GENOMICS

9, :?%-:i:;7

i I!%1

1

Fanconi Anemia:

Evidence for Linkage Heterogeneity on Chromosome 2Oq

R. MANN,* V. 5. VENKATRAJ,” R. G. ALLEN,* QIAN LIU,* DREW A. OLSEN,* BARBARA ADLER-BRECHER,* JEN-I MAO, t BARBARA WEIFFENBACH, t STEPHANIE L. SHERMAN,+ AND ARLEEN D. AUERBACH*,~

WILLIAM

*Laboratory for investigative Dermatology, The Rockefeller University, 1230 York Avenue, New York, New York 10021; tCollaborative Research Inc., Two Oak Park Road, Bedford, Massachusetts 01730; and *Division of Medical Genetics, Department of Pediatrics, Emory University School of Medicine, 2040 Ridgewood Drive, NE, Atlanta, Georgia 30322 Received

July

23,

1990;

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Faneoni Anemia Registry. A significant lod score was obtained between DZOSZO, an anonymous DNA segment from chromosome 2Oq, and Fanconi anemia (Z,,, = 3.04,0,.= = 0.12). However, six other anonymous DNA segments from chromosome 2Oq, including D20S19. which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (x2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at 0 = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q. (’ 199 I .4cademic

Pwss. Inc.

INTRODUCTION

Fanconi anemia (FA) is a rare autosomal recessive disease affecting all races in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies (Swift, 1971; Auerbach and Allen, 1991). Clinically, FA is characterized by progressive pancytopenia and variable phenotypic ahnormalities, including short stature, hyperpigmentation, and radial aplasia (Fanconi, 1967; Schroeder et al., 1976; Alter, 1987). Because the diagnosis of FA on the basis of clinical manifestations alone can often be unreliable (Glanz and Fraser, 1982; Auerbach et al., 1989), an FA carrier frequency estimate of 1300 (Swift, 1971) may be significantly lower than the true frequency. ’ To whom

r~)rrespondence

should

he addressed.

revised

October

5. 1990

Although the molecular basis for FA is unknown, a number of cellular defects have been recognized in cultured FA cells. In addit,ion to an abnormally high frequency of spontaneous chromosomal breakage (Schroeder et al., 1964), these cells are highly sensitive to the clastogenic effect of hifunctional and polyfunctional alkylating agents (Schuler et al., 1969; Sasaki and Tonomura, 1973) and to oxidative stress (Joenje and Oostra, 1983). Cultured FA cells also exhibit a highly specific endogenous cell cycle defect: a G2 phase of about twice the normal length (Dutrillaux et al., 1982; Hoehn et al., 1989). This defect is oxygen sensitive in FA fihrohlasts and can he alleviated by exposure of these cells to low oxygen levels (Hoehn et al., 1989). Nevertheless, no consistent enzyme defect has been identified. At least. two FA complementation groups have been described by Buchwald and co-workers in somatic ceil hybrid studies (Duckworth-Rysiecki et al., 1985). They correspond to phenotypically different classes of cells exhibiting different rates of recovery of semiconservat,ive DNA synthesis after treatment with DNA crosslinking agents in culture (Moustacchi et al., 1987) and different rates of removal of DNA crosslinks as shown by electron microscopy (Rousset et al., 1990). However, these studies have not yet provided a reliable method for determining the complementation group of a given patient, nor is there any apparent correlation het.ween clinical phenotype and genetic class. Hypersensitivity of cultured FA cells to the DNA crosslinking agent diepoxyhutane (DEB) forms the basis of a diagnostic test (Auerhach and Wolman, 1976; Auerhach et al.. 1981,1985,1986, 1989) used in this study to determine the affectation status in FA sibships. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry (IFAR) maintained at The Rockefeller University. The data support the provisional assignment of a gene for FA to chromosome 20q.

330

MANN

MATERIALS

AND

ET

METHODS

Genetic Marker Studies

Patient Identification Diagnosis of FA was based on the presence of clinical manifestations of the disease in the proband (aplastic anemia and/or physical stigmata of FA) and on the unique hypersensitivity of cultured FA cells to the clastogenic effect of DEB. The utility of the DEB test for FA diagnosis has been established in discriminant analysis and segregation analysis studies. Significant clinical differences between DEB-positive and DEB-negative individuals were observed in the former study among 310 individuals enrolled in the IFAR (Auerbach et al., 1989). The latter study demonstrated that whereas DEB-positive IFAR sibships can be considered homogeneous with respect to the mode of inheritance (monogenic autosomal recessive), reports of FA in the literature diagnosed on the basis of clinical findings alone contain an excess of sporadic cases (Rogatko and Auerbach, 1988). The DEB test can be performed both prenatally and postnatally (Auerbach and Wolman, 1976; Auerbach et al., 1981, 1985, 1986, 1989). DEB hypersensitivity is now an accepted criterion in the diagnosis of FA. Thirty-four families informative for linkage analysis were identified through the International Fanconi Anemia Registry (pedigree structures are available upon request). Twelve of the families were consanguineous with one or more affected siblings, 20 nonconsanguineous families were multiplex, and 2 had irregular structures with affected children appearing in collateral or more distant sibships. Table 1 summarizes the number of affected individuals in the consanguineous and nonconsanguineous multiplex families. A total of 68 affected individuals were employed ir. the study, including those from families with irregular structures. Also included in the study were the parents (total number = 62), unaffected siblings (t.otal number = 55), and, when informative, the grandparents and great grandparents (total numbers = 12 and 2, respectively). DNA was obtained from a total of 199 individuals for this study.

TABLE

1

Summary of the Number of Affected 32 of 34 Fanconi Anemia Kindreds Number Families No consanguinity Consanguineous 1st cousin 2nd cousin 3rd cousin -

AL.

Individuals Examined of affected

in

offspring

1

2

:i

0

12

8

4 4 0

1 1 1

1 0 0

parents

DNA was isolated from established lymphoblastoid cell lines, venous blood samples, or cultured fibroblasts either by the “salting out” method of Miller et al. (1988) or according to standard protocols requiring phenol-chloroform extraction (Maniatis et al., 1982). Restriction enzyme digestion was performed following manufacturer’s directions. Gel electrophoresis, transfer to Immobilon-N membranes, random priming, hybridization, stringency washing, and autoradiography were performed as described previously (Mann et al., 1989). In the case of DNA probe IP20K061 [D20S25], the final stringency wash was at 60°C for 20 min in 0.2~ SSC, 0.1% (w/v) SDS. Paternity was confirmed by hybridizing Southern blots with variable number tandem repeat (VNTR) DNA probes. Additionally, random resampling and retyp ing of individuals were performed to ensure genotypic dependability. Data Analysis Two-point linkage analysis was performed using the LIPED program (version 3.0) (Ott, 1974) and the MLINK program (version 4.9) of the LINKAGE package (Lathrop et al., 1984). Multipoint linkage analysis employed the LINKMAP program (version 4.9) of the LINKAGE package (Lathrop et al., 1984). The admixture test for linkage heterogeneity (Eq. [5.20], p. 116; Ott, 1985) was performed using the HOMOG program, kindly provided by Jurg Ott. This was the preferred test because at present there is no reliable method for predividing families into either complementation or clinical groups. For this test, the null hypothesis states that all families are linked to a marker at a particular recombination fraction, i.e., linkage homogeneity. The alternative hypothesis states that some proportion of families ((u) are linked to a marker at a particular recombination fraction and the remainder (1-a) are not linked. Genetic distances on chromosome 20q were based on published (Nakamura et al., 1989; Leppert et al., 1989) and unpublished CEPH data (multipoint analysis kindly supplied by Mark Leppert). No significant differences between male and female recombination frequencies were observed for chromosome 20q (data not shown). Therefore, a sex-averaged genetic map (Fig. 1) was used for all genetic linkage computations on chromosome 2Oq. RESULTS

Pairwise recombination estimates and lod scores calculated between FA and each of the chromosome 20q loci studied (see Table 2) are summarized in Table 3. Marker D20S20 (PIC = 0.34), the first chromo-

FANCONI

ANEMIA:

D20S16 D20S17

ten

LINKAGE

D20S4

HETEROGENEITY

DZOS25

ON

D20S15

331

2Oq

D20S20

D20S19

------014

FIG. 1. Genetic map of chromosome provided by Dr. Mark Leppert).

2Oq, in centimorgans,

0.07

0.19

based on published

some 20q marker examined, gave a significant lod score with FA (Z,,, = 3.04, B,,, = 0.12). There was also a suggestion of linkage between FA and markers D20S4 and D20825 (Z,,, = 1.52, ernBx= 0.21 and Z,,, = 1.71, emsx = 0.19, respectively). Markers D20S17, D20S16, D20S4, D20S25, D20S15, D20S20, and D20S19 excluded linkage (lads < -2.0) at recombination fractions of 0.05,0.20,0.05,0.001,0.15,0.001, and 0.15, respectively. Additionally, the intervals between the seven chromosome 20q markers were excluded for linkage to FA by multipoint linkage analysis (highest lod scores ranged from -3.97 to ~63.24). Thus, paradoxically, two markers (D2OS20 and D20S19) known t,o be tightly linked to each other both in the CEPH families (2 cM, Fig. 1) and the FA kindreds (Iod = 10.38, B = 0.02), produced contradictory result,s when analyzed for linkage with FA. The demonstration by others of at least, two phenotypically different classes of cultured cells from FA patients (Moustacchi et al., 1987; Rousset et al., 1990) and of complementation in FA somatic cell hybrids (Duckworth-.Rysiecki et al., 1985) led us to consider the hypothesis that linkage heterogeneity was responsible for the contradictory results obtained with these t,wo tightly linked markers. Therefore, the admixture t,est for linkage heterogeneity was performed using D20S19 and D20S20 (Tables 4 and 5). The hypotheses of homogeneity and heterogeneity are denoted by H, and H,, respectively. Significant evidence for linkage heterogeneit,y (H, vs H,, Table 5) was obtained with marker D20S19 (P = 0.01) but was only suggestive for D20S20 (P = 0.15). This may be because D20S20 is less informative than D20S19. When multipoint lod scores were obtained for markers D20S19 and D20S20, allowing 2% recombination between these markers (Fig. I), significant heterogeneity was found (P = 0.03). These data suggest that the FA gene is linked to this region in about 10% of the families studied. Two of the largest families, kindreds 1 and 2, gave the highest posterior probabilities for linkage both between FA and the D20S19 locus (0.98 and 0.93, respectively) and between FA and the D20S20 locus (0.93 and 0.95, respectively). The structures of kindreds 1 and 2 are shown in Fig. 2, with the alleles for each of the seven chromosome 20q markers haplotyped based on the minimum number of recombinants in the offspring. Markers D20S19 and D20S20 identified 44 and 21 informative

0.07

data

0.02

(34, 26) and CEPH

data (multipoint

analysis

kindly

meioses, respectively, in these kindreds. Two-point recombination estimates and lod scores between FA and the seven chromosome 20q markers for these two large kindreds are shown in Table 6. At a recombination distance H = 0.001, lod scores for kindred 1 were 2.07, 1.07, and 2.62 with markers D20Sl5, D2OS20, and D20S19, respectively. The corresponding lod scores for kindred 2 were ~0.62, 1.17, and 2.08. If it is assumed that the prior probability of the FA locus being linked to chromosome 20q is 10% in each family studied, six families with posterior probabilities > 10% can be identified in our data set (pp. 105119, Ott, 1985). Multipoint analysis was performed on these six kindreds to refine t.he estimate of the location of the FA locus (Fig. 3). Although t.his analysis is preliminary, it suggests t.hat the most likely position of the FA gene is in the region defined by markers D20S19 and D20S20. As expected, multipoint analysis with the remaining 28 families gave relative location scores more negative than those when all 34 families were examined together. Two-point lod scores for DNA markers on other chromosomes are presented in the Appendix. Collectively, these data exclude approximately 40% of the total genetic map of 3300 CM for linkage to FA. The demonstration of linkage heterogeneity presented here permits us to reevaluate several loci to which linkage of FA could not be ruled out when all of the families were included in the analysis. Studies excluding the six pedigrees that support the assignment of a FA gene to chromosome ZOq are in progress.

TABLE Chromosome 20q Names, Restriction

Marker Map Endonucleases,

Map Locus D”OS1.i D20S16 D”OS4 D2OS2.5 D%OS15

position “oy 2Oq "oql3."

2oq1:3.:3 2oq

D2OS20 ‘Oq D”OS19

20q

2

Probe CRI-Llt’7 CRI-I,1214 pMSl-27 IP’LOKO61 CRI-L:355 pRMR6 pCMM6

a Probe recognizes DNA sequence able number of tandem repeats.

Positions, Probe and PIC Values

Restriction endonuclease

PIG

Ref.

0.3 1 VNTR” 0.36 0.37 0.54

(IS) (15) (9) (41) ( 1 :, )

IbISPI HglII Mspl MpsI HglIl.Ecr1R1 TayI 7’aqI

0.34

(32)

VNTR”

t:U)

polymorphism

hased on vari-

332

MANN

ET

AL.

TABLE Two-Point

Lod

Scores

for Fanconi

Assumed

value

Anemia

of recombination

Locus

0.001

0.05

0.10

0. 15

DWS17 DSOS16 D2OS4 D2OS25 D2OS15 D20S20 D20SlS

-20.048 -99.758 ~31.636 -12.811 -61.888 m-6.709 -83.589

~ :3.089 -24.623 -3.964 -0.413 -12.971 2.348 ~18.807

--0.615 -13.484

0.291 -7.87”

-0.327 1.242 5.573

1.054 -2.227

3.014 -8.770

2.960 -4.009

1.712

DISCUSSION

The enrollment in the IFAR of a sufliciently large number of families informative for genetic linkage analysis enabled us to embark on this study as an alternative experimental approach to ongoing complementation and gene transfer studies in other laboratories (Duckworth-Rysiecki et al., 1985; DiatloffZito et al., 1986; Shaham et al., 1987; Timme and Moses, 1989; Zdzienicka et al., 1989). The feasibility of this approach was supported by the successful mapping to chromosome llq22-23 of several complementation groups in ataxia-telangiectasia, another autosomal recessive syndrome that exhibits mutagen hypersensitivity and chromosomal instability in cultured cells (Gatti et al., 1988). After excluding approximately 40% of the genome for linkage to FA, we obtained a significant lod score with the chromosome 20q marker D20S20 in the 34 FA kindreds examined. Six other chromosome 20q markers revealed only weak or no evidence for linkage in these families by two-point linkage analysis, and the intervals between all chromosome 20q markers examined were excluded for linkage to FA by multipoint analysis. The admixture test for linkage heterogeneity revealed significant, evidence for heterogeneTABLE Components

Hypothesis H,: Heterogeneity

H,: Homogeneity (linked) H,: Homogeneity (not linked)

D”OS19 DBOS20 D20SlS-D2OS20 D2OS19 D2OS20 D20S19~DXOSZO D20SlS D20S20 D20SlS-D20S20

with

Chromosome

fraction

20q Markers

H

0.20

0.30

0.595

0.40

0.5uo

--4.547 1.545

0.174

-1.183 1.387

1.709

-0.0:16 0.685 0.428

1.141

-0.s57 2.632 1.447

(InmA

u..so:~

0.3 10

1.663 o.sss

0.660 0.566

Lx

0.22

0.82

11.45 (I.21 0.19

0.14 I..55

0.:12 0.12

1.71 0.50 :3.04

0.37

0.68

ity in the region defined by markers D2OS19 and D20S20. It was estimated that the FA locus was linked to this region in approximately 10% of the families studied. It is noteworthy that lymphoblastoid cell lines established from two affected individuals in one of the larger kindreds (Fig. 2, KIN 2) have previously been shown to belong to FA complementation group A (Buchwald et al., 1989). We considered three possible explanations for the contradictory lod scores obtained with markers D20S19 and D20S20. The most likely explanation is that they are the result of a combination of linkage heterogeneity and the nature of the pedigree set available for study. The less polymorphic marker D2OS20 was informative in the two large kindreds shown in Fig. 2 but not informative for a proportion of other families, including many of those with consanguineous structures. Thus the information from these two families contributed a large proportion of the overall lod score. This was not true for the highly polymorphic marker D20S19, which was informative in almost every family. Although we did not obtain evidence with this marker for linkage to FA in the whole pedigree set, the large number of informative meioses detected with this marker enabled us to demonstrate linkage heterogeneity. We regard the possibilities of

4

of Test for Linkage Fanconi Anemia LOCUS

3

Heterogeneity

TABLE

in Test for Linkage

Max

(Y

/I

1 .a9

0.10

0.00

3.46

Ohi

0.00

LOD

2.02

0.10

0.00

0.57

I

0.40

3.01 1 .o?J 0 0 0

1 1 U 0 0

0.10 0.30 0.50 0.50 0.50

H, vs H,

H, vs H I,

H, vs H,

5

Heterogeneity

D”OSl9 D”OS20 D”OSlS~D’WS”0 -I _LI D’LOS19 D20S20 1>20s19~1~20s20 D2OSlS D2OSZO D”OS19~D2OS’O

in Fanconi

1 1 1

1 1 1 i> ‘1 2

ti.10 2.04 4.6” 2.61

1 X88 -1.76 x.51 15.92 !I.28

Anemia

FANCONI

ANEMIA:

LINKAGE

HETEROGENEITY

ON

20q

333

Kin 1

D20Sl6

00 3

2--

00

D20S4

2

2

2

2

D20S25

2

2

2

2

D20Sl5

0

0

0

0

2

2

I

y---r--i,g

D20S17

II

III

14

!l

D20S20

~1 12

12

2

1

3

4

3

5

6

f

/

7

2

6

9

10

ii

12

13

1 r 2

4

2

2

2

2

2

2

2

2

2

2

1

Kin 2 12

----@I3

,~!, , , IIII II 2

2

4

3

12 12

II

3

2

2

1

3

o*ahv3

1

2

12

FIG. 2. descending recombinants

4

3

4

5

12

6

7

1

2

2

3

2

3

1

4

2

1

2

2

2

11

11

14

1

4

2

12

2

2

12

41

21

6

9

10

11

12

Structures of two large kindreds included in the study. order: D20S17, D20S16, D20S4, D20S25, D20S15, D20S20, in the offspring and is represented hy shading. Genotype

4

41

Individuals’ genotypes for chromosome 20q markers are shown and D20Sl9. Haplotype analysis is based on the minimum number of 0 indicates that genotype was not determined.

in ot

334

MANN

ET

AL

TABLE Two-Point

Lod

Scores

for Fanconi

Anemia

with

6 chromosome

Assumed Locus

Kindred

D20S17

1 2 1 2 1 2 1 2 1 9 1 2 1 2

D%OSl6 D%O% D20S25 D”OSl5 D20S20 D20Sl9

0.001

0.05

1.771

0.10

1.537 -1.183

-4.2134 -0.028 4.59, -1 -3.042 0.672 0.997 0."7')I &. 2.070 -0.624 1.067 I.173 2.620 2.075

1.301

0.548 0.458 0.837 0.218

0.247 1.835 0.888 0.970 1.064 2.372 1.897

1.701

-10

10

-----

30 -i

50 ---,

70 ~.

~.

fraction

0 0.:10

0.841 -0.313

0.424 ~4.120 0.808 -0.086 0.595

1.128 -0.261 0.709 0,'1'~‘~ < u1 0.658 0.150

1.850 1.488

1 and 2

0.20

1.250

1.448 0.947 0. 7 7 5 0.82"

0.872 0.946 2.11:3

in Kindreds

-0.425 0.683 0.39% 0.750

0.186

1.59” 0.986

Genetic Distance (CM) -30

1.068 -0.477

1.319 -0.693

1.263

-1.211 0.156 0.519 0.920

of recombination 0.15

-0.730

misdiagnosis and/or inaccurate genotyping unlikely explanations for these results. Diagnosis of FA was based on accepted clinical criteria and on the highly specific DEB test. Genotypic dependability was maximized by random resampling and retyping and the use of VNTR markers to confirm paternity. A number of genes that may have pertinence to FA have been localized to the distal region of chromosome 20q. For example, two genes playing a central role in DNA synthesis are the genes encoding proliferating cell nuclear antigen (PCNA, also known as

-50

value

20q Markers

1.106 0.845 0.678 0.690 1.586 I.261

0.178 0.460 0.079 0.640 0..538 0.479 0.410 1.063 0.764

0.40

0.11.5 -0.028 0.429 - 0.017 0.349 0.054 0.242 0.0‘22 0.24% 0.187 0.259 0.1:',6 0.543 0.263

cyclin) (Ku et al., 1989) and topoisomerase I (TOPI) (Juan et al., 1988). The distribution of cellular topoisomerase I activity has been reported to be abnormal in FA placentae and some fibroblast cultures (Wunder et al., 1981; Wunder, 1984) but not in others (Auer et al.. 1982). The genes encoding protein phosphotyrosyl phosphatase 1B (PTPlB) (Brown-Shimer et al., 1990), hemopoietic cell kinase (HCK) (Quintrell et al., 1987), and src (Parker et al., 1985) also map to this region. A lesion in these genes resulting in abnormal cellular levels of phosphotyrosine could lead t.o neoplasia. A gene encoding a phospholipase C subtype (PLCl), another gene involved in signal transduction, has also been mapped to this region (Bristol et al., 1988). It is also noteworthy that deletions of the distal part of chromosome 2Oq, encompassing the location of these genes, have been reported in patients with myelodysplasia or acute myelogenous leukemia (Davis et al., 1984; Mitelman, 1984). This report of the regional placement of an FA-determining gene to chromosome 20q is the first step toward the identification of an FA gene. ACKNOWLEDGMENTS

DZOSZO

D&4

FIG. 3. Linkage of Fanconi anemia to chromosome 20q markers in six kindreds whose posterior probability for linkage was greater than (Y in the test for linkage heterogeneity with both D20Sl9 and D20S20. Computations were performed using the LINKMAP program (version 4.9) of the LINKAGE package (25). Distances between the markers were fixed according to published data (34. 26) and CEPH data (multipoint analysis kindly provided hy Dr. Mark Leppert). D20Sl9 was arbitrarily placed at 0.00 genetic distance, and the other markers were positioned around it.

We thank Mark Leppert and Ray White for their encouragement and helpful discussions and for providing us with a panel o! DNA markers; Collaborative Research, Inc.. for providing us with a panel of DNA markers; Franqois Rouyer for providing us with probe IPZOKO61 [D2OS25]; and Bronya Keats for helpful suggestions in the preparation of this manuscript. We extend our sincere gratitude to the families who participated in this investigation, for their willing and continuing cooperation. We also gratefully acknowledge the contribution of the many physicians who were involved in this study. This study was supported in part by grant.s to A.D.A. from the National Institutes of Health (HL329871, the March of Dimes Birth Defects Foundation (6.521), and the Fanconi Anemia Research Fund, by a General Clinical Research

FANCONI

ANEMIA:

LINKAGE

HETEROGENEITY

ON

X35

“0~1

APPENDIX Lod Scores for Loci That Assumed

Locus DlS58 L* DlS49 DlSSJ DlShl DlS81 DlS48 DZS.50 D2S61 D2S5.1 Lx%21 ms39 D2S30 DZS43 D”S:14 D2S:%=, D’S”0 D”S60 D2S“7 D2S48 DBS4.5 CRYGPI D3S”6 D3S47 D3Sl:i D3S12 LXIS 14 D3S 16 L14S105 L)4slo:< D4Sl:IO D-IS35 LX%% D:iS6:3 DiiSXL’ DSSX‘4 DSS6?

Map location 1 1 lp 1 lq 1 zq “p “q 2 ” 2 2 ” 2 :! “p 2 “pter-q:v “1) %y:i:i -y:3:, :3 :1 x :ipter-p2 1 :i :I 4 4 4 Ipll--yll 5q2 I --q?Ci 5yl 1.“-qlX.? .5yl4--q21 hq21--yz 5q:+qter

Did Not Show Linkage

With

Fanconi

value of recombination fraction /I

Anemia Assumed

0.0s

0.10

020

0.30

0.40

m-6.03 -5.06 -5.06 -9.53 -17.73 -7.9% -3.44 -1.17 8.74 6.75 6.44 -2.6,5 4.27 4.30 -8.16 6.10 -5.10 ps.17 -2.15 -:3239 6.76 923 - 0.47 -8.61 ~ 6.45 -9.%-t -5.09 -2.99 -4.08 0.74 -3.07 -3.40 4.74 -0.40 - 4.9s 0.44

-3.15 -2.30 -2.40 -5.06 -10.1:1 -4.27 ~0.88 -0.58 -3.78 -3.45 -3.12 ~0.56 -1.80 -1.63 -m4.06 3.07 -2.42 -1.77 -0.59 - 1.64 3.42 -4.62 0.47 -0.98 -3.19 -0.96 -2.43 0.01 1.81 1.10 -0.85 -0.98 -1.90 0.22 -2.40 0.97

-1.02 -0.56 --Cl..56 1.66 - 3.83 -1.55 0.39 PO.14 -0..52 -1.11 -1.:10 OX3 ~0.46 0.00 -1.21 -0.96 -- 0.70 0.21 0.:30 -0.16 -1.03 --1.21 0.69 (I.45 1 .06 -0.:10 -0.35 1.41 pO.:i6 0.87 0.3” 0.87 -O.“Ti.,I 0.30 +).7x 0.96

-0.40 -0.05 -0.05

-0.06 -0.03 0.0%

-0.48 -1.87 -0.54 0.:19

--0.09 o.:iti 0.10 0.14

-0.i) 1 0.X

I,ocus

0.01

0.16 -0.39 -0.12 -0.5% -0.14 0.X3 0.06 -0.06 0.0% 0.24 0.11 -0.35 --0.0x -0.31 ~0.04 -O.%O -0.03 0.42 0.20 0.:3:3 0.11 0.14 0.08 -0.28 0.03 m-U.16 0.06 0.40 0.13 0.49 0.‘2’1 -0.4 1 PO.14 0.:39 0.19 -0.0% 0.07 1.09 0.4 1 -0.03 0.09 0.‘4s il. 13 0.43 026 0.40 0.15 0.01 0.00 0.1:s 0.0” -0.28 0.14 0.66 0.3”

Map location

D6SlY D6SlO 116848 DWIX D6S’l D6S:W D6S44 D6SXS 1>7S16 D7S65 D7S56 L< D7S6’ D7S87 L)7S?) L 1 D7S104 D9Sl6 DllSl 14 DllS144 L)l’Sl’i D12S8 L)l:3s:io I)l?lS% LJ14SXi L)IIS16 L)1*5Sl L115S46 Dlf,S49

6p fip 6 fp f({ 6p GpZl-yter 6p21--yter 7y”l my”” 7pter q2 7qX-yter 7pter~qZ 7q’l-:i.‘, Spter-q2:! sy:11 +35 Yy llq”“.:l-q~L:i.:l 1 lq”“.:i-q”:~.;l l”q 12yl4&~trr 1 :iy l:!yl-i.:! $1 I-ly 14 lT,ql-L~y”l 1 .5

Dl7S:l; Dl7S7.1 APOC” D19SS L)lSSX I’KU; D 19S”o D2lSI 1:i D”%S:v

1; 17q l!~ql:~.:! 19cen-ql2 19ylX:! 19plX-qter 19p ‘1(4’2:! t’Zql1.2-yter

0.0s

. . , iF~ r)lhbn

value of recombination fraction H 0.10

0.20

0.30

1 .0x -4.7.; -4.68 02 1 m~O.74 1 .“Y --l.“? -4.5x -~0.1:1 --0.20 0.04 1.21 1.28 --(0.X -~(I.69 4.9” 0. 19 O:lti 0.:i.r 0.M ox 0.29 1 .x2

-027 -0.0s -0. I1 0.0:1 0.06 0.0’ o.:u 0.1% o.I):1 0.1J9 0.W o.:~.’ 4.‘6 - 0.01 0.02 ().I)7 ~~0.01 0.02 -o.oR 0.06 0.11 0.02 PO.49 -4). 19 PO.45 0.10 -0.19 .- 0.10 -0.12 0.1K~ Il.%:1 0.06 (1.1.5 (1.0” 029 0.07 ().:!I) 0.10 Il.49 0.3) 0.4 1 I).16 0.06 -O.I)O ~026 11.1-l O.: