipoprotein C1 (APOC1) gene (Lusis et al.
Offprint requests to: D. J. Shaw
The 44 families with myotonic dystrophy, used in this study, have formed the basis of previous studies (Shaw et al. 1985, 1986; Harley et al. 1988). All families have been clinically assessed, including EMG and opthalmological examination when necessary. Homogeneity tests have been performed on this set of families using the HOMOG programme (Ott 1983) and no detectable heterogeneity was found using the closely linked marker APOC2 [H. Harley, unpublished data; the lod score for APOC2-DM is > 90 at 0 = 0.02-0.03 (Le Beau et al. 1989)]. Only unequivocally affected DM individuals, plus unrelated normal spouses, were scored when establishing linkage between the DM locus and the markers, APOC1 and CYP2A. Therefore, complete penetrance was assumed when calculating the lod scores for APOC1-DM and CYP2A-DM. No individuals at risk of developing myotonic dystrophy were included in these calculations, with the exception of 11 individuals whose affected siblings had the congenital form of myotonic dystrophy. These 11 individuals had all reached puberty without any symptoms of myotonic dystrophy and are therefore considered not to be gene carriers by the criteria of Bundey (1974), O'Brien et al. (1983a) and M.C. Koch, T. Grimm, H. G. Harley, P. S. Harper (in preparation). Isolation of DNA, Southern transfer and DNA hybridisation were performed according to standard protocols (Maniatis et al. 1982). When using the APOC1 cDNA probe, sheared cold total human DNA (10 gg/ml) was added to the prehybridisation buffer, to reduce the background hybridisation with this probe. The probes
306
Table 1. Segregation of genetic markers in four somatic cell hybrids containing chromosome 19 fragments Marker
D19S7 GPI D19S9 MSK37 TGFI3 CYP2A D19S8 ATPIA3 D19S19 APOC1 APOC2 CKMM
Hybrid line TVBID
J640
WILFM2
G175C4
+ + + + + + + + + + +
+ + + + + + -
+ + + + + + + + +
+ + + -
Results
Regional localisation of C Y P 2 A and A P O C 1
Fig.1. a DraI polymorphism detected by the APOC1 probe. Allele 1 7.6kb, 2.6kb, frequency 0.75; allele 2 10.2kb, frequency 0.25. b BglI polymorphism detected by the APOC1 probe. Allele I 8.7 kb, frequency 0.06; allele 2 6.2kb, frequency 0.94. e SstI polymorphism detected by the CYP2A probe. Allele 1 9.3 kb, frequency 0.55; allele 2 5,2kb, 4.1 kb, frequency 0.45
A P O C 1 was present in the cell lines T V B I D and W I L F M 2 b u t was in n e i t h e r G 1 7 5 C 4 n o r J640. C Y P 2 A d e t e c t e d h u m a n E c o R I f r a g m e n t s in T V B I D a n d W I L F M 2 , b u t n o t in G 1 7 5 C 4 , as p r e v i o u s l y r e p o r t e d by B r o o k et al. (1987a). C Y P 2 A was also p r e s e n t in J640. M a r k e r s m a p ping to the long a r m of c h r o m o s o m e 19 can be subdivided o n the basis of their segregation p a t t e r n s in these cell lines. T a b l e 1 s u m m a r i s e s the localisation data of A P O C 1 and C Y P 2 A , together with o t h e r long a r m markers. As can be seen from this T a b l e , C Y P 2 A has the same segregation p a t t e r n as M S K 3 7 a n d TGFI3, w h e r e a s A P O C 1 shows the same segregation as A P O C 2 , C K M M a n d D19S19 ( L D R 1 5 2 ) .
Linkage analysis were labelled using a commercial multi-prime kit (Amersham International). Blots were washed at 65~ to a final stringency of 1 • SSC for APOC1 and 0.1 x SSC for CYP2A. The data were analysed for linkage using the MLINK programme (Lathrop et al. 1984). As mentioned above, complete penetrance was assumed when calculating the lod score data between the DM locus and the markers used in this study. The linkage data were also calculated on the basis of equal rates of male and female recombination, and equal rates of mutation in males and females of zero (i.e. there are no new mutations).
Somatic cell hybrids APOC1 and CYP2A were regionally localised on chromosome 19 using a panel of rodent-human somatic cell hybrids that have been fully described elsewhere: TVBID (Brook et al. 1987a), J640 (J. D. Brook, S. Knight, S. H. Roberts, H. G. Harley, K.V. Walsh, S. A. Rundle, K. Freyne, M. C. Koch, N. D. Epstein, B. Wieringa, D. Schonk, B. Smeets, K. Haddingham, M.J. Siciliano, D. K. Palmer, J. S. Miles, C. R. Wolf, C. Fonatsch, D. J. Shaw, submitted for publication), G175C4 (Brook et al. 1987b) and WILFM2 (Brook et al. 1986, 1987b). TVBID contains region 19q12-qter. J640, G175C4 and WILFM2 are cell lines containing fragments of chromosome 19.
A P O C 1 was i n f o r m a t i v e for linkage analysis with the DraI R F L P in 10 families, a n d with the BglI R F L P in 5 families; 2 of these families were i n f o r m a t i v e with b o t h polymorphisms. O n l y o n e r e c o m b i n a n t (phase u n k n o w n , male meiosis) out of 44 meioses was o b s e r v e d b e t w e e n A P O C 1 a n d D M , giving a m a x i m u m lod score of 9.29 at 0 m a x = 0.02 (see T a b l e 2). I n the D M - A P O C 1 r e c o m b i n a n t i n d i v i d u a l (Fig. 2, pedigree A , II 9), D M has also r e c o m b i n e d with D19S8 a n d C Y P 2 B (a gene that m a p s to within 350 kb of C Y P 2 A ; Miles et al. 1989). C Y P 2 A - D M gives a m a x i m u m lod score of 8.55 at 0max = 0.04 (see T a b l e 2), based o n data from 19 informative families. T h r e e r e c o m b i n a n t s (all phase u n k n o w n , female meioses) out of 64 meioses were observed. T w o of these r e c o m b i n a n t s were also D 19S19-DM crossovers a n d were in the same family, placing C Y P 2 A o n the same side of the D M locus as D19S19. F i g u r e 3 shows the pedigree c o n t a i n i n g the o t h e r C Y P 2 A - D M rec o m b i n a n t (individual II 1, p e d i g r e e B). This i n d i v i d u a l is an u n a f f e c t e d sib in a c o n g e n i t a l m y o t o n i c d y s t r o p h y
307 Table 2. Genetic linkage analysis of APOC1 and CYP2A to myotonic dystrophy (DM)
Loci
0 (Of = 0m)
APOC1-DM CYP2A-DM APOC1-CYP2A
0.01
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
9.25 7.85 8.90
8.92 8.55 9.09
7.92 7.96 8.56
6.78 6.94 7.63
5.59 5.77 6.53
4.40 4.54 5.34
3.23 3.32 4.09
2.13 2.19 2.83
1.15 1.21 1.59
0.38 0.46 0.55
max
Z~ax
0.02 0.04 0.05
9.29 8.55 9.09
Table 3. Data for the APOC1 locus. DM, DM chromosome; N, A
(21)
11
B
(12)
22
normal chromosome; Z2 values in a, b and c are not significant a Number of observed DM/APOC1 (DraI) haplotypes (~2 = 0.423)
C
ii
il 21
21
21
21
21
22
21
21
11
12
12
12
22
12
22
12
22
22
APOC1 (DraI) allele 1
2
28 105
6 31
13
III
lii Fig. 2. Pedigree A. Individual 11 9 is a (APOC1, D19S8, CYP2B) X DM crossover. A APOC1 (DraI), B CYP2B (MspI), C D19S8
I
2
A
11)
12
B
BC
AB
C
I!!0
Fig. 3. Pedigree B. Individual H 1 is a (CYP2A, APOC2, CKMM) X DM crossover; individual H 3 is a CYP2A X (APOC2, CKMM, DM) crossover. A CYP2A, B APOC2 (haplotype), C CKMM (haplotype)
sibship, so does not c o n f o r m to the strict criteria set out by Griggs et al. (1989) for scoring r e c o m b i n a n t s in D M families. H o w e v e r , according to B u n d e y (1974), O ' B r i e n et al. (1983a) and M. C. K o c h , T. G r i m m , H. G. H a r l e y , P. S. H a r p e r (in p r e p a r a t i o n ) , an unaffected individual in a congenital sibship is unlikely to be a D M carrier if they reach p u b e r t y w i t h o u t showing any symptoms of m y o t o n i c dystrophy. Individual II 1, pedigree B, is 40 years old, and has therefore b e e n a s s u m e d to be n o r m a l in o u r linkage analysis. In this individual, D M has r e c o m b i n e d with C Y P 2 A , A P O C 2 and C K M M . W h e n the typings for A P O C 2 and C K M M are considered t o g e t h e r with C Y P 2 A in pedigree B, r e c o m b i n a tion b e t w e e n C Y P 2 A and these m a r k e r s has to be inv o k e d . B a s e d on the assumption that the p a t e r n a l genotype giving the least n u m b e r of r e c o m b i n a n t s is the correct o n e (i.e. the o n e given in Fig. 3), then individual II 3 is the m o s t likely C Y P 2 A - ( A P O C 2 , C K M M ) r e c o m binant. T h e data given in Fig. 3 also suggest that this in-
DM Normal
b Number of observed DM/APOC1 (BglI) haplotypes (Z2 = 1.299) APOC1 (BglI) allele
DM Normal
1
2
1 7
33 71
c Number of observed and expected DM/APOC1 (DraI + BglI) haplotypes (?(2 = 1.769) Haplotype DM/DraI/BglI DM11 Nll DM21 N21 DM12 N12 DM22 N22
Observed 1 3 0 0 25 43 5 13
Expected 1.10 2.10 0.28 0.52 23.70 45.10 5.92 11.28
dividual is a D M - C Y P 2 A r e c o m b i n a n t , giving C Y P 2 A (APOC2, CKMM, DM). A P O C 1 and C Y P 2 A are also linked to each other, a m a x i m u m lod score of 9.09 at 0max = 0.05 being o b t a i n e d (see Table 2). T e n pedigrees were informative for b o t h loci and three r e c o m b i n a n t s (one female and two male meioses) were o b s e r v e d in 49 meioses. All were phase u n k n o w n and in individuals w h e r e it was not possible to relate the r e c o m b i n a n t event to the affected D M gene. In the families informative for linkage analysis, the rarer allele of the m a r k e r s a p p e a r e d to be inherited with the affected allele of the D M gene m o r e often than exp e c t e d given the allele frequencies; this suggests that there m a y be linkage disequilibrium b e t w e e n these loci and the D M gene. Chi-squared tests were p e r f o r m e d o n
308 Table 4. Number of observed APOC1 DraI/BglI haplotypes. Z2 = 1.047; this ~2 value is not significant
DraI allele
BglI allele 1
2
l
4
2
0
68 18
Table 5. Number of observed DM/CYP2A haplotypes. Z2 = 1.803: this X2 value is not significant CYP2A allele DM Normal
1
2
14 90
19 73
all 44 DM families (including those uninformative for linkage analysis), using only one DM chromosome per pedigree (where phase could be ascertained) plus all unrelated normal chromosomes that had been genotyped. Table 3 gives the data for the APOC1 locus. Chi-squared analysis shows that there is no significant disequilibrium between either the DraI or the BglI polymorphisms, or the haplotype of the 2 RFLPs and the DM locus. There is also no disequilibrium between the two polymorphisms for APOC1 (Table 4). The bias towards inheritance of DM with the rarer allele in those families that were informative for linkage analysis appears to have arisen because 3 of the 13 families are of French-Canadian origin and are interrelated. To take this into account, only one D M chromosome from the French-Canadian pedigrees was included in the chi-squared analysis. Therefore, the families informative for linkage did not accurately reflect the pattern in the population as a whole. Similarly, statistical analysis of the C Y P 2 A data (see Table 5) suggests that there is no significant disequilibrium between the C Y P 2 A and DM loci. Again, the bias observed in the informative families appears to be the result of the large number of FrenchCanadian families in this group (7/19). Disequilibrium has been demonstrated in a French-Canadian population between DM and A P O E (Laberge et al. 1985), A P O C 2 and D M (MacKenzie et al. 1989), and also between DM and BCL3 (Korneluk et al. 1989), presumably resulting from a "founder effect" (Laberge 1989).
Discussion
We have shown that the loci APOC1 and C Y P 2 A are linked to DM. APOC1 may be a suitable marker for use in prenatal diagnosis especially when A P O C 2 and CKMM, the closest markers to DM (Shaw et al. 1985, 1986; Shaw and Eiberg 1988; Brunner et al. 1989; H. G. Harley, K.V. Walsh, J. D. Brook, S.A. Rundle, M. Sarfarazi, M. C. Koch, J. L. Floyd, P. S. Harper, D.J. Shaw, submitted for publication) are uninformative. CYP2A is
less suitable for this purpose. Although our data put it only 4 cM from DM, data from multipoint analysis in DM and C E P H pedigrees place CYP2A further from DM than several other markers (the marker order from multipoint analysis is CYP2A-ATPIA3-D19S8-D19S19BCL3-APOC2-CKMM-DM; H. G. Harley, K.V. Walsh, J. D. Brook, S. A. Rundle, M. Sarfarazi, M. C. Koch, J. L. Floyd, P. S. Harper, D.J. Shaw, submitted for publication; H . G . Harley, S.A. Rundle, J.D. Brook, M. Sarfarazi, K. J. Johnson, B. Wieringa, P. S. Harper, D. J. Shaw, in preparation). The genes for APOC1, APOC2 and A P O E are known to be clustered, evidence for this coming from linkage data (Humphries et al. 1984; Myklebost et al. 1984) and somatic cell hybridisation studies (Das et al. 1985; Scott et al. 1985; Tata et al. 1985; Lusis et al. 1986). Moreover, APOC1 and A P O E have been isolated on the same genomic clone from lambda libraries (Das et al. 1985; Davison et al. 1986; Myklebost and Rogne 1986; Lusis et al. 1986). This clustering has also been demonstrated by pulsed-field gel analysis, which suggests the gene order A P O E - A P O C 1 - A P O C 2 (Myklebost and Rogne 1988) and indicates that the cluster spans about 50 kb, an estimate supported by Smit et al. (1988). Our data showing APOC1 to be closely linked to DM, as is APOC2, is consistent with their being clustered. APOC1 and APOC2 also show close genetic linkage, our data giving a maximum lod score of 10.27 at 2 cM (H. G. Harley, K.V. Walsh, J. D. Brook, S. A. Rundle, M. Sarfarazi, M. C. Koch, J. L. Floyd, P. S. Harper, D. J. Shaw, submitted for publication). Despite their close physical linkage, we have observed an APOC1-APOC2 recombinant. This recombinant individual is at present not affected with DM, although at risk, so we are unable to orientate the apolipoprotein gene cluster with respect to the DM gene. However, Brunner et al. (1989) have recently reported an A P O E - A P O C 2 recombinant allowing this gene cluster to be orientated, placing APOC2 closest to DM, and hence giving the order: A P O E - A P O C 1 APOC2-DM. CYP2A has been shown to be linked to PEPD (Davis et al. 1987), a gene also known to be linked to DM (O'Brien et al. 1983b) and A P O C 2 (Ball et al. 1985). Pep4, the mouse homologue of PEPD, is linked to the Coh locus on mouse chromosome 7. A phenobarbital-inducible cytochrome P450 gene family maps to the Coh locus (Simmons and Kasper 1983; Simmons et al. 1985), and thus, the CYP2A and Coh loci are thought to be equivalent (Phillips et al. 1985a; Davis et al. 1987; Miles et al. 1989). Other chromosome 19 markers that have homologues mapping within the Pep4-Coh linkage group on mouse chromosome 7 include GPI, TGFI3, LHI3, ATP1A3 and A P O E . Since the conserved linkage group includes markers covering most of 19q, it is likely that there is also a homologue of the DM gene on mouse chromosome 7. The presence of C Y P 2 A and APOC1 in T V B I D confirm the localisation of these markers to the long arm of chromosome 19. The fragmented cell lines WILFM2, J640 and G175C4 allow long arm markers to be subdivided. APOC1 shows the same pattern as APOC2,
309 C K M M a n d D19S19 ( B r o o k et al. 1987a). H o w e v e r , C Y P 2 A s e g r e g a t e s with TGF[3 a n d M S K 3 7 ( B r o o k et al. 1987a; J. D . B r o o k , S. K n i g h t , S. H . R o b e r t s , H . G . H a r ley, K . V . W a l s h , S . A . R u n d l e , K . F r e y n e , M . C . K o c h , N. D. E p s t e i n , B. W i e r i n g a , D. S c h o n k , B. S m e e t s , K. H a d d i n g h a m , M . J . Siciliano, D . K. P a l m e r , J. S. Miles, C. R. W o l f , C. F o n a t s c h , D . J. S h a w , s u b m i t t e d for p u b l i c a t i o n ) . T h e p r e s e n c e o f C Y P 2 A in J640 suggests t h a t it is p r o x i m a l to t h e a p o l i p o p r o t e i n g e n e c l u s t e r a n d to D M , as this cell line also c o n t a i n s t h e p r o x i m a l m a r k e r D19S7. This is in a g r e e m e n t with S c h o n k et al. (1989) who also localised C Y P 2 A p r o x i m a l to the a p o l i p o p r o t e i n genes using s o m a t i c cell h y b r i d s . T h e ( C Y P 2 A , A P O C 2 , C K M M ) - D M r e c o m b i n a n t (Fig. 3, i n d i v i d u a l II 1) also indicates that C Y P 2 A is p r o x i m a l to D M , as A P O C 2 a n d C K M M a r e n o w k n o w n to m a p p r o x i m a l to D M ( S h a w et al. 1989; H . G . H a r l e y , K . V . W a l s h , J. D . B r o o k , S . A . R u n d l e , M. S a r f a r a z i , M. C. K o c h , J. L. F l o y d , P. S. H a r p e r , D . J. S h a w , s u b m i t t e d for p u b l i c a t i o n ) ; t h e C Y P 2 A ( A P O C 2 , C K M M , D M ) r e c o m b i n a n t (Fig. 3, i n d i v i d u a l I I 3) p l a c e s C Y P 2 A as t h e m o s t p r o x i m a l o f t h e s e m a r k ers. This p r o x i m a l l o c a t i o n of C Y P 2 A is also s u p p o r t e d b y l i n k a g e d a t a f r o m t h e C E P H p e d i g r e e s (H. G . H a r l e y , S. A . R u n d l e , J. D . B r o o k , M. S a r f a r a z i , K. J. J o h n s o n , B . W i e r i n g a , P. S. H a r p e r , D . J . S h a w , in p r e p a r a t i o n ) . T h e r e f o r e , t h e m o s t l i n k e l y o r d e r is C Y P 2 A - ( A P O E ICIlC2)-DM.
Acknowledgements. We would like to thank D r . E . A . Shephard for the CYP2A probe, Dr.T.J. Knott for the APOC1 probe, Dr.M.-C.Thibault for providing the Canadian family material, and Dr. M. C. Koch for family sampling and clinical assessment. This work was supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland, the Muscular Dystrophy Association (USA), the Medical Research Council, and the Wellcome Trust.
References Ball SP, Donald JA, Corney G, Humphries SE (1985) Linkage between the loci for peptidase D and apolipoprotein CII on chromosome 19. Ann Hum Genet 49 : 129-134 Brook JD, Shaw D J, Thomas NST, Meredith AL, Cowell J, Harper PS (1986) Mapping genetic markers on human chromosome 19 using subchromosomal fragments in somatic cell hybrids. Cytogenet Cell Genet 41 : 30-37 Brook JD, Skinner M, Roberts SH, Rettig WJ, Almond JW, Shaw DJ (1987a) Further mapping of markers around the centromere of human chromosome 19. Genomics 1 : 320-328 Brook JD, Beresford HR, Shaw D J, Old LJ, Rettig WJ (1987b) Localisation on human chromosome 19 of three genes for cell surface antigens defined by monoclonal antibodies. Cytogenet Cell Genet 45 : 156-162 Brunner HG, Smeets H, Lambermon HMM, Coerwinkel-Driessen M, Oost BA van, Wieringa B, Ropers H-H (1989) A multipoint linkage map around the locus for myotonic dystrophy on chromosome 19. Genomics 5 : 589-595 Bundey S (1974) Detection of heterozygotes for myotonic dystrophy. Clin Genet 5 : 107-109 Das HK, McPherson J, Bruns GAP, Karathanasis SK, Breslow JL (1985) Isolation, characterization, and mapping to chromosome 19 of the human apolipoprotein E gene. J Biol Chem 260 : 6240-6247 Davis MB (1987) Linkage between the loci for peptidase D and cytochrome P450 (CYP1) on chromosome 19. Ann Hum Genet 51 : 9-12
Davis MB, West LF, Shephard EA, Phillips IR (1986) Regional localization of a human cytochrome P450 (CYP1) to chromosome 19q13.1-13.3. Ann Hum Genet 50:237-240 Davison PJ, Norton P, Wallis SC, Gill L, Cook M, Williamson R, Humphries SE (1986) There are two gene sequences for human apolipoprotein C1 (APOC1) on chromosome 19, one of which is 4 kb from the gene for APOE. Biochem Biophys Res Commun 136 : 876-884 Friedrich U, Brunner H, Smeets D, Lambermon E, Ropers H-H (1987) Three-point linkage analysis employing C3 and 19cen markers assigns the myotonic dystrophy gene to 19q. Hum Genet 75 : 291-293 Frossard PM, Coleman RT, Malloy MJ, Kane JP, Levy-Wilson B, Appleby VA (1987a) Human apolipoprotein C1 (APOC1) gene locus: DraI dimorphic site. Nucleic Acids Res 15 : 1884 Frossard PM, Lim DW, Coleman RT, Funke H, Assmann G, Malloy MJ, Kane JP (1987b) Human apolipoprotein C1 (APOC1) gene locus: BglI dimorphic site. Nucleic Acids Res 15 : 1344 Griggs RC, Wood DS, the Working Group on the Molecular Defect in Myotonic Dystrophy (1989) Criteria for establishing the validity of genetic recombination in myotonic dystrophy. Neurology 39 : 420-421 Harley HG, Brook JD, Jackson CL, Glaser T, Walsh KV, Sarfarazi M, Kent R, Lager M, Koch M, Harper PS, Levenson R, Housman DE, Shaw DJ (1988) Localization of a human Na +, K+-ATPase a subunit gene to chromosome 19q12-q13.2 and linkage to the myotonic dystrophy locus. Genomics 3:380384 Harper PS (1989) Myotonic dystrophy, 2nd edn. Saunders, Philadelphia Hulsebos T, Wieringa B, Hochstenbach R, Smeets D, Schepens J, Oerlemans F, Zimmer J, Ropers H-H (1986) Toward early diagnosis of myotonic dystrophy: construction and characterization of a somatic cell hybrid with a single human der(19) chromosome. Cytogenet Cell Genet 43 : 47-56 Humphries SE, Berg K, Gill L, Cumming AM, Robertson FW, Stalehoef AFH, Williamson R, Borresen AL (1984) The gene for apolipoprotein CII is closely linked to the gene for apolipoprotein E on chromosome 19. Clin Genet 26 : 389-396 Knott TJ, Robertson ME, Priestley LM, Urdea M, Wallis S, Scott J (1984) Characterisation of mRNAs encoding the precursor for human apolipoprotein C1. Nucleic Acids Res 12:39093915 Korneluk RG, MacLeod HL, McKeithan TW, Brook JD, MacKenzie AE (1989) A chromosome 19 clone from a translocation breakpoint shows close linkage and linkage disequilibrium with myotonic dystrophy. Genomics 4:146-151 Laberge C (1989) Myotonic dystrophy in Quebec: geographical distribution and concept of genetic homogeneity. Can J Neurol Sci 16 : 123-128 Laberge C, Gaudet D, Morisette J, Moorjani S, Thibault MC (1985) Linkage of myotonic dystrophy and APOE in a FrenchCanadian isolate (abstract). (8th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 40 : 675 Lathrop GM, Lalouel JM, Julier C, Ott J (1984) Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 81 : 3443-3446 Le Beau MM, Ryan D, Pericak-Vance MA (1989) Report of the committee on the genetic constitution of chromosomes 18 and 19. (10th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 51 : 338-357 Lusis AJ, Heinzmann C, Sparkes RS, Scott J, Knott TJ, Geller R, Sparkes MC, Mohandas T (1986) Regional mapping of human chromosome 19: organization of genes for plasma lipid transport (APOC1, - C 2 , and - E and LDLR) and the genes C3, PEPD, and GPI. Proc Natl Acad Sci USA 83 : 3929-3933 MacKenzie AE, MacLeod HL, Hunter AGW, Korneluk RG (1989) Linkage analysis of the apolipoprotein C2 gene and myotonic dystrophy on human chromosome 19 reveals linkage disequilibrium in a French-Canadian population. Am J Hum Genet 44 : 140-147
310 Maniatis T, Fritsch EF, Sambrook J (eds) (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Miles JS, Bickmore W, Brook JD, McLaren AW, Meehan R, Wolf CR (1989) Close linkage of the human cytochrome P450IIA and P450IIB gene subfamilies: implications for the assignment of substrate specificity. Nucleic Acids Res 17 : 29072917 Myklebost O, Rogne S (1986) The gene for human apolipoprotein CI is located 4.3 kilobases away from the apolipoprotein E gene on chromosome 19. Hum Genet 73 : 286-289 Myklebost O, Rogne S (1988) A physical map of the apolipopro 7 tein gene cluster on human chromsome 19. Hum Genet 78: 244-247 Myklebost O, Rogne S, Olaisen B, Gedde-Dahl T, Prydz H (1984) The locus for apolipoprotein CII is closely linked to the apolipoprotein E locus on chromosome 19 in man. Hum Genet 67 : 309-312 O'Brien T, Newcombe RG, Harper PS (1983a) Outlook for a clinically normal child in a sibship with congenital myotonic dystrophy. J Pediatr 103 : 762-763 O'Brien T, Ball S, Sarfarazi M, Harper PS, Robson EB (1983b) Genetic linkage between the loci for myotonic dystrophy and peptidase D. Ann Hum Genet 47 : 117-121 Ott J (1983) Linkage analysis and family classifications under heterogeneity. Ann Hum Genet 47:311-320 Phillips IR, Shephard EA, Povey S, Davis MB, Kelsey G, Monteiro M, West LF, Cowell J (1985a) A cytochrome P450 gene family mapped to human chromosome 19. Ann Hum Genet 49 : 267-274 Phillips IR, Shephard EA, Ashworth A, Rabin BR (1985b) Isolation and sequence of a human cytochrome P450 cDNA clone. Proc Natl Acad Sci USA 82 : 983-987 Schonk D, Coerwinkel-Driessen M, Dalen I van, Oerlemans F, Schepens J, Hulsebos T, Cockburn D, Boyd Y, Davis M, Rettig W, Shaw D, Roses A, Ropers H-H, Wieringa B (1989) Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q. Genomics 4:384-396 Scott J, Knott TJ, Shaw DJ, Brook JD (1985) Localization of genes encoding apolipoproteins CI, CII, and E to the p13-cen region of human chromosome 19. Hum Genet 71 : 144-146 Shaw D J, Eiberg H (1988) Report of the committee on the genetic constitution of chromosomes 17, 18 and 19. (9th International
Workshop on Human Gene Mapping) Cytogenet Cell Genet 46 : 242-256 Shaw D J, Meredith AL, Sarfarazi M, Huson SM, Brook JD, Myklebost O, Harper PS (1985) The apolipoprotein CII gene: subchromosal localisation and linkage to the myotonic dystrophy locus. Hum Genet 70:271-273 Shaw D J, Meredith AL, Sarfarazi M, Harley HG, Huson SM, Brook JD, Bufton L, Litt M, Mohandas T, Harper PS (1986) Regional localisations and linkage relationships of seven RFLPs and myotonic dystrophy on chromosome 19. Hum Genet 74: 262-266 Shaw D J, Harley HG, Brook JD, McKeithan TW (1989) Longrange restriction map of a region of human chromosome 19 containing the apolipoprotein genes, a CLL-associated translocation breakpoint and two polymorphic MluI sites. Hum Genet 83 : 71-74 Simmons DL, Kasper CB (1983) Genetic polymorphisms for a phenobarbital-inducible cytochrome P450 map to the Coh locus in mice. J Biol Chem 258 : 9585-9588 Simmons DL, Lalley PA, Kasper CB (1985) Chromosomal assignments of genes coding for components of the mixed function oxidase system in mice. Genetic localization of the cytochrome P450 PCN and P450PB gene families and the NADPH-cytochrome P450 oxidoreductase and epoxide hydratase genes. J Biol Chem 260 : 515-521 Smit M, Kooij-Meijs E van der, Frants RR, Havekes L, Klasen EC (1988) Apolipoprotein gene cluster on chromosome 19. Hum Genet 78: 90-93 Stallings RL, Olson E, Strauss AW, Thompson LH, Bachinski LL, Siciliano MJ (1988) Human creatine kinase genes on chromosomes 15 and 19, and proximity of the gene for the muscle form to the genes for apolipoprotein C2 and excision repair. Am J Hum Genet 43 : 144-151 Tata F, Henry I, Markham AF, Wallis SC, Weil D, Grzeschik KH, Junien C, Williamson R, Humphries SE (1985) Isolation and characterisation of a cDNA clone for human apolipoprotein CI and assignment of the gene to chromosome 19. Hum Genet 69 : 345-349 Wainwright B J, Watson EK, Shephard EA, Phillips IR (1985) RFLP for a human cytochrome P450 gene at 19q13.1-qter (HGM8 provisional designation CYP1). Nucleic Acids Res 13 : 4610
9 Springer-Verlag 1990
Linkage relationships of the apolipoprotein C1 gene and a cytochrome P450 gene (CYP2A) to myotonic dystrophy Kathryn V. Walsh, Helen G. Harley, J. David Brook, Shelley A. Rundle, Mansoor Sarfarazi, Peter S. Harper, and Duncan J. Shaw Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK Received September 30, 1989 / Revised January 9, 1990
Summary. We have studied the genetic linkage of two markers, the apolipoprotein C1 ( A P O C 1 ) gene and a cytochrome P450 (CYP2A) gene, in relation to the gene for myotonic dystrophy (DM). A peak lod score of 9.29 at 2 cM was observed for A P O C 1 - D M , with a lod score of 8.55 at 4 cM for C Y P 2 A - D M . These two markers also show close linkage to each other (0max= 0.05, Zmax = 9.09). F r o m examination of the genotypes of the recombinant individuals, C Y P 2 A appears to map proximal to D M because in one recombinant individual C Y P 2 A , A P O C 2 and C K M M had all recombined with DM. Evidence from another C Y P 2 A - D M recombinant individual places C Y P 2 A proximal to A P O C 2 and CKMM. Localisation of C Y P 2 A on a panel of somatic cell hybrids also suggests that it is proximal to DM and A P O C 2 / C U E gene cluster.
1986) and a phenobarbital-inducible cytochrome P450 gene, C Y P 2 A (formerly C Y P I : Phillips et al. 1985a; Davis et al. 1986; Hulsebos et al. 1986). We have also confirmed the localisation of these genes to 19q by use of a panel of somatic cell hybrids.
Materials and methods cDNA probes The cDNA probes for APOC1 (Knott et al. 1984) and CYP2A (pHP45011]; Phillips et al. 1985b) detect the following restriction fragment length polymorphisms (RFLPs): APOC1, DraI (Frossard et al. 1987a), BglI (Frossard et al. 1987b); CYP2A, SstI (Wainwright et al. 1985); see Fig. 1.
Family material and linkage analysis
Introduction Myotonic dystrophy (DM) is a relatively c o m m o n autosomal dominant disorder characterised by myotonia with progressive muscular weakness and wasting, plus a variety of other symptoms (Harper 1989). The underlying biochemical defect is still unknown. However, the D M gene has been localised to the long arm of chromosome 19, region 19q13.2-q13.3, by virtue of its linkage to several chromosome 19 markers (Shaw et al. 1985, 1986; Friedrich et al. 1987; B r o o k et al. 1987a; Stallings et al. 1988). The search for closely linked D N A markers is the first stage in the isolation of a disease gene by the "reverse genetics" approach; these markers are also of practical benefit in presymptomatic diagnosis. We have investigated the genetic linkage to D M of two genes previously assigned to the proximal long arm of chromosome 19: the apolipoprotein C1 (APOC1) gene (Lusis et al.
Offprint requests to: D. J. Shaw
The 44 families with myotonic dystrophy, used in this study, have formed the basis of previous studies (Shaw et al. 1985, 1986; Harley et al. 1988). All families have been clinically assessed, including EMG and opthalmological examination when necessary. Homogeneity tests have been performed on this set of families using the HOMOG programme (Ott 1983) and no detectable heterogeneity was found using the closely linked marker APOC2 [H. Harley, unpublished data; the lod score for APOC2-DM is > 90 at 0 = 0.02-0.03 (Le Beau et al. 1989)]. Only unequivocally affected DM individuals, plus unrelated normal spouses, were scored when establishing linkage between the DM locus and the markers, APOC1 and CYP2A. Therefore, complete penetrance was assumed when calculating the lod scores for APOC1-DM and CYP2A-DM. No individuals at risk of developing myotonic dystrophy were included in these calculations, with the exception of 11 individuals whose affected siblings had the congenital form of myotonic dystrophy. These 11 individuals had all reached puberty without any symptoms of myotonic dystrophy and are therefore considered not to be gene carriers by the criteria of Bundey (1974), O'Brien et al. (1983a) and M.C. Koch, T. Grimm, H. G. Harley, P. S. Harper (in preparation). Isolation of DNA, Southern transfer and DNA hybridisation were performed according to standard protocols (Maniatis et al. 1982). When using the APOC1 cDNA probe, sheared cold total human DNA (10 gg/ml) was added to the prehybridisation buffer, to reduce the background hybridisation with this probe. The probes
306
Table 1. Segregation of genetic markers in four somatic cell hybrids containing chromosome 19 fragments Marker
D19S7 GPI D19S9 MSK37 TGFI3 CYP2A D19S8 ATPIA3 D19S19 APOC1 APOC2 CKMM
Hybrid line TVBID
J640
WILFM2
G175C4
+ + + + + + + + + + +
+ + + + + + -
+ + + + + + + + +
+ + + -
Results
Regional localisation of C Y P 2 A and A P O C 1
Fig.1. a DraI polymorphism detected by the APOC1 probe. Allele 1 7.6kb, 2.6kb, frequency 0.75; allele 2 10.2kb, frequency 0.25. b BglI polymorphism detected by the APOC1 probe. Allele I 8.7 kb, frequency 0.06; allele 2 6.2kb, frequency 0.94. e SstI polymorphism detected by the CYP2A probe. Allele 1 9.3 kb, frequency 0.55; allele 2 5,2kb, 4.1 kb, frequency 0.45
A P O C 1 was present in the cell lines T V B I D and W I L F M 2 b u t was in n e i t h e r G 1 7 5 C 4 n o r J640. C Y P 2 A d e t e c t e d h u m a n E c o R I f r a g m e n t s in T V B I D a n d W I L F M 2 , b u t n o t in G 1 7 5 C 4 , as p r e v i o u s l y r e p o r t e d by B r o o k et al. (1987a). C Y P 2 A was also p r e s e n t in J640. M a r k e r s m a p ping to the long a r m of c h r o m o s o m e 19 can be subdivided o n the basis of their segregation p a t t e r n s in these cell lines. T a b l e 1 s u m m a r i s e s the localisation data of A P O C 1 and C Y P 2 A , together with o t h e r long a r m markers. As can be seen from this T a b l e , C Y P 2 A has the same segregation p a t t e r n as M S K 3 7 a n d TGFI3, w h e r e a s A P O C 1 shows the same segregation as A P O C 2 , C K M M a n d D19S19 ( L D R 1 5 2 ) .
Linkage analysis were labelled using a commercial multi-prime kit (Amersham International). Blots were washed at 65~ to a final stringency of 1 • SSC for APOC1 and 0.1 x SSC for CYP2A. The data were analysed for linkage using the MLINK programme (Lathrop et al. 1984). As mentioned above, complete penetrance was assumed when calculating the lod score data between the DM locus and the markers used in this study. The linkage data were also calculated on the basis of equal rates of male and female recombination, and equal rates of mutation in males and females of zero (i.e. there are no new mutations).
Somatic cell hybrids APOC1 and CYP2A were regionally localised on chromosome 19 using a panel of rodent-human somatic cell hybrids that have been fully described elsewhere: TVBID (Brook et al. 1987a), J640 (J. D. Brook, S. Knight, S. H. Roberts, H. G. Harley, K.V. Walsh, S. A. Rundle, K. Freyne, M. C. Koch, N. D. Epstein, B. Wieringa, D. Schonk, B. Smeets, K. Haddingham, M.J. Siciliano, D. K. Palmer, J. S. Miles, C. R. Wolf, C. Fonatsch, D. J. Shaw, submitted for publication), G175C4 (Brook et al. 1987b) and WILFM2 (Brook et al. 1986, 1987b). TVBID contains region 19q12-qter. J640, G175C4 and WILFM2 are cell lines containing fragments of chromosome 19.
A P O C 1 was i n f o r m a t i v e for linkage analysis with the DraI R F L P in 10 families, a n d with the BglI R F L P in 5 families; 2 of these families were i n f o r m a t i v e with b o t h polymorphisms. O n l y o n e r e c o m b i n a n t (phase u n k n o w n , male meiosis) out of 44 meioses was o b s e r v e d b e t w e e n A P O C 1 a n d D M , giving a m a x i m u m lod score of 9.29 at 0 m a x = 0.02 (see T a b l e 2). I n the D M - A P O C 1 r e c o m b i n a n t i n d i v i d u a l (Fig. 2, pedigree A , II 9), D M has also r e c o m b i n e d with D19S8 a n d C Y P 2 B (a gene that m a p s to within 350 kb of C Y P 2 A ; Miles et al. 1989). C Y P 2 A - D M gives a m a x i m u m lod score of 8.55 at 0max = 0.04 (see T a b l e 2), based o n data from 19 informative families. T h r e e r e c o m b i n a n t s (all phase u n k n o w n , female meioses) out of 64 meioses were observed. T w o of these r e c o m b i n a n t s were also D 19S19-DM crossovers a n d were in the same family, placing C Y P 2 A o n the same side of the D M locus as D19S19. F i g u r e 3 shows the pedigree c o n t a i n i n g the o t h e r C Y P 2 A - D M rec o m b i n a n t (individual II 1, p e d i g r e e B). This i n d i v i d u a l is an u n a f f e c t e d sib in a c o n g e n i t a l m y o t o n i c d y s t r o p h y
307 Table 2. Genetic linkage analysis of APOC1 and CYP2A to myotonic dystrophy (DM)
Loci
0 (Of = 0m)
APOC1-DM CYP2A-DM APOC1-CYP2A
0.01
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
9.25 7.85 8.90
8.92 8.55 9.09
7.92 7.96 8.56
6.78 6.94 7.63
5.59 5.77 6.53
4.40 4.54 5.34
3.23 3.32 4.09
2.13 2.19 2.83
1.15 1.21 1.59
0.38 0.46 0.55
max
Z~ax
0.02 0.04 0.05
9.29 8.55 9.09
Table 3. Data for the APOC1 locus. DM, DM chromosome; N, A
(21)
11
B
(12)
22
normal chromosome; Z2 values in a, b and c are not significant a Number of observed DM/APOC1 (DraI) haplotypes (~2 = 0.423)
C
ii
il 21
21
21
21
21
22
21
21
11
12
12
12
22
12
22
12
22
22
APOC1 (DraI) allele 1
2
28 105
6 31
13
III
lii Fig. 2. Pedigree A. Individual 11 9 is a (APOC1, D19S8, CYP2B) X DM crossover. A APOC1 (DraI), B CYP2B (MspI), C D19S8
I
2
A
11)
12
B
BC
AB
C
I!!0
Fig. 3. Pedigree B. Individual H 1 is a (CYP2A, APOC2, CKMM) X DM crossover; individual H 3 is a CYP2A X (APOC2, CKMM, DM) crossover. A CYP2A, B APOC2 (haplotype), C CKMM (haplotype)
sibship, so does not c o n f o r m to the strict criteria set out by Griggs et al. (1989) for scoring r e c o m b i n a n t s in D M families. H o w e v e r , according to B u n d e y (1974), O ' B r i e n et al. (1983a) and M. C. K o c h , T. G r i m m , H. G. H a r l e y , P. S. H a r p e r (in p r e p a r a t i o n ) , an unaffected individual in a congenital sibship is unlikely to be a D M carrier if they reach p u b e r t y w i t h o u t showing any symptoms of m y o t o n i c dystrophy. Individual II 1, pedigree B, is 40 years old, and has therefore b e e n a s s u m e d to be n o r m a l in o u r linkage analysis. In this individual, D M has r e c o m b i n e d with C Y P 2 A , A P O C 2 and C K M M . W h e n the typings for A P O C 2 and C K M M are considered t o g e t h e r with C Y P 2 A in pedigree B, r e c o m b i n a tion b e t w e e n C Y P 2 A and these m a r k e r s has to be inv o k e d . B a s e d on the assumption that the p a t e r n a l genotype giving the least n u m b e r of r e c o m b i n a n t s is the correct o n e (i.e. the o n e given in Fig. 3), then individual II 3 is the m o s t likely C Y P 2 A - ( A P O C 2 , C K M M ) r e c o m binant. T h e data given in Fig. 3 also suggest that this in-
DM Normal
b Number of observed DM/APOC1 (BglI) haplotypes (Z2 = 1.299) APOC1 (BglI) allele
DM Normal
1
2
1 7
33 71
c Number of observed and expected DM/APOC1 (DraI + BglI) haplotypes (?(2 = 1.769) Haplotype DM/DraI/BglI DM11 Nll DM21 N21 DM12 N12 DM22 N22
Observed 1 3 0 0 25 43 5 13
Expected 1.10 2.10 0.28 0.52 23.70 45.10 5.92 11.28
dividual is a D M - C Y P 2 A r e c o m b i n a n t , giving C Y P 2 A (APOC2, CKMM, DM). A P O C 1 and C Y P 2 A are also linked to each other, a m a x i m u m lod score of 9.09 at 0max = 0.05 being o b t a i n e d (see Table 2). T e n pedigrees were informative for b o t h loci and three r e c o m b i n a n t s (one female and two male meioses) were o b s e r v e d in 49 meioses. All were phase u n k n o w n and in individuals w h e r e it was not possible to relate the r e c o m b i n a n t event to the affected D M gene. In the families informative for linkage analysis, the rarer allele of the m a r k e r s a p p e a r e d to be inherited with the affected allele of the D M gene m o r e often than exp e c t e d given the allele frequencies; this suggests that there m a y be linkage disequilibrium b e t w e e n these loci and the D M gene. Chi-squared tests were p e r f o r m e d o n
308 Table 4. Number of observed APOC1 DraI/BglI haplotypes. Z2 = 1.047; this ~2 value is not significant
DraI allele
BglI allele 1
2
l
4
2
0
68 18
Table 5. Number of observed DM/CYP2A haplotypes. Z2 = 1.803: this X2 value is not significant CYP2A allele DM Normal
1
2
14 90
19 73
all 44 DM families (including those uninformative for linkage analysis), using only one DM chromosome per pedigree (where phase could be ascertained) plus all unrelated normal chromosomes that had been genotyped. Table 3 gives the data for the APOC1 locus. Chi-squared analysis shows that there is no significant disequilibrium between either the DraI or the BglI polymorphisms, or the haplotype of the 2 RFLPs and the DM locus. There is also no disequilibrium between the two polymorphisms for APOC1 (Table 4). The bias towards inheritance of DM with the rarer allele in those families that were informative for linkage analysis appears to have arisen because 3 of the 13 families are of French-Canadian origin and are interrelated. To take this into account, only one D M chromosome from the French-Canadian pedigrees was included in the chi-squared analysis. Therefore, the families informative for linkage did not accurately reflect the pattern in the population as a whole. Similarly, statistical analysis of the C Y P 2 A data (see Table 5) suggests that there is no significant disequilibrium between the C Y P 2 A and DM loci. Again, the bias observed in the informative families appears to be the result of the large number of FrenchCanadian families in this group (7/19). Disequilibrium has been demonstrated in a French-Canadian population between DM and A P O E (Laberge et al. 1985), A P O C 2 and D M (MacKenzie et al. 1989), and also between DM and BCL3 (Korneluk et al. 1989), presumably resulting from a "founder effect" (Laberge 1989).
Discussion
We have shown that the loci APOC1 and C Y P 2 A are linked to DM. APOC1 may be a suitable marker for use in prenatal diagnosis especially when A P O C 2 and CKMM, the closest markers to DM (Shaw et al. 1985, 1986; Shaw and Eiberg 1988; Brunner et al. 1989; H. G. Harley, K.V. Walsh, J. D. Brook, S.A. Rundle, M. Sarfarazi, M. C. Koch, J. L. Floyd, P. S. Harper, D.J. Shaw, submitted for publication) are uninformative. CYP2A is
less suitable for this purpose. Although our data put it only 4 cM from DM, data from multipoint analysis in DM and C E P H pedigrees place CYP2A further from DM than several other markers (the marker order from multipoint analysis is CYP2A-ATPIA3-D19S8-D19S19BCL3-APOC2-CKMM-DM; H. G. Harley, K.V. Walsh, J. D. Brook, S. A. Rundle, M. Sarfarazi, M. C. Koch, J. L. Floyd, P. S. Harper, D.J. Shaw, submitted for publication; H . G . Harley, S.A. Rundle, J.D. Brook, M. Sarfarazi, K. J. Johnson, B. Wieringa, P. S. Harper, D. J. Shaw, in preparation). The genes for APOC1, APOC2 and A P O E are known to be clustered, evidence for this coming from linkage data (Humphries et al. 1984; Myklebost et al. 1984) and somatic cell hybridisation studies (Das et al. 1985; Scott et al. 1985; Tata et al. 1985; Lusis et al. 1986). Moreover, APOC1 and A P O E have been isolated on the same genomic clone from lambda libraries (Das et al. 1985; Davison et al. 1986; Myklebost and Rogne 1986; Lusis et al. 1986). This clustering has also been demonstrated by pulsed-field gel analysis, which suggests the gene order A P O E - A P O C 1 - A P O C 2 (Myklebost and Rogne 1988) and indicates that the cluster spans about 50 kb, an estimate supported by Smit et al. (1988). Our data showing APOC1 to be closely linked to DM, as is APOC2, is consistent with their being clustered. APOC1 and APOC2 also show close genetic linkage, our data giving a maximum lod score of 10.27 at 2 cM (H. G. Harley, K.V. Walsh, J. D. Brook, S. A. Rundle, M. Sarfarazi, M. C. Koch, J. L. Floyd, P. S. Harper, D. J. Shaw, submitted for publication). Despite their close physical linkage, we have observed an APOC1-APOC2 recombinant. This recombinant individual is at present not affected with DM, although at risk, so we are unable to orientate the apolipoprotein gene cluster with respect to the DM gene. However, Brunner et al. (1989) have recently reported an A P O E - A P O C 2 recombinant allowing this gene cluster to be orientated, placing APOC2 closest to DM, and hence giving the order: A P O E - A P O C 1 APOC2-DM. CYP2A has been shown to be linked to PEPD (Davis et al. 1987), a gene also known to be linked to DM (O'Brien et al. 1983b) and A P O C 2 (Ball et al. 1985). Pep4, the mouse homologue of PEPD, is linked to the Coh locus on mouse chromosome 7. A phenobarbital-inducible cytochrome P450 gene family maps to the Coh locus (Simmons and Kasper 1983; Simmons et al. 1985), and thus, the CYP2A and Coh loci are thought to be equivalent (Phillips et al. 1985a; Davis et al. 1987; Miles et al. 1989). Other chromosome 19 markers that have homologues mapping within the Pep4-Coh linkage group on mouse chromosome 7 include GPI, TGFI3, LHI3, ATP1A3 and A P O E . Since the conserved linkage group includes markers covering most of 19q, it is likely that there is also a homologue of the DM gene on mouse chromosome 7. The presence of C Y P 2 A and APOC1 in T V B I D confirm the localisation of these markers to the long arm of chromosome 19. The fragmented cell lines WILFM2, J640 and G175C4 allow long arm markers to be subdivided. APOC1 shows the same pattern as APOC2,
309 C K M M a n d D19S19 ( B r o o k et al. 1987a). H o w e v e r , C Y P 2 A s e g r e g a t e s with TGF[3 a n d M S K 3 7 ( B r o o k et al. 1987a; J. D . B r o o k , S. K n i g h t , S. H . R o b e r t s , H . G . H a r ley, K . V . W a l s h , S . A . R u n d l e , K . F r e y n e , M . C . K o c h , N. D. E p s t e i n , B. W i e r i n g a , D. S c h o n k , B. S m e e t s , K. H a d d i n g h a m , M . J . Siciliano, D . K. P a l m e r , J. S. Miles, C. R. W o l f , C. F o n a t s c h , D . J. S h a w , s u b m i t t e d for p u b l i c a t i o n ) . T h e p r e s e n c e o f C Y P 2 A in J640 suggests t h a t it is p r o x i m a l to t h e a p o l i p o p r o t e i n g e n e c l u s t e r a n d to D M , as this cell line also c o n t a i n s t h e p r o x i m a l m a r k e r D19S7. This is in a g r e e m e n t with S c h o n k et al. (1989) who also localised C Y P 2 A p r o x i m a l to the a p o l i p o p r o t e i n genes using s o m a t i c cell h y b r i d s . T h e ( C Y P 2 A , A P O C 2 , C K M M ) - D M r e c o m b i n a n t (Fig. 3, i n d i v i d u a l II 1) also indicates that C Y P 2 A is p r o x i m a l to D M , as A P O C 2 a n d C K M M a r e n o w k n o w n to m a p p r o x i m a l to D M ( S h a w et al. 1989; H . G . H a r l e y , K . V . W a l s h , J. D . B r o o k , S . A . R u n d l e , M. S a r f a r a z i , M. C. K o c h , J. L. F l o y d , P. S. H a r p e r , D . J. S h a w , s u b m i t t e d for p u b l i c a t i o n ) ; t h e C Y P 2 A ( A P O C 2 , C K M M , D M ) r e c o m b i n a n t (Fig. 3, i n d i v i d u a l I I 3) p l a c e s C Y P 2 A as t h e m o s t p r o x i m a l o f t h e s e m a r k ers. This p r o x i m a l l o c a t i o n of C Y P 2 A is also s u p p o r t e d b y l i n k a g e d a t a f r o m t h e C E P H p e d i g r e e s (H. G . H a r l e y , S. A . R u n d l e , J. D . B r o o k , M. S a r f a r a z i , K. J. J o h n s o n , B . W i e r i n g a , P. S. H a r p e r , D . J . S h a w , in p r e p a r a t i o n ) . T h e r e f o r e , t h e m o s t l i n k e l y o r d e r is C Y P 2 A - ( A P O E ICIlC2)-DM.
Acknowledgements. We would like to thank D r . E . A . Shephard for the CYP2A probe, Dr.T.J. Knott for the APOC1 probe, Dr.M.-C.Thibault for providing the Canadian family material, and Dr. M. C. Koch for family sampling and clinical assessment. This work was supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland, the Muscular Dystrophy Association (USA), the Medical Research Council, and the Wellcome Trust.
References Ball SP, Donald JA, Corney G, Humphries SE (1985) Linkage between the loci for peptidase D and apolipoprotein CII on chromosome 19. Ann Hum Genet 49 : 129-134 Brook JD, Shaw D J, Thomas NST, Meredith AL, Cowell J, Harper PS (1986) Mapping genetic markers on human chromosome 19 using subchromosomal fragments in somatic cell hybrids. Cytogenet Cell Genet 41 : 30-37 Brook JD, Skinner M, Roberts SH, Rettig WJ, Almond JW, Shaw DJ (1987a) Further mapping of markers around the centromere of human chromosome 19. Genomics 1 : 320-328 Brook JD, Beresford HR, Shaw D J, Old LJ, Rettig WJ (1987b) Localisation on human chromosome 19 of three genes for cell surface antigens defined by monoclonal antibodies. Cytogenet Cell Genet 45 : 156-162 Brunner HG, Smeets H, Lambermon HMM, Coerwinkel-Driessen M, Oost BA van, Wieringa B, Ropers H-H (1989) A multipoint linkage map around the locus for myotonic dystrophy on chromosome 19. Genomics 5 : 589-595 Bundey S (1974) Detection of heterozygotes for myotonic dystrophy. Clin Genet 5 : 107-109 Das HK, McPherson J, Bruns GAP, Karathanasis SK, Breslow JL (1985) Isolation, characterization, and mapping to chromosome 19 of the human apolipoprotein E gene. J Biol Chem 260 : 6240-6247 Davis MB (1987) Linkage between the loci for peptidase D and cytochrome P450 (CYP1) on chromosome 19. Ann Hum Genet 51 : 9-12
Davis MB, West LF, Shephard EA, Phillips IR (1986) Regional localization of a human cytochrome P450 (CYP1) to chromosome 19q13.1-13.3. Ann Hum Genet 50:237-240 Davison PJ, Norton P, Wallis SC, Gill L, Cook M, Williamson R, Humphries SE (1986) There are two gene sequences for human apolipoprotein C1 (APOC1) on chromosome 19, one of which is 4 kb from the gene for APOE. Biochem Biophys Res Commun 136 : 876-884 Friedrich U, Brunner H, Smeets D, Lambermon E, Ropers H-H (1987) Three-point linkage analysis employing C3 and 19cen markers assigns the myotonic dystrophy gene to 19q. Hum Genet 75 : 291-293 Frossard PM, Coleman RT, Malloy MJ, Kane JP, Levy-Wilson B, Appleby VA (1987a) Human apolipoprotein C1 (APOC1) gene locus: DraI dimorphic site. Nucleic Acids Res 15 : 1884 Frossard PM, Lim DW, Coleman RT, Funke H, Assmann G, Malloy MJ, Kane JP (1987b) Human apolipoprotein C1 (APOC1) gene locus: BglI dimorphic site. Nucleic Acids Res 15 : 1344 Griggs RC, Wood DS, the Working Group on the Molecular Defect in Myotonic Dystrophy (1989) Criteria for establishing the validity of genetic recombination in myotonic dystrophy. Neurology 39 : 420-421 Harley HG, Brook JD, Jackson CL, Glaser T, Walsh KV, Sarfarazi M, Kent R, Lager M, Koch M, Harper PS, Levenson R, Housman DE, Shaw DJ (1988) Localization of a human Na +, K+-ATPase a subunit gene to chromosome 19q12-q13.2 and linkage to the myotonic dystrophy locus. Genomics 3:380384 Harper PS (1989) Myotonic dystrophy, 2nd edn. Saunders, Philadelphia Hulsebos T, Wieringa B, Hochstenbach R, Smeets D, Schepens J, Oerlemans F, Zimmer J, Ropers H-H (1986) Toward early diagnosis of myotonic dystrophy: construction and characterization of a somatic cell hybrid with a single human der(19) chromosome. Cytogenet Cell Genet 43 : 47-56 Humphries SE, Berg K, Gill L, Cumming AM, Robertson FW, Stalehoef AFH, Williamson R, Borresen AL (1984) The gene for apolipoprotein CII is closely linked to the gene for apolipoprotein E on chromosome 19. Clin Genet 26 : 389-396 Knott TJ, Robertson ME, Priestley LM, Urdea M, Wallis S, Scott J (1984) Characterisation of mRNAs encoding the precursor for human apolipoprotein C1. Nucleic Acids Res 12:39093915 Korneluk RG, MacLeod HL, McKeithan TW, Brook JD, MacKenzie AE (1989) A chromosome 19 clone from a translocation breakpoint shows close linkage and linkage disequilibrium with myotonic dystrophy. Genomics 4:146-151 Laberge C (1989) Myotonic dystrophy in Quebec: geographical distribution and concept of genetic homogeneity. Can J Neurol Sci 16 : 123-128 Laberge C, Gaudet D, Morisette J, Moorjani S, Thibault MC (1985) Linkage of myotonic dystrophy and APOE in a FrenchCanadian isolate (abstract). (8th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 40 : 675 Lathrop GM, Lalouel JM, Julier C, Ott J (1984) Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 81 : 3443-3446 Le Beau MM, Ryan D, Pericak-Vance MA (1989) Report of the committee on the genetic constitution of chromosomes 18 and 19. (10th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 51 : 338-357 Lusis AJ, Heinzmann C, Sparkes RS, Scott J, Knott TJ, Geller R, Sparkes MC, Mohandas T (1986) Regional mapping of human chromosome 19: organization of genes for plasma lipid transport (APOC1, - C 2 , and - E and LDLR) and the genes C3, PEPD, and GPI. Proc Natl Acad Sci USA 83 : 3929-3933 MacKenzie AE, MacLeod HL, Hunter AGW, Korneluk RG (1989) Linkage analysis of the apolipoprotein C2 gene and myotonic dystrophy on human chromosome 19 reveals linkage disequilibrium in a French-Canadian population. Am J Hum Genet 44 : 140-147
310 Maniatis T, Fritsch EF, Sambrook J (eds) (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Miles JS, Bickmore W, Brook JD, McLaren AW, Meehan R, Wolf CR (1989) Close linkage of the human cytochrome P450IIA and P450IIB gene subfamilies: implications for the assignment of substrate specificity. Nucleic Acids Res 17 : 29072917 Myklebost O, Rogne S (1986) The gene for human apolipoprotein CI is located 4.3 kilobases away from the apolipoprotein E gene on chromosome 19. Hum Genet 73 : 286-289 Myklebost O, Rogne S (1988) A physical map of the apolipopro 7 tein gene cluster on human chromsome 19. Hum Genet 78: 244-247 Myklebost O, Rogne S, Olaisen B, Gedde-Dahl T, Prydz H (1984) The locus for apolipoprotein CII is closely linked to the apolipoprotein E locus on chromosome 19 in man. Hum Genet 67 : 309-312 O'Brien T, Newcombe RG, Harper PS (1983a) Outlook for a clinically normal child in a sibship with congenital myotonic dystrophy. J Pediatr 103 : 762-763 O'Brien T, Ball S, Sarfarazi M, Harper PS, Robson EB (1983b) Genetic linkage between the loci for myotonic dystrophy and peptidase D. Ann Hum Genet 47 : 117-121 Ott J (1983) Linkage analysis and family classifications under heterogeneity. Ann Hum Genet 47:311-320 Phillips IR, Shephard EA, Povey S, Davis MB, Kelsey G, Monteiro M, West LF, Cowell J (1985a) A cytochrome P450 gene family mapped to human chromosome 19. Ann Hum Genet 49 : 267-274 Phillips IR, Shephard EA, Ashworth A, Rabin BR (1985b) Isolation and sequence of a human cytochrome P450 cDNA clone. Proc Natl Acad Sci USA 82 : 983-987 Schonk D, Coerwinkel-Driessen M, Dalen I van, Oerlemans F, Schepens J, Hulsebos T, Cockburn D, Boyd Y, Davis M, Rettig W, Shaw D, Roses A, Ropers H-H, Wieringa B (1989) Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q. Genomics 4:384-396 Scott J, Knott TJ, Shaw DJ, Brook JD (1985) Localization of genes encoding apolipoproteins CI, CII, and E to the p13-cen region of human chromosome 19. Hum Genet 71 : 144-146 Shaw D J, Eiberg H (1988) Report of the committee on the genetic constitution of chromosomes 17, 18 and 19. (9th International
Workshop on Human Gene Mapping) Cytogenet Cell Genet 46 : 242-256 Shaw D J, Meredith AL, Sarfarazi M, Huson SM, Brook JD, Myklebost O, Harper PS (1985) The apolipoprotein CII gene: subchromosal localisation and linkage to the myotonic dystrophy locus. Hum Genet 70:271-273 Shaw D J, Meredith AL, Sarfarazi M, Harley HG, Huson SM, Brook JD, Bufton L, Litt M, Mohandas T, Harper PS (1986) Regional localisations and linkage relationships of seven RFLPs and myotonic dystrophy on chromosome 19. Hum Genet 74: 262-266 Shaw D J, Harley HG, Brook JD, McKeithan TW (1989) Longrange restriction map of a region of human chromosome 19 containing the apolipoprotein genes, a CLL-associated translocation breakpoint and two polymorphic MluI sites. Hum Genet 83 : 71-74 Simmons DL, Kasper CB (1983) Genetic polymorphisms for a phenobarbital-inducible cytochrome P450 map to the Coh locus in mice. J Biol Chem 258 : 9585-9588 Simmons DL, Lalley PA, Kasper CB (1985) Chromosomal assignments of genes coding for components of the mixed function oxidase system in mice. Genetic localization of the cytochrome P450 PCN and P450PB gene families and the NADPH-cytochrome P450 oxidoreductase and epoxide hydratase genes. J Biol Chem 260 : 515-521 Smit M, Kooij-Meijs E van der, Frants RR, Havekes L, Klasen EC (1988) Apolipoprotein gene cluster on chromosome 19. Hum Genet 78: 90-93 Stallings RL, Olson E, Strauss AW, Thompson LH, Bachinski LL, Siciliano MJ (1988) Human creatine kinase genes on chromosomes 15 and 19, and proximity of the gene for the muscle form to the genes for apolipoprotein C2 and excision repair. Am J Hum Genet 43 : 144-151 Tata F, Henry I, Markham AF, Wallis SC, Weil D, Grzeschik KH, Junien C, Williamson R, Humphries SE (1985) Isolation and characterisation of a cDNA clone for human apolipoprotein CI and assignment of the gene to chromosome 19. Hum Genet 69 : 345-349 Wainwright B J, Watson EK, Shephard EA, Phillips IR (1985) RFLP for a human cytochrome P450 gene at 19q13.1-qter (HGM8 provisional designation CYP1). Nucleic Acids Res 13 : 4610
