GENOMICS

6,352-357

(1990)

Deletion and Linkage Mapping of Eight Markers from the Proximal Short Arm of Chromosome 6 H. Y. ZoaiBl,*etql C. M. BALLANTYNE,$ W. E. O’BRm,tsS A. E. MCCALL‘S T. J. KWIATKOWSKI, JR.,§ 5. A. LEDBETTER,t AND A. L. BEAUDET*‘t’!j *Department

of Pediatrics, tlnstitute for Molecular Genetics, and *Department of Medicine, and SHoward Hughes Medical Institute, Houston, Received

July 19, 1989;

revised

October

of Medicine, Texas 77030

Baylor College

20, 1989

workers (1986) using three genomic DNA sequences (D6S7, D6S8, D6SlO), the cDNA sequencesfor D6S5, HLA-B, HLA-DR-a, and HLA-D&-a, and electrophoretie assays for glyoxalase 1 (GLOl). As part of efforts aimed at identifying highly polymorphic DNA markers that are closely linked to the HLA-linked form of spinocerebellar ataxia, we have identified new restriction fragment length polymorphisms (RFLPs) at different chromosome 6p loci and established the linkage-relationships among these loci using the Centre d’Etude Polymorphisme Humain (CEPH) reference families (Dausset, 1986). In this study, we report the results of deletion mapping and linkage analyses using two of the marker loci used by Leach and co-workers (D6S5 and HLA-A-BDQ-DR) and six additional markers from the short arm of chromosome 6.

Eight chromosome 6p markers (MUT, D6S4, DGSS, D6S19, D6S29, PIM, HLA, and F13A) were regionally mapped using somatic cell hybrid deletion cell lines that retained different regions of chromosome 6p. New restriction fragment length polymorphisms were identified at the D6SS and PIM loci using newly isolated genomic clones at these loci. Genetic linkage among the eight loci was determined using the 40 CEPH reference families. Linkage analyses showed that these loci are in one linkage group spanning 48 CM in males and 128 CM in females. Using both the deletion mapping data and multipoint linkage analyses, chromosomal order for these loci was determined as centromere-(MUT, D6S4)-(D6S5, D6SlS)(D6S29, PIM)-HLA-FlSA-telomere. Analyses of sex-specific recombination frequencies revealed a higher rate of recombination in females in the region between D6S4 and D6S29, while the recombination rate in males was higher for the interval between D6S29 and the HLA loci. c 1990 ACSB~~IDICPR=, I~C.

METHODS

Subjects, Probes, and Genotype Determinations As part of an ongoing study (Zoghbi et al., 1989), six unrelated American black individuals were chosen as the source of DNA for detection of polymorphisms with a variety of restriction enzymes. The DNAs were extracted from blood or established lymphoblastoid cell lines according to published protocols (Zoghbi et al., 1988a). For further characterization of the polymorphisms in Caucasians and for linkage studies, we used DNAs provided by CEPH from the 40 reference families. Seven cloned DNA sequences, the cDNA for methylmalonyl-CoA mutase (MMCM, MUT), CRIL171 (D6S19), pAGB6 (D6S5), HHH157 (D6S29), 4Cll (D6S4), pHPIM5RI (PIM), and the cDNA for coagulation factor XIIIA (F13A), were used to genotype human genomic DNA sequencesby means of Southern analysis. These DNA sequences were kindly provided by other investigators. The clone for CRI-L171 was purchased from Collaborative Research Inc. (Bedford,

INTRODUCTION

The availability of thousands of polymorphic DNA markers has allowed the construction of genetic maps for several human chromosomes. Genetic maps that are developed using highly informative DNA markers that are evenly spaced on a chromosome in a known order will be extremely valuable for mapping genes responsible for human genetic diseases. Such maps already exist for several human chromosomes (O’Connell et al., 1987; Farrer et al., 1988; Tanzi et al., 1988). A preliminary genetic map of markers for the short arm of chromosome 6 was first developed by Leach and co1 To whom reprint requesta should be addressed at the Institute for Molecular Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

o&323-7543/90

$3.00

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

352

EIGHT

CHROMOSOME

TABLE Newly

Identified

Probe

Locus

353

6p MARKERS

1

Polymorphisms

on the Short

Enzyme

Allele (size kb)

Arm

of Chromosome

6

Allele frequency’

size of chromosomes)

SaMpk

(No.

PIM

pHPIM5RI

TaqI

1.3 1.45

0.72 (C) 0.28

44

PIM

HZ-PIM

MspI

1.9 1.6 4.1 2.3

0.9 (B) 0.1 0.08 (B) 0.92

30

0.71 (C) 0.29 0.3 (B) 0.7

35

Ban1

D6S5

HZ-AGBGb

19 -20 19 -20

SphI SphI

26

30

’ C, Caucasian; B, American black. The TaqI RFLP at the PIM locus was not observed in American blacks at the PIM locus were not observed in Caucasians. b Caucasian and American black frequencies for D6S5 are significantly different (Xf = 10.9, P < 0.005).

MA). To increase the information content at the D6S5 locus and the PIM locus, we used the probes pAGB6 and pHPIM5RI to screen a human genomic DNA library which we constructed in the bacteriophage vector X-DASH (Stratagene, San Francisco, CA). The human DNA contained in the library was isolated from peripheral blood. Two phage clones, HZ-AGB6 and HZPIM, were isolated and were used to search for RFLPs. For genotype determinations, human DNA samples were digested with the appropriate restriction endo-

and the MspI

and Ban1 RFLPs

nuclease and fractionated in 0.8% agarose. Southern transfer, hybridizations, and washes were carried out according to previously published protocols (Zoghbi et al., 1988a). Probes were labeled in low-melt agarose according to the method of Feinberg and Vogelstein (1983). When the whole insert of HZ-AGB6 or HZPIM was used, the labeled probe was prehybridized with sonicated total human placental DNA to block repetitive sequencesaccording to the method of Sealey et al. (1985). To detect RFLPs using pHPIM5R1, HZ-

a. Cell Line 1-7

II-

6.3.6-46

II-

3.1 .&A I+,-.

b.

I

, I D9s4 MUT

FIG. markers.

1.

II D6S5 D6Sl9 D6S29 PM

, HLA

III

,

CALLAS-

-9-3

Iv

F13A

Deletion map of chromosome 6p. (a) A schematic diagram of the hybrid cell lines used for regional (b) The four defined regions of chromosome 6p and the results of the regionai mapping of markers.

mapping

of chromosome

6p

354

ZOGHBI

ET

AL.

at F13A. Genotypic data for the BgZII RFLP at D6S4 and the MspI RFLP at D6S5 and the serologic typing data for HLA-A-B-DQ-DR were obtained from CEPH. Hybrid Cell Lines and Regional Mapping To regionally localize chromosome 6p markers, we used a somatic cell hybrid panel that retained different fragments of chromosome 6. This somatic cell hybrid panel divides chromosome 6p into four different regions. The I-7 somatic hybrid cell line contains the short arm of chromosome 6 as the only human chromosome; it was derived in our laboratory by repeated, nonselective subcloning of the somatic cell hybrid CF 34-2-10111. CF 34-2-10111 was provided by T. Mohandas and retained human chromosomes 11, 13, and 6p (Mohandas et al., 1980). Twenty-eight cells from the resulting subcloned hybrid were analyzed by G-11 staining (Alhadeff et al., 1977). The somatic hybrid cell lines 3.1.0-A and 6.3.6-46 contain chromosome 6p breakpoints that have been characterized by enzyme markers, HLA markers, and polymorphic DNA sequences (Gladstone et aZ., 1982; Zoghbi et al., 1988b). The somatic cell hybrid cell line CALLAg-1-9-3 retains the most distal fragment of chromosome 6p from 6p23pter (Naylor et al., 1983).

FIG. 2.

Physical mapping of CRI-L171 (D6S19). This figure illustrates a Southern blot analysis of HindIII-digested DNA from human-Chinese hamster hybrids (see Fig. l), human cells, and hamster cells probed with CRI-L171 (D6S19). The cell lines used as a source of DNA are depicted above the autoradiograph.

PIM, and HZ-AGBG, we screened blots of restriction enzyme-digested genomic DNA from six unrelated American black individuals. The following restriction enzymes were used: Apa& AuaII, BarnHI, Bard, BanII, BclI, BgZI, B&II, DruI, EcoRI, EcoRV, Fnu4H1, HincII, HindIII, &x21, MboI, MspI, PSSI, PstI, PUUII, RSUI, ScaI, SphI, S&I, StuI, TaqI, and XbaI. In addition, pHPIM5RI and HZ-AGB6 were hybridized to Southern blots containing TuqI- and SphI-digested DNAs from unrelated CEPH parents to Cal&ate the gene frequencies of these two polymorphisms in Caucasians. For linkage analysis all the informative CEPH families were genotyped in our laboratory for the Hind111 RFLP at MUT locus, TuqI RFLP at D6S19, BumHI RFLP at D6S29, TuqI RFLP at PIM, and Bc21 RFLP

d

Telomere

63

7 15 27

k CellZrmere FIG. 3. Male and female mosome 6. HLA-A-B-DQ-DR Genetic distances are given mapping function.

d genetic maps of the short arm of chrohaplotypes were uaed at the HLA loci. in centimorgans assuming Haldane’s

EIGHT

CHROMOSOME

(F13A locus, Zoghbi et al., 1988a). Previously ported polymorphisms were identified using pHPIM5R1, HZ-PIM, and HZ-AGBG. Table scribes the newly identified polymorphisms used study.

Linkage Studies Pairwise linkage analyses for all pairs of markers were performed using the MLINK program (Lathrop et al., 1984). The lod scores were calculated for a set of recombination frequencies that varied from 0 to 0.50 in 0.01 increments. Sex-specific recombination frequencies were determined for all the pairs of the eight markers tested using the ILINK program. Multilocus linkage analyses were performed using the LINKAGE programs (Lathrop et al., 1984), version 4.7, and incorporating the results of the pairwise analyses. To estimate the relative likelihood of the various possible marker orders and to obtain the most accurate map distance, we used ILINK of the LINKAGE program and performed multipoint calculations involving the analysis of three to four loci simultaneously. We started the analysis by using two markers whose position relative to each other was known by physical mapping, and subsequently used a third marker and calculated the difference in log,, likelihood for the three possible orders. Once a significant likelihood order (log,, likelihood difference > 3 relative to the next best order) was favored, we added a fourth marker to the linkage group of the three existing markers and calculated the most likely marker order for this new group. Different permutations and orientations for each subgroup of three and four markers were tested, and the highest relative likelihood compared to the alternative order within each subgroup was determined.

Characterization of the I-7 somatic cell hybrid line by G-11 staining showed that 26 of 28 cells retained 6p as the only identifiable human chromosome; the other two cells did not retain any human chromosomes. We used the I-7 somatic cell hybrid line and a panel of somatic cell hybrids containing defined regions of the short arm of chromosome 6 (Zoghbi et al., 1988b) to determine the physical locations for D6S4, MUT, D6S19, D6S29, PIM, HLA-DP, and F13A. Regional mapping of D6S5, HLA-B, HLA-DQ, and HLA-DR on the short arm of chromosome 6 has been reported by Leach and co-workers (1986). The results of the regional mapping from our study are shown in Fig. 1. D6S4 and MUT map to the most proximal portion of chromosome 6p between the centromere and 6~12 (Region I). D6S5, D6S19, D6S29, and PIM map to the region between 6~12 and 6~21 (Region II). Region III is marked by the HLA-DP locus on the centromeric end and 6~23 at the telomeric end, F13A maps to this region. Region IV extends from 6~23 to the telomere. Figure 2 shows the regional localization of one of the DNA markers, D6S19, using the panel of somatic cell hybrids. Linkage Analysis Pairwise linkage analyses showed significant evidence of linkage between several markers (Table 2). To determine the ratio of the recombination frequency in females versus that in males, we used the ILINK program to compare the estimates for the recombination rates under the models of constant and variable female/male distance ratio as well as under the model of equal recombination in females and males. The female and male genetic maps of the short arm of chro-

Sequence Polymorphisms

For most of the markers used in the linkage analysis, the DNA sequence polymorphisms have been reported previously. These include 4Cll (D6S4 locus, Blanche et al., 1987), MMCM (MUT locus, Zoghbi et al, 1988b), pAGB6 (D6S5 locus, Leach et al., 1986), CRI-L171 (D6S19 locus, Donis-Keller et al., 1987), HHH157 (D6S29 locus, Hoff et aZ., 1988), and pIC19H-12-1 TABLE Pairwise

2

Linkage Recombination

Lod score

D6S4

D6S4 MUT D6S5 D6S19 D6S29 PIM HLA F13A

22.9 0.84 10.27 6.77 1.5 6.68 0.91

u Sex-averaged

recombination.

Data fraction”

MUT

D6S5

D6S19

D6S29

0.01

0.25 0.168

0.15 0.04 0.001

0.16 0.20 0.11 0.05

2.12 8.26 4.2 1.42 3.45 0.58

5.71 1.04 1.42 5.49 0.45

unreclones 1 dein this

Regional Localizations

RESULTS

DNA

355

6p MARKERS

15.79 3.53 19.14 2.1

4.5 28.35 2.05

PIM 0.23 0.14 0.001 0.001 0.001 9.6 0.04

HLA 0.28 0.28 0.15 0.16 0.07 0.06 10.64

F13A 0.39 0.38 0.38 0.32 0.30 0.41 0.29

356

ZOGHBI

mosome 6 are shown in Fig. 3. When the null hypothesis of no sex effect was tested against the hypothesis of a variable female/male distance ratio along the chromosome, the null hypothesis was rejected (~2 = 30.1, P < 0.005). Similarly when the hypothesis of a constant female/male distance ratio was tested against the hypothesis of a variable female/male distance ratio, the constant ratio also was rejected (x; = 1.98,0.01> P > 0.005). The distance ratios estimated under the variable sex-difference model suggested an excess of female over male recombination in the region from D6S4 through D6S29 and an excess of male over female recombination in the region from D6S29 through HLA. Multipoint calculations were performed using ILINK to determine the likelihood of the various possible marker orders. The first set of likelihood calculations involved the use of markers D6S4, D6S19, and D6S29. The chromosomal orientation of D6S4 and D6S29 is known from mapping data using the somatic cell hybrids. The likelihood for the various possible positions of D6S19 relative to D6S4 and D6S29 was subsequently calculated using ILINK. The data provided very strong support for the order D6S4-D6S19-D6S29 versus the order D6S19-D6S4-D6S29 (log,, likelihood difference = 6.7). Due to the extremely close linkage between D6S4 and MUT, the relative order of these two markers could not be obtained. Similarly the close linkage between D6S19 and D6S5, and between D6S29 and PIM, did not permit the determination of the relative order of D6S5 and D6S19 or of D6S29 and PIM. Four-point analyses comparing the orders D6S4-D6S19-D6S29HLA, D6S4-HLA-D6Slg-D6S29, and D6S4-D6S19HLA-D6S29 provided strong support for the first order versus the second order (log,, likelihood difference = 18) and for the first versus the second order (log,, likelihood difference = 9). A log,, likelihood difference of 48 supported the order D6S29-HLA-F13A versus the second best order, D6S29-F13A-HLA. DISCUSSION

We have regionally mapped eight markers from the short arm of chromosome 6 using somatic cell hybrids retaining various fragments of chromosome 6p, and we subsequently analyzed the linkage relationships of these markers using all the CEPH reference families. The primary set of markers evaluated in this study forms a continuous linkage group that spans approximately 48 CM in males and 128 CM in females. Two maps of markers for the short arm of chromosome 6 have been published. The first map was reported by Leach and co-workers (1986). The investigators used data from GLOl, D6S5, D6S7, D6S8, D6S10, and HLAB, -DR-(w, and -D&-a using a subset of the CEPH panel. The second map published by Donis-Keller and col-

ET

AL.

leagues (1987) used the same markers as Leach and co-workers and three additional markers, CRI-L171, CRI-L320, and CRI-R125, again using a subset of the CEPH panel. In this study, we used serology data at the HLA loci in a haplotype form which provides highly informative meioses. We also used one of the markers used by Leach et al. (D6S5) and 4Cll at the D6S4 locus, and we genotyped the whole CEPH panel for D6S19, MUT, D6S29, PIM, and F13A. Newly identified RFLPs at the D6S5 and PIM loci are also reported. The results of regional mapping using human-hamster hybrids, pairwise linkage analysis, and multilocus linkage analysis provide support for a primary map of the five highly informative anchor loci in the following order: cen-D6S4-D6S19-D6S29-HLA-Fl3A-tel. Tight linkage between D6S4 and MUT (2 = 22.93, 0 = 0.01) did not allow the ordering of these two markers. Similarly the order of D6S5 with respect to D6S19 and the order of PIM with respect to D6S29 could not be determined becauseof close linkage between these pairs of markers and because D6S5 and PIM were not informative for many CEPH families. Linkage analyses using no sex difference, a constant sex difference, and a varying sex difference showed a significant difference in the recombination rates in female meiosescompared to male meioses. This finding has been described for several human chromosomes or chromosome regions. We found an increased rate of recombination in female meioses in the region between D6S4 and D6S29, while the rate of recombination in male meioses was increased in the region between D6S29 and the HLA loci. The extremely high ratio of 218 for female/male recombination rate between D6S19 and D6S29 is a biased ratio because there were no recombinants in male meioses in that region. However, taking the five most informative loci simultaneously, the differences between the effect of varying sex difference and no sex difference or between varying and constant sex differences are significant, with P < 0.005 and P < 0.01, respectively. The map by Donis-Keller et al. (1987) revealed an increase in the recombination frequencies in female meioses compared to male meioses; however, a detailed statistical analysis of the data was not presented. Leach and colleagues (1986) reported the sexspecific recombination frequencies based on the imposition of a constant ratio between male and females; using this method of calculation they did not detect significant sex differences in recombination. Ultimately, the goal will be to combine the data for all the markers that have been genotyped from chromosome 6p in order to obtain more accurate estimates of the genetic distances along the short arm of chromosome 6. ACKNOWLEDGMENTS We thank Dr. Lodewyk concerning the methodology

Sandkuyl for his very helpful suggestions of the linkage analysis. We are grateful

EIGHT

CHROMOSOME

to Drs. Carlos Croce, Fred LedIey, Hans Kupper, Yusuki Nakamura, Lydia Villa-Komaroff, and Robert Williamson for providing DNA probes. Drs. D. Pious, T. Mohandas, and Carl-Heinz Grexchik were extremely generous in providing valuable cell lines. We thank Grace Watson for the preparation of this manuscript. This work was supported by Grant Kll HDO6684 from the National Institutes of Health. Christie Ballantyne was supported by a Bugher grant from the American Heart Association. REFERENCES 1. ALHADEFF, B., VELIVASAKIS, M., AND SINASCALCO, M. (1977). Simultaneous identification of chromatid replication and of human chromosomes in metaphases of man-mouse somatic cell hybrids. Cytogenet. Cell Genet. 19: 236-239. 2. BLANCHE, H., ZUNEC, R., GILLAM, C., HARTLEY, D., WILLIAMSON, R., DAUSSET, J., AND CANN, H. M. (1987). A human anonymous low copy number clone, 4Cll (D6S4) localized to 6p126~21, detecta 2 RFLPs, one of which is moderately polymorphic. Nucleic Acids Res. 16: 5902. 3. DAUSSET, J. (1986). Le centre d’etude du polymorphisme humain. La Presse Medicale 13: 1801. 4. DONIS-KELLER, H., GREEN, P., HELMS, C., CARTINHOUR, S., WEIFFENBACH, B., STEPHENS, K., KEITH, T. P., BOWDEN, D. W., SMITH, D. R., LANDER, E. S., BOTSTEIN, D., AKOTS, G., REDIKER, K. S., GRAVIUS, T., BROWN, V. A., RISING, M. B., PARKER, C., POWERS, J. A., WATT, D. E., KAUFFMAN, E. R., BFUCKER, A., PHIPPS, P., MULLER-KAHLE, H., FULTON, T. R., NG, S., SCHUMM, J. W., BRAMAN, J. C., KNOWLTON, R. G., BARKER, D. F., CROOKS, S. M., LINCOLN, S. E., DALY, M. J., AND ABRAHAMSON, J. (1987). A genetic linkage map of the human genome. CeU 61: 319-337. 5. FARRER, L. A., CASTIGLIONE, C. M., KIDD, J. R., MYERS, S., CARSON,N., SIMPSON, N. E., AND KIDD, K. K. (1988). A linkage group of five DNA markers on human chromosome 10. Genomics 3: 72-77. 6. FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Bbchem. 132: 6-13. 7. GLADSTONE, P., FUERESZ,L., AND PIOUS, D. (1982). Gene dosage and gene expression in the HLA region: Evidence from deletion variants. Proc. Natl. Acad. Sci. USA 79: 1235-1239. a. HOFF, M., NAKAMURA, Y., HOLM, T., GILLILAN, S., O’CONNELL, P., LEPPERT, M., LATHROP, G. M., LALOUEL, J.-M., AND WHITE,

357

6p MARKERS

R. (1988). Isolation and mapping of a polymorphic DNA sequence (pHHH157) on chromosome 6p [D6S29]. Nucleic Acids Res. 16: 5217. 9. LATHROP, G. M., LALOUEL, J.-M., JULIER, C., AND OTT, J. (1984). Strategies for multilocus linkage analysis in humans. Proc.

Natl.

Acad.

Sci

USA

81: 3443-3446.

10. LEACH, R., DEMARS, R., HA~STEDT, S., AND WHITE, R. (1986). Construction of a map of the short arm of human chromosome 6. Proc. Natl. Acad. Sci. USA 83: 3909-3913. 11. MOHANDAS, T., SPARKES, R. S., SHULKIN, J. D., SPARKES, M. C., AND MOEDJONO, S. (1980). Assignment of PGM3 to the long arm of human chromosome 6: Studies using Chinese hamster X human hybrids containing a human 6/15 translocation. Cytogenet. Cell Genet. 28: 116-120. 12. NAYLOR, S. L., SAKAGUCHI, A. Y., SHOWS, T. B., GRZESCHIK, S.-H., HOLMES, M., AND ZASLOFF, M. (1983). Two nonahelic tRNAMa genes are located in the p23 + q12 region of human chromosome 6. Proc. Natl. Acad. Sci. USA 80: 5027-5031. 13. O’CONNELL, P., LATHROP, G. M., LAW, M., LEPPERT, M., NAKAMURA, Y., HOFF, M., KUMLIN, E., THOMAS, W., ELSNER, T., BALLARD, L., GOODMAN, P., AZEN, E., SADLER, J. E., CAI, G. Y ., LALOUEL, J.-M., AND WHITE, R. (1987). A primary genetic linkage map for human chromosome 12. Genomics 1: 93-102. 14. SEALEY, P. G., WHITTAKER, P. A., AND SOUTHERN,E. M. (1985). Removal of repeated sequences from hybridization probes. Nucleic Acids Res. 13: 1905-1922. 15. TANZI, R. E., HAINES, J. L., WATKINS, P. C., STEWART, G. D., WALLACE, M. R., HALLEWELL, R., WONG, C., WEXLER, N. S., CONNEALLY, P. M., AND GUSELLA, J. F. (1988). Genetic linkage map of human chromosome 21. Genomics 3: 129-136. 16. ZOGHBI, H. Y., DAIGER, S. R., MCCALL, A., O’BRIEN, W. E., AND BEAUDET, A. L. (1988a). Extensive DNA polymorphism at the factor XIIIa (F13A) locus and linkage to HLA. Amer. J. Hum.

Genet.

42: 877-883.

17. ZOGHBI, H. Y., O’BRIEN, W. E., AND LEDLEY, F. D. (198813). Linkage relationships of the human methylmalonyl CoA mutase to the HLA and D6S4 loci on chromosome 6. Genomics 3: 396398.

18. ZOGHBI, H. Y., SANDKUYL, L. A., OTT, J., DAIGER, S. P., POLLACK, M., O’BRIEN, W. E., AND BEAUDET, A. L. (1989). ASsignment of autosomal dominant spinocerebelhu ataxia (SCAl) centromeric to the HLA region on the short arm of chromosome 6, using multilocus linkage analysis. Amer. J. Hum. Genet. 44: 255-263.