GENOMICS 14, 715-720 (1992)

A Genetic Linkage Map of Human Chromosome 9q LAURIE J. OZELIUS,*"~ DAVID J. KWIATKOWSKI,~: DEBORAH E. SCHUBACK,* XANDRA O. BREAKEFIELD,*'§ NANCY S. WEXLER,II'¶ JAMES F. GUSELLA,*'~" AND JONATHAN L. HAINES*'** *Neurogenetics Laboratory, Massachusetts General Hospital, and Departments of tGenetics, *,*Neurology, and §Neuroscience, Harvard Medical School, Boston, Massachusetts 02115; SDivision of Experimental Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02155; IIHereditary Disease Foundation, Santa Monica, California 90404; and ¶Columbia University College of Physicians and Surgeons, New York, New York 10027

ReceivedMay 18, 1992 A genetic linkage map of human chromosome 9q, s p a n n i n g a s e x - e q u a l d i s t a n c e o f 1 2 5 cM, h a s b e e n developed by genotyping 26 loci in the Venezuelan Reference Pedigree. The loci include 12 anonymous microsat e l l i t e m a r k e r s r e p o r t e d b y K w i a t k o w s k i et al. ( 1 9 9 2 ) , s e v e r a l c l a s s i c a l s y s t e m s p r e v i o u s l y a s s i g n e d to c h r o mosome 9q, and polymorphisms for the genes tenacin (HXB), g e l s o l i n (GSN), a d e n y l a t e k i n a s e 1 (AK1), arginosuecinate synthetase (ASS), A B L o n c o g e n e (ABL1), A B O b l o o d g r o u p (ABO), a n d d o p a m i n e ~ - h y d r o x y l a s e (DBI-I). O n l y a m a r g i n a l l y s i g n i f i c a n t s e x d i f f e r e n c e is f o u n d a l o n g t h e e n t i r e l e n g t h o f t h e m a p and results from one interval, between D9S58 and D9S59, t h a t d i s p l a y s a n e x c e s s o f f e m a l e r e c o m b i n a t i o n . A c o m p a r i s o n o f t h e g e n e t i c m a p to t h e e x i s t i n g p h y s i c a l d a t a s u g g e s t s t h a t t h e r e is i n c r e a s e d r e c o m b i nation in the 9q34 region with a recombination event occurring every 125-400 kb. This map should be useful in further characterizing the relationship between p h y s i c a l d i s t a n c e a n d g e n e t i c d i s t a n c e , as w e l l a s f o r g e n e t i c l i n k a g e s t u d i e s o f d i s e a s e s t h a t m a p to c h r o m o some 9q, including multiple self-healing squamous epit h e l i o m a (MSSE), G o r l i n s y n d r o m e ( N B C C S ) , x e r o d e r m a p i g m e n t o s u m (XPA), n a i l - p a t e l l a s y n d r o m e (NPS1), t o r s i o n d y s t o n i a (DYT1), a n d t u b e r o u s s c l e r o s i s (TSC1). © 1992 AcademicP. . . . Inc.

INTRODUCTION G e n e t i c l i n k a g e m a p s of t h e h u m a n g e n o m e are p o w e r ful r e s o u r c e s for l o c a l i z i n g d i s e a s e g e n e s t h r o u g h l i n k a g e a n a l y s i s a n d for u s e as r e f e r e n c e p o i n t s for b u i l d i n g a n d verifying physical maps. Microsatellite polymorphism m a p s a r e a n e x c e l l e n t set of h i g h l y i n f o r m a t i v e m a r k e r s t h a t are v a l u a b l e for g e n e t i c l i n k a g e a n a l y s i s . H o w e v e r , as t h e s e m a r k e r s a r e m o s t l y a n o n y m o u s p r o b e s , t h e i r l o c a t i o n s r e l a t i v e to m a p p e d g e n e s a n d o t h e r c h r o m o somal l a n d m a r k s are u n k n o w n . W e i n i t i a l l y d e v e l o p e d a g e n e t i c m a p of c h r o m o s o m e 9 q 3 2 - q 3 4 t o h e l p localize t h e d y s t o n i a g e n e ( D Y T 1 ) w i t h i n t h i s r e g i o n ( O z e l i u s e t al., 1989; K r a m e r e t al.,

1990). T h e s e m a r k e r s , a l t h o u g h n o t all h i g h l y p o l y m o r phic, i n c l u d e m a n y of t h e o r i g i n a l m a r k e r s m a p p e d o n c h r o m o s o m e 9q ( L a t h r o p e t al., 1988), as well as s e v e r a l genes t h a t have been m a p p e d using physical techniques ( C o o k e t al., 1978; C a r r i t t a n d P o v e y , 1979; H e i s t e r k a m p e t al., 1983; S u e t al., 1984; A l l d e r d i c e e t al., 1986; C r a i g e t al., 1988; K w i a t k o w s k i e t al., 1988; G u l c h e r e t al., 1990). These data have been combined with the recently publ i s h e d d i n u c l e o t i d e r e p e a t p o l y m o r p h i s m m a p of c h r o m o s o m e 9q ( K w i a t k o w s k i e t al., 1992), w h i c h w a s g e n e r a t e d u s i n g a s u b s e t of t h e s a m e r e f e r e n c e p e d i g r e e s . B y m e r g i n g t h e s e t w o g e n e t i c m a p s , we h a v e b e e n a b l e t o r e l a t e t h e r a n d o m m i c r o s a t e l l i t e m a r k e r s to loci w i t h k n o w n c h r o m o s o m a l positions a n d provide a more useful a n d c o m p r e h e n s i v e m a p o f 9q.

MATERIALS AND METHODS

D N A methods. Table 1 provides a detailed description of the probes and polymorphisms used to construct the map. For RFLP genotyping, genomic DNA (5 #g) was digested with the appropriate enzyme according to the manufacturer's instructions. Agarose gel electropheresis, DNA transfer, hybridization, and autoradiography were performed as described in Ozelius et al. (1989). Probes were radiolabeled by the oligonucleotide priming method of Feinberg and Vogelstein (1984). To generate a dinucleotide repeat polymorphism at the dopamine fl-hydroxylase (DBH) locus, a total genomic phage library in EMBL3A was screened with a cDNA for human DBH (gift of J. Mallet, CNRS). Positive clones were digested with Sau3A, subcloned into BlueScript (Stratagene), and probed with poly(dGT) (Pharmacia). GT-repeat-containingclones were sequenced using Sequenase (USB), and oligonucleotide primers were selected to flank the repeat sequence (PCR primers: 5'-ccaggtgagctgtctcagaa-3' and 5'-cgtgcatatgtgtaca-3'; EMBL Accession No. Zl1653). PCR analysis of all microsatellite polymorphisms was carried out on genomic DNA as described (Ozelius et al., 1992a), using oligonucleotide primer pairs previously reported (Kwiatkowski et al., 1992; Ozelius et al., 1992b; and above). In cases of missing or questionable serum typing, the ABO locus was retyped using PCR analysis (Yamamoto et al., 1990). The PCR products were cut with BstEII and fractionated on 3% Nusieve agarose gels, and the DNA was visualized with UV light after staining with ethidium bromide. Linkage analysis. The loci marked with an asterisk (*) in Table 1 were typed in the original 17 Venezuelan Reference Pedigrees (VRP) (Haines et al., 1990). These consist of 190 individuals with a total of 715

0888-7543/92 $5.00 Copyright © 1992by AcademicPress, Inc. All rights of reproductionin any form reserved.

716

OZELIUS ET AL.

TABLE 1 Chromosome 9q Markers Locus

Clone

Polymorphism

Cal. Het.

Reference

D9S15" D9S56" D9S29 D9S58" D9S59" D9S107" HXB D9S26 GSN D9S60" AK1 D9S61" D9S62" D9S63" D9S65" ASS ABLI D9S64" ABO DBH D9S10 D9S66" D9S14" D9S30 D9S67" D9S7

MCT112 1AE1 LAMP92 1AC3 3AF10 LAMP95 p31 L659 M1D lAD2 hAK1B3.25 lAD7 lAG5 lAB9 4AB7 ASSG3 ABL3 1AA7

GT n GTn TaqI, PvuII GT n GT~

0.66 0.80 0.69 0.88 0.70 0.48 0.79 0.45 0.39 0.81 0.33 0.80 0.44 0.89 0.71 0.92 0.72 0.82 0.48 0.61 0.50 0.79 0.36 0.48 0.67 0.75

24 24 35, 45 24 24 47 34 8 23 24 3 24 24 24 24 24, 31 2, 24 24 27 38 27 24 27 30 24 27

DBH1.9 MCT136 4AF10 MCT96.1 MHZ21 3AEll EFD126.3

MspI GT

TaqI StuI, BclI GT n

TaqI GTn GT n GTn GT~ GTn, PstI GTn, StuI GT~ Protein GT~, XbaI

PstI GT~

RsaI PstI GT~

MspI

Physical loc.

Reference

9q13-q21.1

39

9q31

35

9q32 9q32-q34 9q33-q34 9q33

47 13 47 22

9q34.1

9q34.1 9q34.1 9q34-qter 9q34.1-q34.2 9q34.3 9q34.3 9q34-qter 9q34 9q34 9q34-qter 9q34

1, 6, 29

5, 29, 40 18 24 6 7 47 24 47 47 24 47

* Markers typed in original 17 VRP sibships only. 303 potentially informative meioses. Two of these sibships are also part of the CEPH panel of families (CEPH families 102 and 104). Data were generated on all other markers in an expanded version of the VRP containing 47 large interrelated sibships representing over 888 potentially informative meioses (Tanzi et al., 1992). All data were entered into a computer file using the LIPIN data management program (Trofatter et al., 1986). The program CHROMLOOK (Haines, 1992) was employed to identify male and female recombinants. Observations of chromosomes containing two or more recombinants were suggestive of genotyping error, and in these cases the entire sibship was retyped to ensure data integrity. Linkage data were analyzed using the program M A P M A K E R (version 1.0) (Lander et al., 1987) employing the strategy described in Haines et al. (1990) and briefly outlined here. All possible 2-point led scores were generated and examined to determine a set of three highly informative, well-spaced markers. These markers were then tested for all possible orders. If one order had odds of at least 1000:1 better than any other order, it was chosen as the "framework" onto which all other markers were mapped. Each additional marker was tested in every possible position and added if one placement was favored by odds of at least 1000:1 over all other positions, thus extending the map in a sequential manner. As a final check for accuracy, the map was decomposed into overlapping sets of 5 markers and all 60 possible orders of each set were tested. We considered the map confirmed if every marker appeared in at least one 5-point analysis with at least 1000:1 odds over any other order. Although this method is conservative in the number of markers that can be placed, it also reduces the number of false orders. Those markers that could not be placed with 1000:1 odds were regionally localized to determine their 1000:1 odds boundaries (Fig. 1). Sex differences in recombination fraction were tested by calculating the - 2 in(likelihood) difference between the sex-equal and the sexspecific maps and using the likelihood ratio test (Haines et al., 1990). Because errors in the genotypic data can have a major effect on the resulting map, we instituted several error checking procedures. All data were checked for Mendelian inheritance and rechecked for accuracy after being entered into the computer file. Once an initial locus

order was determined, the data were analyzed to identify individual chromosomes with multiple recombinants. Markers were retyped when these recombinants occurred in loci that were within 20 cM of each other. As a final check of accuracy, we examined the effect of each marker on overall map length (Lasher et al., 1991; Patterson, 1991). Each marker was dropped from the analysis one at a time and the map was recalculated. If errors exist in marker genotyping, they are likely to result in an apparent excess of recombinants and subsequent map inflation. Thus, a drop in map length with the removal of a marker is an indication of potentially unresolved errors. We have arbitrarily chosen > +1 SD as indicative of such problems.

RESULTS We have constructed a genetic linkage map spanning a s e x - e q u a l d i s t a n c e o f 125 c M o n t h e l o n g a r m o f c h r o m o s o m e 9. T h e m a p w a s m a d e u s i n g 31 d i s t i n c t p o l y m o r p h i s m s : 14 R F L P s , 16 ( d G d T ) n r e p e a t s , a n d 1 p r o t e i n m a r k e r . T h e s e m a r k e r s d e f i n e 26 s e p a r a t e l o c i i n cluding the genes for tenacin (HXB), gelsolin (GSN), adenylate kinase 1 (AK1), arginosuccinate synthetase (ASS), ABL oncogene (ABL1), ABO blood group (ABO), a n d d o p a m i n e / ~ - h y d r o x y l a s e ( D B H ) ( T a b l e 1). T h e m a p i n c l u d e s 16 u n i q u e r e f e r e n c e p o i n t s t h a t c o u l d b e o r d e r e d w i t h o d d s o f a t l e a s t 1000:1 ( F i g . 1). I n s e v e r a l cases, two markers could be mapped into a single interv a l w i t h 1000:1 o d d s , b u t c o u l d n o t b e o r d e r e d w i t h i n that interval. For example, D9S29 can be replaced by D9S56; AK1 with D9S61; ABO with D9S66 or D9S10; and D9S30 by D9S7. Making these switches does not either alter the relative order of the other markers or

717

G E N E T I C M A P OF H U M A N 9q

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34.2 34.3 q arm

1 ................. --2

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] DBH/D9810/DgS66

10 I D9814 D987

F I G . 1. Genetic linkage map of chromosome 9q showing sex-equal, male and female maps. Distances are indicated in cM (Kosambi m a p p i n g function). Physical locations for some of the markers are denoted by the bars on the far left of the idiogram. Markers with only regional localizations are displayed on the far right. T h e bars delimit their 1000:1 odds boundaries.

m a k e a significant difference in the overall m a p length. T h e choice of m a r k e r s for the 1000:1 m a p was b a s e d on several factors, including which m a r k e r s m a p p e d w i t h the highest relative odds b e t w e e n flanking m a r k e r s a n d w h e t h e r a p h y s i c a l p o s i t i o n h a d b e e n reported. T w o p a i r s of m a r k e r s , D9S62/D9S63 a n d ASS/ABL, are given t h e s a m e m a p p l a c e m e n t s because no recombin a t i o n events were o b s e r v e d with either p a i r in 132 a n d 437 m u t u a l l y i n f o r m a t i v e events, respectively. T h e p e a k 2-point lod scores b e t w e e n t h e s e m a r k e r s are 39.74 (D9S62 vs D9S63) a n d 131.55 (ASS vs ABL) at 0 = 0.00. Several o t h e r m a r k e r s could n o t be placed on the m a p with 1000:1 odds either b e c a u s e t h e y were not very inf o r m a t i v e in our d a t a set (D9S107, D9S14) or because of t h e i r close p r o x i m i t y to o t h e r m a r k e r s . Figure 1 shows t h e i r m o s t likely positions. D9S56 m a p s in the interval b e t w e e n D9S15 a n d D9S58 with t h e p r e f e r r e d position distal to D9S29 b u t w i t h odds of only 3:1. D9S61 m a p s on either side of AK1, a g a i n the m o s t likely position is distal with odds of 251:1. D9S14 a n d D9S7 m a p n e a r D9S30; D9S14 b e i n g p r o x i m a l to D9S30 with odds of 2.5:1 a n d D9S7being distal w i t h odds of 2.3:1. F o u r o t h e r m a r k e r s , ABO, DBH, D9S10, a n d D9S66, m a p at virtually the s a m e location. T h e odds favoring a n y one order over the o t h e r s are negligible. No crosses h a v e b e e n seen b e t w e e n ABO a n d t h e o t h e r t h r e e m a r k e r s or

b e t w e e n D9S10 a n d D9S66, b u t one cross has b e e n observed b e t w e e n DBH a n d b o t h D9S10 a n d D9S66, suggesting t h a t DBH is p r o x i m a l to these two m a r k e r s . T h e position for D9S107 is the least well defined. Its m o s t p r e f e r r e d p l a c e m e n t is b e t w e e n GSN a n d D9S60 b u t t h e s u p p o r t for this is only 1.1:1. D9S107 could lie a n y w h e r e in the interval b e t w e e n D9S59 a n d D9S60. T h e overall female to m a l e ratio of r e c o m b i n a t i o n fraction along the entire m a p is 1.3:1. T h i s difference is only m a r g i n a l l y significant (x2(15) = 25.3 ( P < 0.05)). W h e n sex differences were e x a m i n e d for specific intervals, the only significant difference f o u n d was b e t w e e n D9S58 a n d D9S59 (X2(1) = 7.96 ( P < 0.005)). T o assess the integrity of the g e n o t y p i n g d a t a underlying the finished m a p , a n e r r o r checking p r o c e d u r e was p e r f o r m e d , w h e r e b y each m a r k e r in t h e m a p was sequentially d r o p p e d a n d the m a p was recalculated. T h e results, depicted graphically in Fig. 2, are e n c o u r a g i n g since only HXB a n d GSN caused s u b s t a n t i a l decreases in overall m a p length w h e n removed. DISCUSSION

W e have a n a l y z e d the linkage relationships of 26 D N A m a r k e r s on c h r o m o s o m e 9q. In g e n e r a t i n g this m a p , we h a v e i n c o r p o r a t e d all 14 m a r k e r s r e p o r t e d b y

718

OZELIUS ET AL. 140

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120

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110

(cM) 100 $15

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S29

$58

$59

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i $26

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i $60

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i $65

i $62

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ABO $ 6 7

$30

LOCUS Chromosome

9

FIG. 2. A plot of the effect of eliminating a single marker on the overall map length. Points falling above or below the + 1 SD lines are indicative of potential unresolved errors in the data for that marker. K w i a t k o w s k i et al. (1992). T h e order of the m a r k e r s rem a i n s c o n s i s t e n t b u t the overall length of the m a p has b e e n e x p a n d e d f r o m a sex-equal distance of 90 to 125 cM. S o m e of this difference (9 cM) results f r o m the addition of a telomeric m a r k e r , b u t m o s t of the difference is seen in the interval b e t w e e n D 9 S 1 5 a n d D9S29. Because these m a r k e r s s p a n a large genetic distance, a n d the dist a n c e b e t w e e n D 9 S 2 9 a n d D 9 S 5 6 is 18 cM, the overall length e s t i m a t e s are r a t h e r crude. In the interval bet w e e n D 9 S 5 8 a n d D9S65, the two m a p s are not significantly different. T h e increase in m a p length is p r o b a b l y due to the addition of several m o r e m a r k e r s . T h e dist a n c e in the region b e t w e e n D 9 S 6 2 / 6 3 a n d D 9 S 6 7 rem a i n s unchanged. T w o o t h e r genetic linkage m a p s of c h r o m o s o m e 9 h a v e b e e n p u b l i s h e d (Donis-Keller et al., 1987; L a t h r o p et al., 1988). T h e p r e s e n t m a p includes seven loci in comm o n with the L a t h r o p et al. (1988) m a p a n d t h r e e loci in c o m m o n w i t h t h e m a p r e p o r t e d b y D o n i s - K e l l e r et al. (1987). B o t h of these m a p s were g e n e r a t e d using the C E P H pedigrees. T h e order of m a r k e r s for all t h e m a p s is entirely consistent. T h e distance e s t i m a t e s f r o m a sex-average L a t h r o p et al. m a p are n o t significantly diff e r e n t f r o m t h o s e of our sex-equal m a p , a l t h o u g h differences b e t w e e n individual m a r k e r s can be seen. F o r instance, we see no r e c o m b i n a t i o n s b e t w e e n A B O a n d D 9 S 1 0 while L a t h r o p et al. r e p o r t a distance of 4 c M in m a l e s a n d 5 c M in females. Likewise, D o n i s - K e l l e r et al. (1987) r e p o r t a distance of 20 c M b e t w e e n A B L 1 a n d A B O , which is m u c h g r e a t e r t h a n the distance r e p o r t e d in our m a p or in the L a t h r o p et al. (1988) m a p a n d m a y be due to the smaller n u m b e r of families t y p e d b y DonisKeller et al. (1987). W e have f o u n d only one interval, b e t w e e n D 9 S 5 8 a n d D 9 S 5 9 , with a significant sex difference in r e c o m b i n a tion rates. T h i s region showed no r e c o m b i n a n t s in males, b u t 7% r e c o m b i n a t i o n in females; t h u s the exact f e m a l e / m a l e ratio is undefined. L a t h r o p et al. (1988) also r e p o r t e d an excess of female r e c o m b i n a t i o n b u t e n c o m passing a m u c h larger i n t e r v a l c o r r e s p o n d i n g to D 9 S 1 5 A K 1 . T w o possible e x p l a n a t i o n s for this result are t h a t our lack of significance is the result of r a n d o m fluctua-

tion a r o u n d the true sex differences a n d t h a t t h e r e is true p o p u l a t i o n v a r i a t i o n in the r e c o m b i n a t i o n fraction. W e p l a n to explore these possibilities once a n index m a r k e r m a p for each pedigree set is available. B o t h d a t a sets agree t h a t t h e r e are no r e c o m b i n a t i o n differences b e t w e e n males a n d females in t h e telomeric region of 9q as h a s b e e n seen on o t h e r c h r o m o s o m e s (Buetow et al., 1991; W e i f f e n b a c h et al., 1991) b u t we do n o t k n o w how close the m o s t distal m a r k e r s are to the telomere. T h e physical locations of several of the loci on the p r e s e n t m a p have b e e n r e p o r t e d previously (Table 1). M o s t of these locations were assigned on the basis of in situ hybridization results or b y m a p p i n g the m a r k e r s versus a chronic m y e l o g e n o u s l e u k e m i a (CML) cell line with a P h i l a d e l p h i a c h r o m o s o m e . T h e s e cell lines h a v e a t r a n s l o c a t i o n b e t w e e n the 5' e n d of the A B L oncogene a n d the 3' end of the B C R gene on c h r o m o s o m e 22 (Grosveld et al., 1986; W e s t b r o o k et al., 1988). T h i s t r a n s l o c a tion can be used to m a p p r o b e s p r o x i m a l or distal to the A B L oncogene. In this w a y it has b e e n s h o w n t h a t A S S is p r o x i m a l to A B L ( L e v e r s h a et al., 1991; H e n s k e personal c o m m u n i c a t i o n ) . Recently, H a r r i s et al. (1991) have r e p o r t e d using a c o m b i n a t i o n of fluorescence in situ hybridization a n d pulsed-field gel electropheresis ( P F G E ) to c o n s t r u c t a physical m a p of 9q32-q34. T h i s m a p c o n t a i n s seven m a r k e r s in c o m m o n with t h e genetic m a p r e p o r t e d here. T h e y confirm the p l a c e m e n t of D B H p r o x i m a l to D 9 S 1 0 b y physically m a p p i n g these m a r k e r s on the s a m e 350-kb M l u I f r a g m e n t . However, t h e y place H X B distal to G S N b u t not on similar P F G f r a g m e n t s . T h i s conflict b e t w e e n the genetic a n d the physical locations of these two m a r k e r s r e m a i n s to be resolved. T h e only area for direct c o m p a r i s o n is the region e n c o m p a s s ing A S S to D B H / D 9 S I O , where n e a r b y m a r k e r s are totally linked on P F G f r a g m e n t s . T h i s interval has a genetic distance of 8 c M b u t is p r e s e n t on c o m m o n P F G f r a g m e n t s totalling only 3.15 Mb. On average across t h e h u m a n genome, 1 c M is a s s u m e d to be equal to 1000 kb b u t in this region one r e c o m b i n a t i o n occurs approxim a t e l y every 400 kb, or 2~ t i m e s as f r e q u e n t l y as the average. T h e exact p l a c e m e n t of the p r o b e s within the P F G f r a g m e n t s is n o t known; therefore, this 3.15 M b r e p r e s e n t s the largest possible physical distance bet w e e n these m a r k e r s b u t the actual distance could be even smaller, m a k i n g the r e c o m b i n a t i o n frequency even higher. H e n s k e et al. (1992) h a v e g e n e r a t e d a r a d i a t i o n reduced s o m a t i c cell h y b r i d line c o n t a i n i n g the region f r o m D 9 S 6 0 to the A B O / D B H / D 9 S 1 0 / D 9 S 6 6 cluster as its only h u m a n c o m p o n e n t . T h e y e s t i m a t e t h a t the hybrid contains 5 M b of h u m a n D N A , b u t this region genetically is 21 cM on our sex-equal m a p . G i v e n the above physical distance b e t w e e n A S S a n d A B O , a r e c o m b i n a tion e v e n t m a y occur as frequently as once in every 125 kb in the region b e t w e e n D 9 S 6 0 a n d A S S . T h e s e d a t a suggest t h a t t h e r e is a n increased rate of r e c o m b i n a t i o n in 9q34. I n c r e a s e d r e c o m b i n a t i o n rates have b e e n n o t e d at the t e l o m e r e s of o t h e r c h r o m o s o m e s (Buetow et aI., 1991; T a n z i et al., 1988) a n d m a y explain the d i s p a r i t y b e t w e e n the genetic a n d t h e physical distances.

GENETIC MAP OF HUMAN 9q A m a j o r d i f f i c u l t y i n u s i n g p u b l i s h e d l i n k a g e m a p s is determining the confidence with which order and distance have been generated. Uncorrected genotyping errors can have a significant effect upon both order and d i s t a n c e ( B u e t o w e t al., 1991; L a s h e r e t al., 1991), m a k ing it important that some level of error checking be used in map generation. The method proposed by Chakrav a r t i ( P a t t e r s o n , 1991) is a s i m p l e g r a p h i c a l r e p r e s e n t a tion of the effects of each marker on the overall map l e n g t h a n d p r o v i d e s a q u i c k q u a l i t a t i v e m e t h o d f o r determining the confidence one may have in the resulting m a p (Fig. 2). D e s p i t e o u r e x t e n s i v e e r r o r c h e c k i n g , u n determined errors may remain in HXB and GSN, and therefore the distances in those intervals may be slightly overestimated. D9S67 also shows a decrease in map l e n g t h b u t i t is d i f f i c u l t t o d e t e r m i n e w h e t h e r e r r o n e o u s g e n o t y p e s o c c u r i n D 9 S 6 7 a n d D 9 S 3 0 b e c a u s e t h e r e is n o marker flanking D9S30 with which to accurately determine phase. The map presented here incorporates highly polymorphic microsatellite probes with classical markers and genes of known physical location. This combination provides a unique framework of index markers that can b e u s e d t o a s s i g n n e w d i s e a s e loci t o t h i s c h r o m o s o m e . M o r e o v e r , t h o s e d i s e a s e g e n e s a l r e a d y m a p p e d t o 9q c a n be better localized using this map. These include, multip l e s e l f - h e a l i n g s q u a m o u s e p i t h e l i o m a ( M S S E ) 9q31 ( G o u d i e e t al., 1991), G o r l i n s y n d r o m e ( N B C C S ) 9q31 ( G a i l a n i e t al., 1992), x e r o d e r m a p i g m e n t o s u m ( X P A ) 9 q 3 2 - q 3 4 . 1 ( H e n n i n g e t al., 1990), n a i l p a t e l l a s y n d r o m e ( N P S 1 ) 9q34.1 ( R e n w i c k a n d L a w l e r , 1955), t o r s i o n d y s t o n i a ( D Y T 1 ) 9q34.1 ( O z e l i u s e t al., 1992a), a n d t u b e r o u s s c l e r o s i s ( T S C 1 ) 9 q 3 4 . 2 - q 3 4 . 3 ( H a i n e s e t al., 1991). T h i s f r a m e w o r k m a p c a n a l s o b e u s e d a s a s t a r t i n g point to compare and contrast the physical and genetic maps eventually leading to the study of various factors t h a t i n f l u e n c e r e c o m b i n a t i o n , s u c h a s t h e i m p a c t o f sex, age, a n d i n d i v i d u a l v a r i a t i o n . T h e p r e s e n t m a p is a g o o d beginning toward the goal of a 2-cM map of every chromosome proposed by the Human Genome Project. The map reflects the overall dearth of probes in the proximal 9q r e g i o n a n d h i g h l i g h t s t h e a r e a s w h e r e a c o n c e n t r a t e d e f f o r t is n e e d e d t o a c h i e v e a 2 - c M m a p . ACKNOWLEDGMENTS The authors thank the National Tuberous Sclerosis Association. This work was supported by grants from the Dystonia Medical Research Foundation to X.O.B. and J.F.G. and NIH Grants NS28384 to X.O.B. and D.J.K., HG000324 to J.L.H., HG00169 to J.F,G., HG00598 to D.J.K. and NS22031 N.S.W. REFERENCES 1. Allderdice, P. W., Kaita, H., Lewis, M., McAlpine, P. J., Wong, P., Anderson, J., and Giblett, E. C. (1986). Segregation of marker loci in families with an inherited paracentric insertion of chromosome 9. Am. J. Hum. Genet. 39: 612-617. 2. Baumann, R., and Smith, M. (1990). StuI RFLP in the human ABL locus. Nucleic Acids Res. 18: 693.

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