0021-972x/92/7501~0201103.00/0 .Journal of Clinical Endocrinology and Me&holism CopyrIght it 1992 by The Endocrme Smety

Multilocus Mapping Rickets Gene* MICHAEL MARGARET

Vol. 75, No. 1 Printed in U.S.A.

of the X-Linked

J. ECONS, DAVID F. BARKER, MARCY C. SPEER, A. PERICAK-VANCE, PAMELA R. FAIN, AND MARC

Hypophosphatemic

K. DREZNER

Departments of Medicine and Cell Biology and The Sarah W. Stedman Center for Nutritional Studies (M.J.E., M.C.S., M.A.P.-V., M.K.D.), Duke University Medical Center, Durham, North Carolina; and Department of Medical Genetics (D.F.B., P.R.F.), The University of Utah, Salt Lake City, Utah ABSTRACT X-linked hypophosphatemic rickets (HYP), the most common form of familial hypophosphatemic (vitamin D-resistant) rickets, is an Xlinked dominant disorder characterized by decreased renal tubular phosphate reabsorption and consequent hypophosphatemia. Despite the application of a wide variety of biochemical and cell biology techniques, controversy exists regarding whether a primary renal abnormality underlies the abnormal phosphate transport or if this defect is secondary to the effects of a hormonal/metabolic factor. Thus localization of the HYP gene and its ultimate cloning may be necessary to elucidate the pathophysiology of the disorder. In order to map the human HYP gene we investigated several new polymorphic probes for linkage to HYP and constructed a map of markers around the gene. The database used to ascertain linkage and perform mapping included 5 large HYP kindreds, 40 Centre d’Etudie Polymorphisms Humain

reference pedigrees, and 19 kindreds which had been obtained for other disease linkage studies. Two point LOD scores (odds of linkage, log,,,) indicate that the probes DXS365, DXS257, DXS451, and DXS41 are tightly linked to the HYP locus. Indeed, there were no cross-overs between DXS365 and HYP with a peak LOD score of 13.98 [recombination fraction (0) = O.OO]. Moreover, multipoint analysis reveals a probable locus order of: Xtel-DXS315-DXS43-DXS257-HYP-DXS41DXS451-Xcen. The likelihood of HYP occurring between DXS257 and DXS41 is 407:l over the next most likely position. DXS365 is located between DXS41 and DXS43 but could not be located with respect to HYP and DXS257. Regardless, we have located the HYP gene between the flanking markers DXS257 (telomeric) and DXS41 (centromeric) which are 3.5 centiMorgans apart. Thus, the results of this study will facilitate attempts to further localize and eventually clone the gene. (J Clin Endocrinol Metub 75: 201-206, 1992)

X

iments reported by Meyer et al. (8, 9) and Nesbitt et al. (10) indicate that the effect of a humoral factor on the brush border membranesunderlies the abnormality. Thus, localization of the HYP gene and its ultimate cloning may be necessary to elucidate the pathophysiology of this disease. Recently, studies using restriction fragment length polymorphisms have localized the human HYP gene locus to the Xp22.1-~22.2 region (11-14). Unfortunately, these studies have been constrained by the limited number of informative matings available in the diseasekindreds studied, the paucity of polymorphic probes in the region, and the relatively large distances between the flanking markers and the HYP gene locus. As a consequence, investigators have been unable to construct a well defined genetic map of the region or attain tightly linked flanking markers, necessary prerequisites for obtaining the gene by positional cloning strategies.Therefore, in the present study we used several large multigeneration diseasekindreds, as well as reference families, to construct a map of the Xp22.1-~22.2 region with several newly defined markers. Subsequently, using multilocus analysis, we identified tightly linked flanking markers for the diseasegene and located it within the newly defined map.

-LINKED hypophosphatemic rickets (HYP) is the most common form of familial hypophosphatemic (vitamin D-resistant) rachitic disease.Patients frequently present with lower extremity deformities, radiographic evidence of rickets, short stature, bone pain, dental abscesses,bone overgrowth at the sites of muscle attachments and around joints, and osteomalacia.However, there is a great deal of variability in the clinical expression of the diseaseeven between affected family membersof the same sex. Biochemically, the disorder is characterized by hypophosphatemia due to decreasedrenal tubular phosphate reabsorption. The abnormal renal phosphate handling has been indirectly identified in affected patients (l-3) and further defined and characterized in the brush border membranes of the proximal nephron in the Hyp mouse, the murine homolog of the human disease(4, 5). Nevertheless, controversy exists regarding whether a primary renal abnormality exists or if the abnormal phosphate transport is secondary to the effects of a hormonal/metabolic factor. Studies by Bell et al. (6) and Dobre et al. (7) suggest that the deranged phosphate transport results from a primary membrane defect in the proximal tubule. In contrast, experReceived May 31, 1991. Address reprint requests to: Michael Econs, M.D., Box 3298, Duke University Medical Center, Durham, North Carolina 27710. * This study was supported by NIH Grants MOl-RR-SO, AR-27032, DK-38015, and NS-26630, and Research Program Project lPOl-NS26630 (MAP-V). Presented in part at the Annual Meeting of the American Society of Bone and Mineral Research, Atlanta, GA, August 28-31, 1990. Part of this work has appeared in abstract form in J Bone Min Res (Supp 2) 5:S108, 1990.

Materials

and Methods

HYP families We performed linkage studies in five large HYP kindreds (pedigree diagrams available on request). Venous blood was obtained from 96 affected individuals (34 males and 62 females) and 172 nonaffected

201

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202

ECONS

relatives and spouses (87 males and 85 females). We ascertained the presence of disease as previously reported (15). In brief, we established the diagnosis by physical examination and evaluation of the serum phosphorus concentration, and, in some cases, urinary phosphate excre-

tion as well as bone radiographs and bone biopsy. Paternity was verified using the DNA probe DZS44 (16). We performed simulation studies with the SIMLINK computer program (17) to determine the maximum amount of information available within the HYP pedigrees. We assumed that HYP was inherited as a fully penetrant, X-linked dominant disease with an allele frequency of 0.0001 and that the two markers flanking the HYP locus were spaced 3 centiMorgans (CM; 1 CM = 1% recombination) apart. We further assumed that each of the flanking markers were biallelic with equal allele frequencies. Simulation results are based on 200 replications of the pedigrees.

DXS357) to increase the resolution of the genetic map. We determined the most likely order of the 10 markers, as well as the genetic distances between them, using the CRIMAP computer program (25, 26). The BUILD option of CRIMAP selects two of the most highly informative markers in the set and adds additional loci using a user-defined criterion for marker order. Maximum likelihood estimates of the genetic distances between markers are also computed by the BUILD option. We determined the reliability of the final marker order with the FLIPS option of CRIMAP. The FLIPS option permutes the order of a specified number of adjacent markers and compares the likelihood of each of these orders with the most likely order obtained from the BUILD option. We assumed that all of the most likely alternative marker orders could be obtained by permuting four adjacent loci using the FLIPS option.

Linkage Reference families In the generation of the genetic map for the Xp22.1-~22.2 region, we employed samples provided by the Centre d’Etudie Polymorphisms Humain (18), from a group of 40 families in which DNA has been obtained from an average of 8 children, their parents, and, in most cases, both sets of grandparents. In order to increase the number of meioses for constructing the fixed map, we also generated marker data for the

region in kindreds previously obtained for genetic linkage studies of Alports

syndrome

(19; 2 kindreds)

and neurofibromatosis

type I (20; 17

kindreds).

analysis

HYP was analyzed as an X-linked dominant trait with complete penetrance. We calculated two-point (pairwise) LOD scores between the markers and HYP using the LIPED (27) computer program. The LOD score quantifies the probability of linkage between two loci and is equal to the log,, of the odds of linkage at a particular genetic distance between two loci. A LOD (Z) score of 3 or more is evidence of linkage (lOOO:l), and a LOD score of -2 or less is evidence of nonlinkage (1OO:l) (28). The LOD scores were calculated over a range of recombination fractions (e), the genetic distance between two linked markers. The best estimate for the recombin$ion fraction (0) is that value at which the LOD score

is maximized [Z(e)].

Methods Venous blood was obtained in tubes containing ACD solution B (Vacutainer Corp., Rutherford, NJ). DNA was isolated from whole blood by standard methods (21) and 10 fig digested to completion with a 4fold excess of restriction enzyme according to the specifications of the manufacturers (Table 1). Subsequently, samples were separated on 1% agarose gels by electrophoresis and transferred to Gene Screen Plus filters (DuPont-NEN, Boston, MA) by alkaline transfer (22). In the linkage analysis of HYP we used the DNA probes DXS257, DXS365, DXS315, DXS451, DXS41, and DXS43, the allele size and frequency data for which are detailed in Table 1. We radiolabeled these polymorphic probes by oligonucleotide labeling (23) with a commercial kit (Amersham, Arlington Heights, IL) and hybridized them with the filters overnight at 42 C in a hybridization solution containing 50% formamide, 10% dextran sulfate, 1% sodium dodecyl sulfate, 20 rnM NaHzP04, 5~ Denhardt’s solution (24), 1 M NaCl, and 0.1 mg/ml sonicated, boiled salmon sperm DNA. Thereafter, filters were washed in 2X standard saline citrate (SSC) (24), 1% sodium dodecyl sulfate at 60-65 C and autoradiographed at -70 C for l-7 days.

Generation

of the Xp genetic map

In addition to DXS451, DXS315 we used additional

TABLE

JCE & M. 1992 Vol75.Nol

ET AL.

1. RFLP

allele

LOCUS

DXS41, markers

DXS257, DXS444,

DXS43, DXS443,

and and

Results Initially we analyzed pairwise the HYP gene locus. *We found

linkage of each marker to no recombination between

DXS365 and HYP [Z(e) = 13.98 at 8 = 0.00; Table ?I. Single recombinant events separated DXS257AandHYP [Z(0) = 8.09 at 0 = 0.061and DXS451 and HYP [Z(0) = 13.19 at 0 = 0.031 . We found two or more recombinants between HYP and DXS315 [Z(e) = 6.27 at 19= 0.101 and between HFP and the previously tested markers (11-13, 15), DXS41 [Z(e) = 7.31 at 0 = 0.071 and DXS43 [Z(0) = 7.19 at 0 = 0.071.

sizes and frequencies NilIlX

DXS 251

DXS365, (DXS319,

We performed multipoint linkage analysis with the LINKMAP subprogram of the linkage computer package (Version 4.6.) (29). Using the fixed genetic map that we established, HYP was placed sequentially in each interval of the map as well as on either end of the map. The multipoint LOD scores were calculated at various locations of HYP in the fixed map with the peak multipoint LOD score indicating the most likely position of the disease gene. The multipoint LOD score is the log,0 of the ratio of the likelihood of linkage of a disease (or marker) to a fixed genetic map compared to nonlinkage of the disease (or marker) to that map. Support for this position was assessed by comparing the multipoint LOD score at the most likely position with the multipoint LOD score at the next most likely position.

QST-1 H3

Location

Xp21.3-22.2

Enzyme Taq

I

DXS 315

KZO-52 R5

XpZL-22.3

Msp I

DXS 365

RX-314 E4

Xp21.3-22.2

Msp I

DXS 451

QST-80 Hl

Xp21.3-22.2

Taq

DXS41

99.6

xp22.1-22.2

PstI

DXS43

D2

xp22.1-22.2

PvuII

I

Allele Size (kb)

Frequency

Ref.

5.5 4.5 3.8 2.1 8.5 3.5 3.0 2.9 22.0 13.0 6.6 6.0

0.59

39

0.41 0.60 0.40 0.49 0.51 0.42 0.58 0.71 0.29 0.45 0.55

39 39 (Our

unpublished

observations) 40 40

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MULTILOCUS TABLE

2. Pairwise

Marker

linkage 0.00

of each marker 0.001

to the HYP 0.05

MAPPING

OF HYP

203

gene locus

0.10

0.15

0.20

0.40

0.30

Z(B)

s

C.I. (0)

95%

(41)

DXS365 DXS451 DXS257 DXS41 DXS43 DXS315

13.98 m 00 m ~0 00

13.95 11.18 7.07 3.20 2.92 -6.98

12.61 13.11 8.01 7.17 7.13 5.26

11.19 12.15 7.47 6.88 7.10 6.27

9.73 10.89 6.77 6.19 6.65 6.22

Subsequently, we defined a genetic map of the Xp22.1~22.2 region that permitted localization of the HYP gene locus. We analyzed segregation of the various markers employed through the HYP kindreds and the reference families using multipoint analysis with the CRIMAP program. We were able to order six of the 10 markers used in this analysis at criterion odds of 1OOO:l or greater. The most likely order of these markers is Xpter-DXS315-DXS43-DXS365-DXS41DXS45 1-DXS3 19-Xcen. DXS444 and DXS257 mapped to the same interval as DXS315 and DXS365, respectively, and were nonrecombinant with these markers. Therefore, we haplotyped these pairs of loci. The least two informative markers, DXS357 and DXS443, could not be positioned into a single interval defined by the framework map and were not considered further. After haplotyping theApairs of nonrecombinant markers [DXS315 and DXS444, Z(0) = 9.03 at 8 = 0.0; and DXS365 and DXS257, Z(0) = 17.46 at 8 = 0.01 the most likely order is Xpter-(DXS315/DXS444)-DXS43-(DXS257/DXS365) -DXS41-DXS451-DXS319-Xcen. Subsequently, we permuted sets of four adjacent loci from the most likely order and compared its likelihood with that of each alternative order. The most likely order was 1OOO:l or greater more likely than any other order. When this analysis was performed using only the Centre d’Etudie Polymorphisms Humain reference families, we obtained similar results except that the odds positioning DXS43 centromeric to DXS315/DXS444 decreased from 1OOO:l or greater odds to 575:l odds. In order to localize the HYP gene within this genetic map, we analyzed the recombination events in the various HYP kindreds. Such efforts are illustrated in a partial pedigree from a single family (Fig. 1). Patient II-3 is an affected woman who is heterozygous for the markers DXS43, DXS257, and DXS451. Her affected daughter, 111-3, shows segregation of the disease with alleles (b, c, and F) of these markers, as do all but one of the other affected members in the family. This single patient, 111-5, the son of 11-3, exhibits recombination between HYP and DXS43 and DXS257, and consequent segregation of the disease with the B and C alleles of these respective markers. In contrast, there was no recombination between HYP and the more centromeric marker DXS451. Regardless, the diagnosis of HYP in individual III-5 was confirmed on two occasions. This 25-yr-old man, who displays short stature as compared to other family members, valgus deformity of the lower extremities, and multiple tooth abscesses, had marked hypophosphatemia [serum phosphorus of 0.39 and 0.52 mmol/L (normal 0.81-1.45 mmol/L)]. Thus, these observations, coupled with the marker-to-marker order established above, demonstrate that DXS257 and

8.22 9.46 5.97 5.33 6.01 5.73

1.94 2.90 2.18 1.15 2.34 1.58

5.11 6.30 4.18 3.36 4.36 3.99

I

13.98 13.19 8.09 7.31 7.19 6.27

0.00 0.03 0.06 0.07 0.07 0.10

T 1

a

0.00-0.04 0.01-0.09 0.001-0.12 0.02-0.13 0.01-0.18 0.05-0.22

2

2

4

5 DXS315-a DXS43 -e DXS257-C DXS365-D DXS41 -E DXS451-F

IV 1 DXS315-Aa ox%3 -bb DXS.FJ-CC DXS365-dD DXS.41 WE DXS451.F

2 DXS315-A DXS43 -b DXS257-C DXS365-d DXS.4, ‘B DXS451 -I

3 DXSSIS-a DXS43 -b DXS257-C DXS365-d DXS41 -0 DXS451-1

KEY MALE

FEMALE

/

n l

AFFECTED

j

0

NORUAL

0

J

FIG. 1. Partial pedigree demonstrating a cross-over between the teleomerit markers, DXS257 and DXS43, and HYP. Patient II-3 is an affected woman who is heterozygous for the markers DXS43, DXS257, and DXS451. Her affected daughter, 111-3, shows segregation of the disease with alleles (b, c, and F) of these markers, as do all but one of the other affected members in the family. Patient 111-5, the son of II3, exhibits recombination between HYP and DXS43 and DXS257 and consequent segregation of the disease with the B and C alleles of these respective markers. In contrast, there was no recombination with the more centromeric marker DXS451. This mating was not informative for DXS365 and DXS41. All results were confirmed by recollection and reanalysis of the individuals shown.

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ECONS ET AL.

204

DXS43 are telomeric to the HYP gene, whereas DXS451 is centromeric to the gene. Unfortunately, this mating was not informative for DXS365 and DXS41 and consequently did not provide information on the relative orders of these markers and the gene. After these preliminary observations, we attempted to localize the HYP locus within the genetic map with more precision than could be accomplished by analysis of individual cross-overs. We used the previously established genetic order (with DXS365 and DXS257 haplotyped) and multilocus analysis with the LINKMAP program to determine the most likely location of the HYP gene. This analysis generated the multipoint LOD scoresillustrated in Fig. 2. There is a prominent peak between DXS257 and DXS41 with a maximum multipoint LOD score (Z) of 26.26 (73% of the maximum attainable score, 35.99 + 0.31, as calculated by the SIMLINK computer program) and a less prominent peak between DXS43 and DXS257 (Z = 23.65). Thus, HYP is 10z6* (407) times more likely to lie in the 3.5-CM interval between DXS257 and DXS41 than in the 5.5-CM interval between DXS257 and DXS43. Additional peaks (not shown) are located distal to DXS43 (Z = 20.92) and proximal to DXS451 (Z = 20.59). Discussion Although an inborn error of phosphate transport undoubtedly underlies the pathogenesis of HYP, controversy exists regarding the etiology of the disorder. Thus positional cloning may be necessary to elucidate the pathophysiology of the disorder. This approach has been successfully used to 2126.26

2'1 26 25 24 ’ i

23 22 21 20 19 18 -5

0 GENETIC

5

10

DISTANCE

FIG. 2. LINKMAP analysis of the HYP locus. the genetic location in centiMorgans. DXS43 0.00, and the other loci were positioned from previously described genetic map. The vertical LOD score for the placement of HYP at a given

15

CM

The horizontal axis is was arbitrarily set at it according to the axis is the multipoint position on the map.

JCE & M. 1992 Vol75.Nol

identify genetic abnormalities responsible for a variety of different diseases(30-33). It permits localization and characterization of a disease gene without precise knowledge regarding the pathophysiology of the disorder. Requisite to this technique, however, is not only identification of the chromosome on which a diseasegene is localized but successful fine structure mapping of the appropriate genetic region and establishment of tightly linked flanking markers. Such efforts permit precise gene localization and ultimate cloning. Unfortunately, application of this methodology to HYP has been limited despite early clinical studiesthat established familial hypophosphatemic rickets as an X-linked dominant disorder (34-36). In this regard, the lack of sufficient polymorphic probes for the X chromosome, the limited number of patient samples available for linkage analysis, and the failure to extend the database by the use of reference pedigrees have prevented significant progress. Indeed, the previous efforts of Machler et al. (11) and Thakker et al. (13) provided only a limited map of the region around the gene and did not identify tightly linked flanking markers. In the current study, therefore, we used a variety of strategies to markedly improve the genetic map in the area of interest and to successfully localize the HYP gene. Our linkage study of HYP, using multiple previously untested polymorphic markers to define the genetic map of the short arm of the X chromosome, established tight linkage between HYP and DXS365 and DXS257. Additionally, we confirmed previous reports (1l-l 6) of linkage between HYP and DXS41 and DXS43. More importantly, we successfully used multilocus mapping with these markers to define a previously unavailable detailed map of the Xp22.1-~22.2 region and calculate the most likely location for the HYP gene. Within the order Xtel-DXS315-DXS43-(DXS365/ DXS257)-DXS41-DXS451-Xcen, a location of HYP between DXS257 and DXS41 was favored above all other locations. The genetic distancesbetween these flanking markers is 3.5 CM. Although great caution must be exercised when correlating genetic and physical distances, this corresponds to a physical distance of approximately 3.5 million base pairs. In spite of these advances in genetic mapping, we could not determine the position of DXS365 relative to the HYP locus or DXS257. The absence of cross-overs between DXS365 and HYP, and the lack of information provided by DXS365 in the one instance that DXS257 crossedwith HYP, precluded estimation of the relative position of DXS365 and these loci. Thus, further studies may provide additional resolution of the genetic map if the definitive position of DXS365 is determined. Our results are in agreement with previous studies in which the linkage of DXS41 and DXS43 with HYP were reported (11, 13, 15). In addition, our data confirm the results of Thakker et al. (13), who established the relative positions of DXS43 (telomeric) and DXS41 (centromeric) to the HYP gene. Moreover, the distance we determined for the region between these markers (8 CM) compares favorably with the estimate (9 CM) of Alitalo et al. (37). In contrast, Thakker et al. (13) reported a considerably greater distance (24.5 CM)

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MULTILOCUS between DXS43 and DXS41. Although the reason(s) for this apparent discrepancy is not certain, the data set that we used was much larger, and the 95% confidence limits for the distances estimated by Thakker et al. (13) encompass those obtained in our study. Therefore, it appears likely that the genetic distance between DXS41 and DXS43 is considerably less than 24.5 CM. In any case, our studies have established a detailed map of the region around the HYP locus and identified closely linked genetic markers for HYP. These markers may be important tools for genetic counseling and may also aide in the diagnosis of affected children in the first few months of life when HYP may not be apparent (38). Additionally, they may be useful in mapping other disorders with a genetic defect in the Xp22 region. More importantly, the mapping of HYP by our linkage study represents an important step in the precise localization and ultimate cloning of the HYP gene. Such investigations may provide essential information requisite to determining the pathophysiological abnormality that underlies HYP.

MAPPING

12.

13.

14.

15.

16.

17.

18. 19.

Acknowledgments 20. We are indebted to the study subjects who consented to give blood for the linkage studies. We would also like to thank Mrs. Elizabeth Ross and Mrs. Zelda Wood for their assistance in the genealogical studies of the HYP kindreds. We are indebted to Drs. Wu Yen Hung and Larry Yamaoka for their thoughtful advice and guidance. Ms. Claire Flaresheim provided expert technical assistance.

21.

22. 23.

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