A genetic linkage map of the long arm of human chromosome 22

GENOMICS

4, l-6

(1989)

A Genetic Linkage Map of the Long Arm of Human Chromosome 22 GUY A. ROULEAU,* JONATHAN L. HAINES,* ANNE BAZANOWSKI,* ANNETTE COLELLA-CROWLEY,* JAMES A. TRoFArrER,t NANCY S. WEXLER,* P. MICHAEL CoNNEALLy,t AND JAMES F. GUSELLA* *Neurogenetics Laboratory, Massachusetts General Hospitaal and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02 114; tDepartment of Medical Genetics, Indiana University, Indianapolis, Indiana 46223; and *Department of Neurology, Columbia University, New York, New York 10032, and Hereditary Disease Foundation, 606 Wilshire Boulevard, Suite 504, Santa Monica, California 90401 Received

May

16, 1988;

We have used a recombinant phage library enriched for chromosome 22 sequences to isolate and characterize eight anonymous DNA probes detecting restriction fragment length polymorphisms on this autosome. These were used in conjunction with eight previously reported loci, including the genes BCR, IGLV, and PDGFB, four anonymous DNA markers, and the PI blood group antigen, to construct a linkage map for chromosome 22. The linkage group is surprisingly large, spanning 97 CM on the long arm of the chromosome. There are no large gaps in the map; the largest intermarker interval is 14 CM. Unlike several other chromosomes, little overall difference was observed for sex-specific recombination rates on chromosome 22. The availability of a genetic map will facilitate investigation of chromosome 22 rearrangements in such disorders as cat eye syndrome and DiGeorge syndrome, deletions in acoustic neuroma and meningioma, and translocations in Ewing sarcoma. This defined set of linked markers will also permit testing chromosome 22 for the presence of particular disease genes by family studies and should immediately support more precise mapping and identification of flanking markers for NF2, the defective gene causing bilateral acoustic neuroflbromatosis. 0 1989 Academic PTOSS, IUC.

revisedAugust25,

1988

wright et al., 1985), and von Recklinghausen’s neurofibromatosis (Barker et al., 1987; Seizinger et al., 1987a). As the number of available marker loci has grown, it has become feasible to use multipoint analysis to maximize the available information derived from complex diseasepedigrees (Lathrop et al., 1984). Consequently, the efficiency of mapping genetic defects can be improved dramatically by construction of defined genetic linkage maps of DNA markers on each chromosome. Similarly, the comparison of a genetic linkage map with the physical locations of marker loci relative to chromosomal deletions and translocations can yield new insights into disorders involving specific rearrangements. Detailed linkage maps have already been reported for several chromosomes, and rudimentary maps are available for the remainder. Our recent demonstration of chromosome 22 hemizygosity in acoustic neuroma and meningioma (Seizinger et al., 1986,1987b) and assignment to this chromosome of NF2, the bilateral acoustic neurofibromatosis locus (Rouleau et al., 1987), prompted us to generate new RFLP markers and to construct a detailed linkage map for this autosome. MATERIALS

INTRODUCTION

The use of restriction fragment length polymorphisms (RFLPs) as high-quality genetic markers has provided a powerful tool for mapping human genes (Gusella, 1986). Linkage analysis with individual DNA markers has been successful in localizing numerous defects, including those underlying Huntington’s disease (Gusella et al., 1983), adult polycystic kidney disease (Reeders et al., 1985), cystic fibrosis (Tsui et al., 1985; Knowlton et al., 1985; White et al., 1985; Wain-

AND

METHODS

Hybrid Cell Lines and Chromosome

22 Library

DNA was prepared from two somatic cell hybrids, each containing a single human chromosome on a mouse background. WA17 contains only human chromosome 21 (Kozak et al., 1977), and R-G21-J-46 contains only chromosome 22 (Schuchman et al., 1984). The chromosome 22 library (LL22NSOl) was constructed at the Biomedical Sciences Division, Lawrence Livermore National Laboratory (Livermore, CA) under the auspices of the National Laboratory Gene Library Project, which is sponsored by the U.S. Department of oess-7543/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

ROULEAU

Energy. This library contains DNA obtained by flow sorting of chromosomes from a human diploid skin fibroblast line, digested to completion with HindIII and cloned into the bacteriophage vector Charon 21A. Probe Preparation

Phage DNA was prepared using the plate lysate miniprep procedure (Maniatis et al., 1982). Inserts were released from the phage DNA by HindIII digestion and excised from low-melt agarose gels (Seaplaque, FMC Bioproducts) for labeling with 32P by the method of Feinberg and Vogelstein (1983). Physically Mapped Chromosome 22 Loci

PDGFB, the platelet-derived growth factor o-chain locus (homologous to the SIS oncogene), maps to 22q12.3 + q13.1 (Julier et al., 1988; Jhanwar et al., 1984). The BCR (breakpoint cluster region) locus represents the 22qll breakpoint of the Philadelphia chromosome in chronic myelogenous leukemia (Groffen et al., 1984). IGLV, the variable region of the X light chain immunoglobulin locus, also maps to 22qll (Willard et al., 1985; Julier et al., 1988). The locus D22Sl displays a two-allele BglII polymorphism with the probe pMS318 and maps to 22qll--* qter (Barker et al., 19&Q; Julier et al., 1988). D22S9, an anonymous DNA locus detected by probe ~22134, maps to 22qll (McDermid et al., 1986; Willard et al., 1985). Both D22515 and D22SlO are anonymous DNA markers assigned to chromosome 22, but not regionally localized (Rouleau et al., 1988; Hofker et al., 1985). Southern Transfer and Hybridization

Endonuclease digestions were performed as suggested by the supplier (Boehringer Mannheim or New England Bioiabs). Transfer of phage plaques to nitrocellulose filters and hybridization to identify plaques free of human repeat sequences were carried out as previously described (Gusella et al., 1980). Southern blotting and hybridization were carried as described (Gusella et al., 1983). Venezuela Reference Pedigree

The Venezuela Reference Pedigree was established from a large kindred in which the Huntington’s disease gene is segregating (Gusella et al., 1983). The reference pedigree contains over 250 potential meiotic events, and permanent lymphoblastoid cell lines have been established for each member as a source of DNA for RFLP typing. Twenty phenotypic markers, including Pl, and over 150 DNA markers have been typed in this pedigree.

ET AL.

Linkage Analysis

Two-point linkage analysis was performed using the computer program LIPED (version 3) (Ott, 1976). Multipoint analyses were accomplished by using the ILINK and LINKMAP (version 3.5) programs of the LINKAGE package (Lathrop et al., 1984). In most cases, confirmation of order was obtained with either three- or four-point analysis. Odds of >lOOO:l (lod difference of >3) were used as a basis for accepting one order over another. Maximum likelihood estimates of 0 were obtained from five-point analyses. Either a DEC 2060 or a VAX 8800 computer was employed for calculations. RESULTS Isolation of Phage Containing Chromosome 22 DNA

We randomly picked 384 phage clones from the sorted chromosome 22 library into gridded arrays and prepared nitrocellulose replica filters. Clones containing highly repeated sequences were identified by hybridization of the filters with 32P-labeled total human DNA. Of 199 plaques which showed no hybridization, 154 were further analyzed by digestion of phage DNA with Hind111 followed by agarose gel elctrophoresis. Seventy-nine inserts larger than 600 bp were identified and excised from the gels for use as DNA probes. The remainder of the clones contained either no inserts or inserts smaller than 600 bp that could not be visualized clearly under the gel conditions used. To confirm the chromosome 22 location of individual clones and to eliminate those containing infrequent repeat sequences, each labeled insert was hybridized to a Southern blot containing four DNAs: human genomic DNA derived from a normal lymphoblastoid cell line; DNAs from the hybrid cells WA17 and R-GBl-J46, containing human chromosomes 21 and 22, respectively, on a mouse background; and DNA from the mouse cell line RAG. We chose to include a chromosome 21 hybrid cell line since the similar sizes of chromosomes 21 and 22 make the former a likely contaminant in the flow-sorting procedure. Forty inserts (51%) contained some repeat sequences, while 39 (49%) were single copy. Fifteen single-copy inserts mapped to chromosome 22,4 mapped to chromosome 21, and 20 mapped to neither chromosome, based on this restricted mapping panel. Screening for DNA Polymorphisms

To detect RFLPs, each probe was hybridized to genomic DNA from five unrelated individuals: each DNA was digested with 35 different restriction enzymes. Of the 15 single-copy chromosome 22 probes screened, variants were identified for 10 probes, and for 8 of these further characterization was performed to establish

CHROMOSOME

that the individual differences represented true RFLPs (Table 1). Mendelian transmission of each RFLP was confirmed by analyzing the segregation of the alleles in several nuclear families. The polymorphism information content (PIG; Botstein et aI., 1980) was determined by analyzing DNA, digested with the appropriate restriction enzyme, from 34 unrelated individuals of Northern European ancestry. The results are shown in Table 1 along with the locus names corresponding to each probe. Two probes, WllOD and W13E, revealed insertion/ deletion type polymorphisms which could be detected with several different enzymes. Five probes detected multiple polymorphisms which significantly increased the PIC of the corresponding loci, since there was no strong linkage disequilibrium between most sites. Genetic Map

We typed eight of our RFLP markers and eight previously described loci in the Venezuela Reference Pedigree to construct a linkage map of chromosome 22. Included in the analysis were phenotypic data previously generated for the Pl blood antigen. Pairwise lod scores for linkage of the chromosome 22 loci are presented in Table 2.

Description LOCUS symbol

Probe name

of New RFLP

Insert size”

Enzyme

3

22 MAP

To establish order of the loci on chromosome 22 and to optimize recombination frequencies, multipoint linkage analysis using three to six markers was performed with LINKMAP and ILINK of the LINKAGE package. Figure 1 summarizes our findings. The odds in favor of the relative order of adjacent loci are at least 1000~1, except for the following pairs of the very tightly linked markers: D22S9/D22S24, D22Sl/ D22S15, D22S28/D22S29, D22S27/Pl, and D22S21/ D22S23. The most likely order for these tightly linked loci, based on only a limited number of observed recombinations, is also shown in Fig. 1. Sex-specific linkage maps are also presented in Fig. 1. There was little difference in the overall map length from male and female meioses. Although the sex-specific recombination fractions do appear to vary along the chromosome, none of the differences achieve statistical significance. We tested this first by splitting the chromosomal map into three sections and allowing each section to have its own female/male recombination fraction ratio, and then by letting the ratio for each intermarker interval vary independently. The results are presented in Table 3. It may seem surprising that the sex-averaged map is slightly shorter (3 CM) than either sex-specific map. However, this is due primarily to the male-specific estimate of 9 CM between

TABLE

1

Markers

for Human Chromosome Invariant fragments’

22

Allelic fragments“ (frequency)

PIG

D22S21

W13E

3.6

TM

1.05

2.8 (88%), 1.5 (82%),

1.6 (12%) 1.35 (18%)

0.18 0.25

D22S22

WlloD

1.9

TM

2.2

1.9 (36%),

1.8 (64%)

0.35

D22S23

W24F

2.1

sad Tag1 EcoRV

D22S24

W21G

3.6

TM MspI

D22S27

W26D

3.1

D22S28

W23C

D22S29

W22D

D22S30 ’ All sizes are given

W17E in kb.

10.5 (28%), 8.4 (72%) 3.6 (12%), 2.1 + 1.5 (88%) 6.8 (65%), 5.4 (35%) Haplotype

0.31 0.18 0.35 0.70

7.0 (66%), 6.6 (78%). Haplotype

5.9 (34%) 2.8 (22%)

0.35 0.37 0.57

HindIII

8.0 (15%),

3.0 (85%)

0.22

3.0

BglI TM Sac1

8.0 (39%, 6.6 (61%) 5.4 (94%), 3.7 (6%) 5.2 (l%), 4.9 (99%) Haplotype

0.36 0.10 0.02 0.42

10.0

TaqI

4.0, 2.6

5.1 (45%), 4.3 + 0.8 (55%) 6.4 (l%), 2.6 (99%)

0.37 0.02

BclI

4.2

4.6 (34%), Haplotype

3.0 + 1.6 (66%)

0.34 0.44

6.8 (90%),

6.6 (10%)

0.15

2.45

sad

6.6

4

ROULEAU

ET

TABLE Peak Probe

022824

D22S9

IGLV

D22SlO

Two-Point

BCR

D22Sl

Thetas 022915

AL.

2 and Lod D22S28

Scores D22S29

PDGFB

PI

022822

D22S23

0.38 0.50 0.38 0.40 0.19 0.30 0.50 0.20 0.25 0.16 0.13

0.38 0.50 0.50 0.37 0.50 0.50 0.36 0.50 0.35 0.32 0.22 0.15

D22S21

Theta D22S24 D22S9 ZGLV D22SlO BCR D22Sl D22S1.5 D22S28 D22S29 PDGFB PI D22S22 D22S23 D22S21

0.02 8.34 14.56 8.71 8.21 4.09 0.65 0.92 2.71 0.00 0.40 0.28 0.50 0.82

0.10 0.13

2.52 0.02 2.01 0.76 0.00 0.00 0.04 1.20 0.00 0.00 0.00 0.00

15.36 10.20 5.27 1.75 5.09 3.82 1.51 2.05 0.16 0.00 0.34

0.17 0.39 0.03

0.13 0.16 0.08 0.07

10.77 5.33 6.48 3.22 5.29 0.14 1.49 0.08 0.42 0.29

5.95 2.80 6.43 2.44 4.19 0.00 2.43 0.00 0.62

0.21 0.25 0.12 0.12 0.08 2.15 9.21 8.85 0.60 3.16 0.34 0.00 0.00

0.33 0.40 0.19 0.15 0.14 0.00 6.52 8.24 3.30 0.95 0.00 0.31 0.16

0.34 0.50 0.19 0.19 0.15 0.05 0.04 23.59 4.05 3.71 3.61 0.00 0.33

0.30 0.36 0.23 0.21 0.18 0.07 0.09 0.01 4.88 6.14 1.50 0.96 0.55

0.50 0.20 0.26 0.33 0.14 0.21 0.11 0.09 0.14 0.14 2.66 0.70 2.70

0.35 0.50 0.20 0.25 0.50 0.15 0.15 0.14 0.11 0.36 3.40 1.17 1.05

4.58 10.10

0.36 0.50 0.38 0.40 0.35 0.50 0.40 0.35 0.38 0.21 0.25 0.09 0.01

27.56

Lod score

IGLV and D22SlO. This figure is based on very few informative events, has weak support, and consequently carries little weight when used to estimate the sex-averaged map distance. In fact, the sex-averaged estimate of 4 CM for this interval is identical to the corresponding female-specific estimate. If this were used instead of the male-specific estimate, the male map would be slightly shorter than the sex-averaged map, more in line with the maps of other chromosomes. The markers D22527 and D22S30 could not be placed precisely on the map because they were relatively uninformative in the Venezuelan Reference Pedigree. However, D22S27 was linked to PDGFB with a maximum lod score (23 = 2.02 at 8 = 0.07, and D22S30 was linked to Pl with a maximum lod score TABLE Test of Significance Recombination

(3 = 5.38 at 8 = 0.0. D22S30 could be excluded from a position distal to D22S22 by odds of greater than 1OOO:land from a position proximal to D22S29 by odds of 3Oo:l. DISCUSSION We have constructed a genetic map of the long arm of chromosome 22 using 16 polymorphic loci with a

3 for Sex-Specific Estimates

12 1 12.2 12.3 13.1

Test’

-2 In LDb

dfc

P

A vs B B vs C A vs C

3.68 8.81 12.49

3 7 10

>0.25 z-0.25 >0.25

’ The likelihood ratio may be used to test for significant differences by comparing maps constructed with various assumptions concerning sex-specific recombination. The -2 In (likelihood difference) between two maps approximates a x2 distribution with the degrees of freedom equal to the number of parameters estimated. The three maps being tested are A, sex-averaged map (male recombination = female recombination); B, female/male ratio varied among three regions, but constant within a region; and C, female/male ratio varied independently within each intermarker interval. b -2 In (likelihood difference). ’ Degrees of freedom.

13.2

FIG. 1. Human chromosome 22 genetic map. The sex-averaged genetic map (male recombination = female recombination) is shown, flanked by maps constructed on the basis of only male or female meioses, respectively. Marker order was determined by joint analysis of three to six loci as described in the text. Optimal estimates of recombination frequencies were then obtained by multipoint analysis of five to six loci using ILINK. The maps are in centiMorgans (using Haldane’s mapping function to convert recombination frequency). The bracketed regions indicate the physical regions to which particular loci have been assigned.

CHROMOSOME

total of 29 RFLPs. The average distance between markers is 8.1 CM and the maximum distance between any two loci is 14 CM, providing a level of resolution which should permit efficient exclusion (or inclusion) of disease loci from chromosome 22 by family studies. This work also provides a characterization of one of the National Laboratory Gene Library Project chromosome 22 libraries (LL22NSOl). Of the single-copy sequences derived from this library, 38% mapped to chromosome 22 and slightly over one-half of these detected useful RFLPs; 10% of the sequences from the library mapped to chromosome 21, which has a similar size. The remaining 52% of the single-copy sequences presumably map to other regions of the genome and are probably derived from chromosomes that fractured during some aspect of the sorting procedure, therefore contaminating the chromosome 22 fraction. The eight new anonymous polymorphic loci that did map to chromosome 22 display a total of 16 RFLPs. Thus, by making use of a combination of the flow-sorted DNA library and specific somatic cell hybrids to focus effort on chromosome 22, we have doubled the number of polymorphic markers available for linkage studies on this autosome. Chromosome 22 contains about 1.9% of the total haploid genome, or about 57 X lo6 basesof DNA (Korenberg and Engels, 1978). On the basis of this physical length, we anticipate a corresponding genetic map of approximately 60cM, but the observed map length of 97 CM already exceeds this estimate by 60%. The short arm of the chromosome, containing primarily ribosomal genes, is not included in the linkage map. The most distal marker in the linkage group, D22S21, does not have a firmly established physical position relative to the telomere. Consequently, additional genetic distance may be added to the map as more distal probes are found, especially in view of the dramatic increase in recombination observed toward the telomere of chromosome 21, a similarly sized acrocentric (Tanzi et al., 1988). Consequently, the data to date suggest that chromosome 22 undergoes more recombination than average, based only on its physical size. We have also observed this phenomenon on chromosome 21, a physically similar chromosome. The results of our linkage map are consistent with previously published but less detailed maps of chromosome 22. Julier et al. (1988) described a map of chromosome 22 with five polymorphic markers spanning 49 CM. These authors found a distance of 49 CM between IGLV and PDGFB, versus our distance of 48 CM for the same two markers. The chromosome 22 map described by Donis-Keller et al. (1987) also has five markers, spanning 71 CM. In this case, a distance of 37 CM between D22SlO and PDGFB compares to a separation of 43 CM observed in the present study. It has become increasingly clear that genetic linkage

22 MAP

5

maps of all the chromosomes will be used extensively to map both normal and disease genes. The data set accumulated for chromosome 22 markers in the Venezuela Reference Pedigree facilitates the mapping of any new loci on this autosome by providing a defined collection of defined crossovers. In the case of chromosome 22, the genetic map will also be valuable for delineating the extent of deletions seen in acoustic neuromas and meningiomas (Seizinger et al., 1986, 1987b), for determining the detailed nature of chromosome 22 rearrangements in cat eye syndrome (McDermid et al., 1986) and DiGeorge syndrome (de la Chapelle et al., 1981), and for defining the precise breakpoints of the Ewing sarcoma translocation (Aurias et al., 1983) and the frequent constitutional t(11;22) (Fraccaro et ab, 1980). To maximize the utility of RFLP markers in each of these strategies, a 1-CM resolution map would clearly be desirable. Since too few markers are currently available to achieve this goal, we are continuing to identify new markers on chromosome 22 in a effort to provide such a detailed genetic map. ACKNOWLEDGMENTS This work was supported by NINCDS Grants NS16367, NS29012, NS24279, and NS22031 and by grants from the McKnight Foundation, the Julieanne Dorn Fund for Neurological Research, and the Hereditary Disease Foundation. G.A.R. is supported by the Medical Research Council of Canada and the Fonds de Recherches en Sante du Quebec. J.F.G. is a Searle Scholar of the Chicago Community Trust. We thank all members of the Venezuela Collaborative HD Project for their help in collection of blood samples; Drs. R. White, B. White, J.-C. Kaplan, P. L. Pearson, and D. Kumit for providing probes, Dr. R. Desnick for supplying the R-G21-J-46 cell line; and Dr. G. Darlington for supplying the WA17 hybrid. Computing facilities were provided by ICPUI Computing Services.

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