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Fine Mapping of the Cystinosis Gene Using an Integrated Genetic and Physical Map of a Region within Human Chromosome Band 17p13
BIOCHEMICAL AND MOLECULAR MEDICINE ARTICLE NO.
58, 135–141 (1996)
0041
Fine Mapping of the Cystinosis Gene Using an Integrated Genetic and Physical Map of a Region within Human Chromosome Band 17p13 GERALDINE MCDOWELL,* TAKAO ISOGAI,*,† AKIRA TANIGAMI,‡ SENATOR HAZELWOOD,*,§ DAVID LEDBETTER,‡ MIHAEL H. POLYMEROPOULOS,Ø UTA LICHTER-KONECKI\,** DAVID KONECKI,\ MARGARET M. TOWN,†† WILLIAM VAN’T HOFF,‡‡ JEAN WEISSENBACH,*** AND WILLIAM A. GAHL* *Section on Human Biochemical Genetics, Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892; †Fujisawa Pharmaceutical Co., Ltd., Ibaraki 300-26, Japan; ‡Diagnostic Development Branch, National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892; §Howard Hughes Medical Institute, NIH Research Scholar Program; Ø Laboratory of Genetic Disease Research, National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892; \Molecular and Cellular Biology Laboratory, Marshfield Medical Research Foundation Center for Medical Genetics, Marshfield, Wisconsin 54449; **University Children’s Hospital, Heidelberg, Germany; ††Division of Medical and Molecular Genetics, UMDS, Guy’s Hospital, London SE1 9RT, United Kingdom; ‡‡Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom; and ***CNRS URA 1922, Genethon, Human Genome Research Center, 1 rue de l’Internationale, BP60, 91002 Evry, France Received April 18, 1996, and in revised from May 14, 1996
present with renal tubular Fanconi syndrome in the first year of life, rickets and growth retardation in early childhood, and renal glomerular failure by approximately 10 years of age (4,5). Children with cystinosis also suffer photophobia due to corneal crystal accumulation and primary hypothyroidism due to tissue fibrosis. Patients who survive into their twenties and thirties by virtue of a renal allograft may develop other complications (6), including retinal blindness (7), a distal vacuolar myopathy (8), swallowing difficulties (9), and pancreatic insufficiency (10,11). The cystinedepleting agent cysteamine (12), or Cystagon, has proven efficacy in retarding renal damage and enhancing growth (13,14), preventing hypothyroidism (15), and lowering muscle cystine content in cystinosis (16). Given topically, it also dissolves corneal cystine crystals (17,18). Despite documentation of the basic transport defect in cystinosis and extensive knowledge of its clinical consequences, the cystinosis gene product itself has not been isolated. Biochemical approaches are difficult because of the low abundance of the transport protein, its membranous milieu, and its relatively low affinity for ligands (19). Hence, a molecular approach has been pursued, using linkage to map the gene to chromosome 17p13 between markers D17S1583 and D17S1584 (Fig. 1) (20). This was fol-
The cystinosis gene has been reported to reside in a 3.1 cM region of chromosome 17p13 flanked by markers D17S1828 and D17S1798. We created a yeast artificial chromosome (YAC) contig between these markers and report here an integrated genetic and physical map which will aid in the identification of other genes in this area. Using one pertinent YAC clone, 898A10, we identified new polymorphic markers in the cystinosis gene region. One such marker, D17S2167, was localized by radiation hybrid analysis to within 10.2 cR8000 of D17S1828. Haplotype analysis in two separate informative families revealed recombination events which placed the cystinosis gene between markers D17S1828 and D17S2167, an area estimated to be 187–510 kb in size. This dramatic narrowing of the cystinosis gene region permits the creation of a P1 or cosmid contig across the area of interest. The ultimate cloning of the cystinosis gene should eventually reveal how a functional lysosomal transport protein is synthesized, targetted, processed, and integrated into the lysosomal membrane. q 1996 Academic Press, Inc.
Nephropathic cystinosis, an autosomal recessive lysosomal storage disorder, results from impaired transport of free cystine out of cellular lysosomes (1–3). Cystine trapped within lysosomes forms crystals and causes cell and tissue destruction. Clinically, patients 135
1077-3150/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Integrated genetic and physical map of chromosome 17p in the region of the cystinosis gene. (A) Chromosome 17 ideogram showing the approximate location of the cystinosis gene region on band: 17p13. (B) Genetic map of the region flanking the cystinosis gene (25). Sex-averaged distances between markers are given in cM. Linkage and recombinant analyses previously placed the gene between D17S1828 and D17S1583, as indicated by shaded area. (C) Radiation hybrid map of the cystinosis region. Filled circles indicate polymorphic markers and open circles nonpolymorphic markers. Distances between markers are given in cR8000 units. Recombination analysis has placed the cystinosis gene between D17S1828 and D17S2167, as indicated by the shaded area. (D) YAC contig of the cystinosis gene region. A circle signifies the presence of a marker and an X the absence of the marker on CEPH YAC clones 767F9, 898A10, 416F9, and 770F3.
lowed by high-resolution mapping (21) to a region between markers D17S1798 and D17S1828 estimated to span 3.1 cM. Several genes responsible for human diseases have been mapped to chromosome 17p13, including Canavan disease (22), autosomal dominant retinitis pigmentosa (23), and Miller– Dieker syndrome (24). We now report the narrowing of the cystinosis gene locus to a 187–510 kb region using a new polymorphic marker and an integrated genetic and physical map of this region. These data not only provide mileposts for the mapping of other genes in the area, but also allow identification of P1 and cosmid clones whose expressed sequences contain the cystinosis gene itself. MATERIALS AND METHODS Patient Samples DNA was obtained from cystinosis patients who contributed to the previous Cystinosis Collaborative
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Research Group study (20) or were cared for by the authors (U.L.-K., W.A.G.). Genomic DNA was extracted from leucocytes as described (31). Yeast Artificial Chromosomes YAC clones from the CEPH YAC libraries were obtained from NCHGR/NIH, and YAC DNA was extracted as described (32). PCR reactions included total yeast DNA (Ç50 ng) or YAC DNA (0.2 ng), 10 mM Tris – HCl buffer, pH 8.3, 200 mM dNTPs, 1 mM of each primer (Table 1), and 0.25 u Taq DNA polymerase (Boehringer-Mannheim) in a total volume of 10 ml. Amplification consisted of denaturing at 947C for 2 min followed by 35 cycles of 947C for 10 s, 567C for 10 s, and 727C for 40 s, followed by 727C for 7 min. For PCR of marker D17S1583, 40 cycles were used, and the annealing temperature was changed to 557C. Agarose gel electrophoresis was performed as previously described, using 3% NuSieve GTG agarose and 1.5% agarose ME (FMC Bioproducts) (31).
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TABLE 1 Reagents Used for Fine Mapping of the Cystinosis Gene Region Reagent
Source/Ref
YACs 767F9 898A10 416F9 770F3 Markers (polymorphic) D17S1584
NCHGR/NIH NCHGR/NIH NCHGR/NIH NCHGR/NIH 20, 25
D17S1828
20, 25
D17S1583
20, 25
D17S2167
This paper
D17S2169
This paper
Markers (nonpolymorphic) ASPA
22, 27
OLFR1
26
D17S126
27
D17S379 (A34)
27
Primers for SSR search Alu-5* Alu-3* Universal vectorette primer Universal vectorette linker
33 33 34 34
Cloning of New Polymorphic Markers Polymorphic simple sequence repeats (SSRs) were sought on YAC clone 898A10 (28). First, Alu PCR was performed using the Expand Long Template PCR System (Boehringer-Mannheim) and Alu-5* and Alu-3* primers (33). All three combinations of these two primers (Table 1) were used. Each reaction included 500 mM dNTPs, 1 mM of each primer, 0.75 ml enzyme mix (Taq and Pwo DNA polymerase), and 50 mM Tris–HCl buffer, pH 9.2, containing 16 mM ammonium sulfate and 2.25 mM magnesium chloride in a total volume of 50 ml. Cycling conditions were 947C for 2 min followed by 30 cycles of 947C for 10 s, 607C for 30 s, and 687C for 20 min, with 7 min at 687C for extension. Alu PCR products were separated on a 1% agarose gel (1 1 TAE), transferred and cross-linked to nylon filters, and hybridized with the 32P-labeled probes
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5*-AGC TGC TTC TGC AAA GAT G-3* 5*-TAC AAG TCC TGG GCC AC-3* 5*-TGC ACT CAC AGA TTT GCC-3* 5*-TTA AGC CAC TTC GGA TTT G-3* 5*-TGC CCA TGC TGA CAT A-3* 5*-GAC CTG ACT AAA ANA CTC CA-3* 5*-TTC CTA AGT CTC AAA TGA GAC C-3* (forward) 5*-ATC CTA GAA AGA AGC AGT TTG C-3* (reverse) 5*-GAT CAG ATG GAC AAG AGA G-3* (forward) 5*-TCA TGG ACA ACA GCC CAA G-3* (reverse) 5*-CTC TTG ATG GGA AGA CGA TC-3* 5*-ACA CCG TGT AAG ATG TAA GC-3* 5*-TGT GTG TTA TAC AGC AGT GAT TGG-3* 5*-CTG TAC TTT GGA TGT GTG CTA GAT G-3* 5*-GGT CAC ATT AAC CTA TGT CTT TG-3* 5*-CAT ATT GGC ACA CCA ACA CAT CAG-3* 5*-CCT AAC TGA ATG ACA TGG AGG AC-3* 5*-GTT GGA ACA GAA CTA TGA ATA AC-3* 5*-GGA TTA CAG GCG TGA GCC AC-3* 5*-GAT CGC GCC ACT GCA CTC C-3* 5*-C GAA TCG TAA CCG TTC GTA CGA GAA TCG CT-3* 5*-G ATC AAG GAG AGG ACG CTG TCT GTC GAA GGT AAG GAA CGG ACG AGA GAA GGG AGA G-3* 5*-CTC TCC CTT CTC GAA TCG TAA CCG TTC GTA CGA GAA TCG CTG TCC TCT CCT T-3*
(CA)22 , (AAAG)10 , (AAAT)10 , and (AGC)10 . The probes were 5*-end-labeled using g-[32P]ATP and T4 polynucleotide kinase. Hybridization was performed with 51 SSPE, 51 Denhart’s solution, 0.5% SDS, and 100 mg/ml sheared salmon sperm DNA at 657C overnight, followed by washing for 30 min at 657C twice in 21 SSC, 0.1% SDS. Only the (CA)22 probe revealed positive bands, of size 7.2, 5.3, and 3.9 kb. The Alu PCR products were digested with Sau3AI. A universal vectorette linker (Table 1) was ligated to the digested products, and these ligation products served as templates for PCR amplification. Since the primers used included the universal vectorette primer (34) and either G4(GT)12 or G4(CA)12 , amplification occurred in both directions from any GT/CA repeat. PCR was carried out as described for YAC DNA, except that annealing occurred at 657C for 30 s. The PCR products representing both-strand fragments were size fractionated on an agarose gel (3%
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NuSieve, 1.5% agarose ME) in 11 TAE and the small fragments (70–900 bp) cloned into TA cloning vector pCRII (In Vitrogen). Twelve GT-directed clones (70– 800 bp) and 9 CA-directed clones (70–300 bp) were sequenced using Sequenase Version 2.0 (U.S. Biochemicals). Primers were made which corresponded to sequences in 4 CA-direction clones and 6 GT-direction clones. Amplification was carried out as described for YAC DNA using all combinations of CAdirection and GT-direction primers. If a CA-direction and GT-direction pair of primers yielded a PCR product of the size predicted by their clones’ sequences, then the primer pair matched and flanked a CA repeat. Such was the case for the primer pairs which defined the SSRs D17S2167 and D17S2169 (Table 1). Radiation Hybrid Analysis Genotyping was performed for the 83 radiation hybrids in the Stanford G3 panel (Research Genetics, Birmingham, AL) as well as a positive human DNA control and a negative rodent DNA control. Each PCR reaction contained 25 ng hybrid DNA, 1.5 mM MgCl2 , 10 mM Tris, 50 mM KCl, 0.01% (w/v) gelatin, 0.1% Triton X-100, 200 mM each of dATP, dGTP, and dTTP, 2.4 mM dCTP, 2.5 mCi a[32P]dCTP (3000 Ci/mmol), 60 ng of each primer, and 0.45 u Taq DNA polymerase (Boehringer-Mannheim) in a total volume of 15 ml. Amplification consisted of 30 cycles of 947C for 1.5 min, 577C for 1 min, and 727C for 1 min. PCR products were fractionated on 6% acrylamide gels and each hybrid was scored by visual inspection of the resulting autoradiograph. The data were analyzed by multipoint maximum likelihood analysis using the RHMAP statistical package (35). A branch and bound ordering strategy was employed assuming equal retention probabilities for all fragments. Distances between markers were expressed in cR8000 , which correspond to 1% breakage frequency. RESULTS Construction of a Yeast Artificial Chromosome (YAC) Contig Several YAC clones from the CEPH YAC libraries were previously mapped to chromosome 17p13 (A.T.). These YACs were screened by PCR with primers for polymorphic markers D17S1584, D17S1828, D17S1583, and D17S1798 (25), which flank the cystinosis gene. The YACs were also screened for the nonpolymorphic markers ASPA (22), OLFR1 (26),
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D17S379(A34), and D17S126 (27), which are located in the area surrounding the cystinosis gene. A total of four YAC clones (767F9, 898A10, 416F9, and 770F3), forming a contig between markers D17S1584 and D17S379 (Fig. 1), were identified. Because the YAC clone 898A10 was completely contained within the cystinosis region as defined by markers D17S1828 and D17S1798, it was used as a template to identify new SSRs which could be used to further narrow the cystinosis gene region. Recombination Mapping and Identification of New SSRs The placement of polymorphic markers from 17p, including D17S1584, D17S1828, D17S1583, and D17S1798, on a genetic map with an odds ratio of 1000:1 allowed for accurate recombination mapping of the cystinosis gene (25). Haplotypes with six markers from 17p were constructed for 31 cystinosis families assuming the most parsimonious linkage phase. In family 30, recombination occurred in affected individuals between markers D17S1828 and D17S1583 on the paternal chromosome (Fig. 2). This recombination event indicated that the cystinosis gene was telomeric to D17S1828, confirming the findings of others (21). The narrowed region of interest, however, was still 3.1 cM in size, according to the most recent linkage map (25). Since this was too large for the identification of candidate genes, we sought new polymorphic markers in this interval by hybridizing YAC Alu PCR products with a poly(CA/GT) probe under conditions of high stringency (28). Two new SSRs, D17S2167 and D17S2169, were identified which were polymorphic in our cystinosis families. D17S2167 had the polymorphic sequence (TG)8TTTA(TG)6CG(TG)10 , with allele sizes of 161 and 169 bp in our families. D17S2169 consisted of an imperfect AG repeat followed by (AGG)8 ; its alleles were 122 and 131 bp in our families. PCR amplification verified that both D17S2167 and D17S2169 were located on YAC clones 767F9 and 898A10. However, the precise location of these new markers in relation to the previously known markers remained to be determined. Radiation Hybrid Mapping The 10 polymorphic and nonpolymorphic markers used to identify our YAC contig were examined by PCR amplification for their presence or absence in 83 radiation hybrids of the Stanford G3 panel (29). The order of the markers and the distances separating them were determined using the computer pro-
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FIG. 2. Haplotype analysis of cystinosis patients with critical recombination events. Genotypes are indicated for 6 polymorphic microsatellite loci in two cystinosis families. Affected individuals are shaded. Half-shaded individuals are obligate heterozygotes. Haplotypes associated with the cystinosis gene are boxed. The affected individuals in both families 30 and 20 share the region of 17p between markers D17S1828 and D17S2167.
gram RHMAP (Fig. 1). The two best orders, supported by odds of at least 100:1, differed only in the positions of OLFR1 and D17S126. We show in Fig. 1 the order which conforms to previous physical mapping of these markers (30), although inversion of this order and placement of OLFR1 telomeric to D17S126 would be the most parsimonious solution with respect to YAC 767F9. This alternative placement of these markers would not require an interstitial deletion of YAC 767F9. Markers D17S1583 and D17S1798 had identical retention patterns and, therefore, their order could not be determined. Both D17S2167 and D17S2169, as well as ASPA and OLFR1, were located within the cystinosis region flanked by D17S1828 and D17S1583 (Fig. 1). The total distance between D17S1583 and D17S1584 was 85.9 cR8000 , and the distance between D17S1828 and D17S2167 was 10.2 cR8000 . Recombination Mapping Using New SSRs Haplotype analysis, including the new polymorphic markers D17S2167 and D17S2169, was performed in five families previously shown to have obligate recombination between the cystinosis gene and other 17p loci. The genotypes for D17S2169 were uninformative in these families. However, in family 20, recombination occurred in an affected individual between markers D17S1828 and D17S2167 on the paternal chromosome (Fig. 2). This placed the cystinosis gene centromeric to D17S2167 and, together with the recombination event in family 30, defined a 10.2 cR8000 candidate region between D17S1828 and D17S2167.
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DISCUSSION The search for the cystinosis gene was advanced significantly by the Cystinosis Collaborative Research Group’s recent demonstration of linkage to chromosome 17p13 (20). In that report, and in our subsequent studies, there has been no evidence for locus heterogeneity for the cystinosis gene. Apparent inconsistencies in the previously reported family 12 resulted from misidentification of patient samples. Furthermore, two families with juvenile/adolescent cystinosis have now contributed positive lod scores for linkage to chromosome 17p (data not shown), consistent with allelism of this cystinosis variant with classical nephropathic cystinosis. The area of the cystinosis gene was narrowed to a region of approximately 3.1 cM according to a recently published genetic map of the region (21,25). The crucial recombination event defining this interval involved an individual determined to be a cystinosis carrier on the basis of leucocyte cystine levels (21). The authors called for confirmation of this finding, and the present paper provides such verification. A recombination event in an affected individual in our family 30 indicates that the gene lies telomeric to D17S1828 (Fig. 2). We previously demonstrated that the telomeric border of the cystinosis region was D17S1583 (20). Although D17S1583 could not be distinguished from D17S1798 by our radiation hybrid analysis, it appears to lie centromeric to D17S1798, according to the published genetic map generated by genotyping 134 individuals in 8 CEPH families (25), as well as our own analysis of 478 individuals in 37
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CEPH families (data not shown). In addition, two affected Pakistani children of a first-cousin marriage were homozygous for D17S1583, as well as more centromeric markers, but not for D17S1798 (data not shown). However, the frequencies of these different alleles in the Pakistani population are unknown. Our focused search for markers in the cystinosis gene region used the stable YAC clone 898A10 as a template and identified several candidate SSRs. One of these, D17S2167, was only 10.2 cR8000 from D17S1828. A fortuitous recombination event in family 20 indicated that the cystinosis gene lies centromeric to D17S2167 (Fig. 2). Since the gene was shown to be telomeric to D17S1828 through recombination in family 30, these data together place the gene between D17S1828 and D17S2167. Our radiation hybrid mapping estimated the length of the recently reported cystinosis interval (D17S1828–D17S1798) to be 66.5 cR8000 . The new interval reported here (D17S1828–D17S2167) has a length of 10.2 cR8000 , which is 15% of the previous size. Data from chromosome 21 suggest that one cR8000 is equivalent to approximately 50 kb (29). Therefore, the current cystinosis gene region may be 510 kb in size. An alternative size analysis would examine only the specific area of chromosome 17p surrounding the cystinosis gene. For example, the estimated distance between markers OLFR1 and D17S126 is 73 kb (30), and we found that these markers are 4.0 cR8000 apart, providing a conversion factor of 18.3 kb/cR8000 . Using these data, the new cystinosis interval would span only 187 kb. A segment of DNA this size should be suitable for the identification of candidate genes. We report here the physical mapping of four YAC clones which form a contig across the original cystinosis interval of D17S1584 and D17S1583. These data alone should prove useful in the future orientation of various genes in the area. In addition, markers D17S1828 and D17S2167, which flank the cystinosis gene, can be used to screen a chromosome 17-specific cosmid library, as well as P1 clones and BAC clones. These studies, currently underway, will allow creation of a smaller contig across the cystinosis region and the identification of expressed sequences. The ultimate description of the cystinosis gene should provide new insights into the synthesis, targeting, processing, and integration of functional transporters in the lysosomal membrane. ACKNOWLEDGMENTS We thank Drs. Frances L. Harley and Paul Roy for obtaining certain patient DNA samples and Dr. Settara Chandrasekhara-
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ppa for providing helpful YAC clones. Dr. Michael Boehnke graciously offered advice regarding the radiation hybrid analysis. M.M.T. and W.v.H. were supported in part by the Medical Research Council (UK). U.L.-K. was supported by the Deutche Forschungsgemeinschaft (D.F.G.) Grants Li 375/5-1, -2 (D.F.G. Schwerpunktprogramm ‘‘Analyse des menschen Genoms mit molekularbiologischen Methoden’’), Li 375/6.1, 6.2 (Habilitations stipendium).
REFERENCES 1. Gahl WA, Bashan N, Tietze F, Bernardini I, Schulman JD. Cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis. Science 217:1263– 1265, 1982. 2. Gahl WA, Tietze F, Bashan N, Steinherz R, Schulman JD. Defective cystine exodus from isolated lysosome-rich fractions of cystinotic leucocytes. J Biol Chem 257:9570–9575, 1982. 3. Jonas AJ, Smith ML, Schneider JA. ATP-dependent lysosomal cystine efflux is defective in cystinosis. J Biol Chem 257:13185–13188, 1982. 4. Gahl WA. Cystinosis coming of age. Adv Pediatr 33:95–126, 1986. 5. Gahl WA, Schneider JA, Aula P. Lysosomal transport disorders: Cystinosis and sialic acid storage disorders. In The Metabolic and Molecular Bases of Inherited Disease (Scriver CR, Beaudet AL, Sly WS Valle D, Eds.). New York: McGraw–Hill, 1995, pp. 3763–3797. 6. Theodoropoulos DS, Krasnewich D, Kaiser-Kupfer MI, Gahl, WA. Classical nephropathic cystinosis as an adult disease. JAMA 270:2200–2204, 1993. 7. Kaiser-Kupfer M, Caruso RC, Minckler DS, Gahl WA. Longterm ocular manifestations in nephropathic cystinosis postrenal transplantation. Arch Ophthalmol 104:706–711, 1986. 8. Charnas LR, Luciano CA, Dalakas M, Gilliatt RW, Bernardini I, Ishak K, Cwik VA, Fraker D, Brushart TA, Gahl WA. Distal vacuolar myopathy in nephropathic cystinosis. Ann Neurol 35: 181–188, 1994. 9. Sonies BC, Ekman EF, Andersson HC, Adamson M, Kaler S, Markello T, Gahl WA. Swallowing dysfunction in nephropathic cystinosis. N Engl J Med 323:565–570, 1990. 10. Fivush B, Flick JA, Gahl WA. Pancreatic exocrine insufficiency in a patient with nephropathic cystinosis. J Pediatr 112:49–51, 1988. 11. Fivush B, Green OC, Porter CC, Balfe JW, O’Regan S, Gahl WA. Pancreatic endocrine insufficiency in post-transplant cystinosis. Am J Dis Child 141:1087–1089, 1987. 12. Thoene JG, Oshima RG, Crawhall JC, Olson DL, Schneider JA. Cystinosis: Intracellular cystine depletion by aminothiols in vitro and in vivo. J Clin Invest 58:180–189, 1976. 13. Gahl WA, Reed GF, Thoene JG, Schulman JD, Rizzo WB, Jonas AJ, Denman DW, Schlesselman JJ, Corden BJ, Schneider JA. Cysteamine therapy for children with nephropathic cystinosis. N Engl J Med 316:971–977, 1987. 14. Markello TC, Bernardini IM, Gahl WA. Improved renal function in children with cystinosis treated with cysteamine. N Engl J. Med 328:1157–1162, 1993. 15. Kimonis VE, Troendle J, Yang ML, Rose SR, Markello TC, Gahl WA. Effects of early cysteamine therapy on thyroid
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FINE MAPPING OF THE CYSTINOSIS GENE function and growth in nephropathic cystinosis. J. Clin Endocrinol Metab 80:3257–3261, 1995. 16. Gahl WA, Charnas L, Markello T, Bernardini I, Ishak KG, Dalakas M. Parenchymal organ cystine depletion with longterm cysteamine therapy. Biochem Med Metab Biol 48:275– 285, 1992. 17. Kaiser-Kupfer MI, Fujikawa L, Kuwabara T, Gahl WA. Removal of corneal crystals by topical cysteamine in nephropathic cystinosis. N Engl J Med 316:775–779, 1987. 18. Kaiser-Kupfer MI, Gazzo MA, Datiles MB, Caruso RC, Kuehl EM, Gahl WA. A randomized placebo-controlled trial of cysteamine eyedrops in nephropathic cystinosis. Arch Ophthalmol 108:689–693, 1990. 19. Gahl WA, Tietze F, Bashan N, Bernardini I, Schulman JD. Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions. Biochem J 216:393–400, 1983. 20. The Cystinosis Collaborative Research Group. Linkage of the gene for cystinosis to markers on the short arm of chromosome 17. Nature Genet 10:246–248, 1995. 21. Jean G, Fuchshuber A, Town MM, Gribouval O, Schneider JA, Broyer M, van’t Hoff W, Niaudet P, Antignac C. Highresolution mapping of the gene for cystinosis, using combined biochemical and linkage analysis. Am J Hum Genet 58:535–543, 1996. 22. Kaul R, Gao GP, Balamurugan K, Matalon R. Cloning of the human aspartoacylase cDNA and a common missense mutation in Canavan disease. Nature Genet 5:118–123, 1993. 23. Kojis TL, Heinzmann C, Flodman P, Ngo JT, Sparkes RS, Spence MA, Bateman JB, Heckenlively JR. Map refinement of locus RP13 to human chromosome 17p13.3 in a second family with autosomal dominant retinitis pigmentosa. Am J Hum Genet 58:347–355, 1996. 24. Stratton RF, Dobyns WB, Airhart SD, Ledbetter DH. New chromosomal syndrome: Miller–Dieker syndrome and monosomy 17p13. Hum Genet 67:193–200, 1984. 25. Dib C, Faure S, Fizames C, Samson D, Drouot N, Vignal A, Millasseau P, Marc S, Hazan J, Seboun E, Lathrop M, Gyapay G, Morissette J, Weissenbach J. A comprehensive ge-
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26.
27.
28.
29.
30.
31.
32. 33.
34.
35.
141
netic map of the human genome based on 5,264 microsatellites. Nature 380:152–154, 1996. Schurmans S, Muscatelli F, Miot F, Mattei M-G, Vassart G, Parmentier M. The OLFR1 gene encoding the HGMP07E putative olfactory receptor maps to the 17p13–p12 region of the human genome and reveals an MspI restriction fragment length polymorphism. Cytogenet Cell Genet 63:200–204, 1993. Genome Data Base, GDB (TM). The Human Genome Data Base Project, Johns Hopkins University, Baltimore, MD. World Wide Web »URL: http://gdbwww.gdb.org/gdbdoc/ topq.html…, 1995. Rotman G, Vanagaite L, Collins FS, Shiloh Y. Rapid identification of polymorphic CA-repeats in YAC clones. Mol Biotech 3:85–92, 1995. Cox DR, Burmeister M, Price ER, Kim S, Myers RM. Radiation hybrid mapping: A somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes. Science 250:245–250, 1990. Ben-Arie N, Lancet D, Taylor C, Khen M, Walker N, Ledbetter DH, Carrozzo R, Patel K, Sheer D, Lehrach H, North MA. Olfactory receptor gene cluster on human chromosome 17: Possible duplication of an ancestral receptor repertoire. Hum Mol Genet 3: 229–235, 1994. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982. Philippsen P, Stotz A, Scherf C. DNA of Saccharomyces cerevisiae. Meth Enzymol 194:169–182, 1991. Tagle DA, Collins FS. An optimized Alu-PCR primer pair for human-specific amplification of YACs and somatic cell hybrids. Hum Mol Genet 1:121–122, 1992. Riley J, Butler R, Oglivie D, Finniear R, Jenner D, Powell S, Anand R, Smith JC, Markham AF. A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucl Acids Res 18:2887–2890, 1990. Boehnke M, Lange K, Cox DR. Statistical methods for multipoint radiation hybrid mapping. Am J Hum Genet 49:1174–1188, 1991.
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Fine Mapping of the Cystinosis Gene Using an Integrated Genetic and Physical Map of a Region within Human Chromosome Band 17p13 GERALDINE MCDOWELL,* TAKAO ISOGAI,*,† AKIRA TANIGAMI,‡ SENATOR HAZELWOOD,*,§ DAVID LEDBETTER,‡ MIHAEL H. POLYMEROPOULOS,Ø UTA LICHTER-KONECKI\,** DAVID KONECKI,\ MARGARET M. TOWN,†† WILLIAM VAN’T HOFF,‡‡ JEAN WEISSENBACH,*** AND WILLIAM A. GAHL* *Section on Human Biochemical Genetics, Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892; †Fujisawa Pharmaceutical Co., Ltd., Ibaraki 300-26, Japan; ‡Diagnostic Development Branch, National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892; §Howard Hughes Medical Institute, NIH Research Scholar Program; Ø Laboratory of Genetic Disease Research, National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892; \Molecular and Cellular Biology Laboratory, Marshfield Medical Research Foundation Center for Medical Genetics, Marshfield, Wisconsin 54449; **University Children’s Hospital, Heidelberg, Germany; ††Division of Medical and Molecular Genetics, UMDS, Guy’s Hospital, London SE1 9RT, United Kingdom; ‡‡Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom; and ***CNRS URA 1922, Genethon, Human Genome Research Center, 1 rue de l’Internationale, BP60, 91002 Evry, France Received April 18, 1996, and in revised from May 14, 1996
present with renal tubular Fanconi syndrome in the first year of life, rickets and growth retardation in early childhood, and renal glomerular failure by approximately 10 years of age (4,5). Children with cystinosis also suffer photophobia due to corneal crystal accumulation and primary hypothyroidism due to tissue fibrosis. Patients who survive into their twenties and thirties by virtue of a renal allograft may develop other complications (6), including retinal blindness (7), a distal vacuolar myopathy (8), swallowing difficulties (9), and pancreatic insufficiency (10,11). The cystinedepleting agent cysteamine (12), or Cystagon, has proven efficacy in retarding renal damage and enhancing growth (13,14), preventing hypothyroidism (15), and lowering muscle cystine content in cystinosis (16). Given topically, it also dissolves corneal cystine crystals (17,18). Despite documentation of the basic transport defect in cystinosis and extensive knowledge of its clinical consequences, the cystinosis gene product itself has not been isolated. Biochemical approaches are difficult because of the low abundance of the transport protein, its membranous milieu, and its relatively low affinity for ligands (19). Hence, a molecular approach has been pursued, using linkage to map the gene to chromosome 17p13 between markers D17S1583 and D17S1584 (Fig. 1) (20). This was fol-
The cystinosis gene has been reported to reside in a 3.1 cM region of chromosome 17p13 flanked by markers D17S1828 and D17S1798. We created a yeast artificial chromosome (YAC) contig between these markers and report here an integrated genetic and physical map which will aid in the identification of other genes in this area. Using one pertinent YAC clone, 898A10, we identified new polymorphic markers in the cystinosis gene region. One such marker, D17S2167, was localized by radiation hybrid analysis to within 10.2 cR8000 of D17S1828. Haplotype analysis in two separate informative families revealed recombination events which placed the cystinosis gene between markers D17S1828 and D17S2167, an area estimated to be 187–510 kb in size. This dramatic narrowing of the cystinosis gene region permits the creation of a P1 or cosmid contig across the area of interest. The ultimate cloning of the cystinosis gene should eventually reveal how a functional lysosomal transport protein is synthesized, targetted, processed, and integrated into the lysosomal membrane. q 1996 Academic Press, Inc.
Nephropathic cystinosis, an autosomal recessive lysosomal storage disorder, results from impaired transport of free cystine out of cellular lysosomes (1–3). Cystine trapped within lysosomes forms crystals and causes cell and tissue destruction. Clinically, patients 135
1077-3150/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Integrated genetic and physical map of chromosome 17p in the region of the cystinosis gene. (A) Chromosome 17 ideogram showing the approximate location of the cystinosis gene region on band: 17p13. (B) Genetic map of the region flanking the cystinosis gene (25). Sex-averaged distances between markers are given in cM. Linkage and recombinant analyses previously placed the gene between D17S1828 and D17S1583, as indicated by shaded area. (C) Radiation hybrid map of the cystinosis region. Filled circles indicate polymorphic markers and open circles nonpolymorphic markers. Distances between markers are given in cR8000 units. Recombination analysis has placed the cystinosis gene between D17S1828 and D17S2167, as indicated by the shaded area. (D) YAC contig of the cystinosis gene region. A circle signifies the presence of a marker and an X the absence of the marker on CEPH YAC clones 767F9, 898A10, 416F9, and 770F3.
lowed by high-resolution mapping (21) to a region between markers D17S1798 and D17S1828 estimated to span 3.1 cM. Several genes responsible for human diseases have been mapped to chromosome 17p13, including Canavan disease (22), autosomal dominant retinitis pigmentosa (23), and Miller– Dieker syndrome (24). We now report the narrowing of the cystinosis gene locus to a 187–510 kb region using a new polymorphic marker and an integrated genetic and physical map of this region. These data not only provide mileposts for the mapping of other genes in the area, but also allow identification of P1 and cosmid clones whose expressed sequences contain the cystinosis gene itself. MATERIALS AND METHODS Patient Samples DNA was obtained from cystinosis patients who contributed to the previous Cystinosis Collaborative
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Research Group study (20) or were cared for by the authors (U.L.-K., W.A.G.). Genomic DNA was extracted from leucocytes as described (31). Yeast Artificial Chromosomes YAC clones from the CEPH YAC libraries were obtained from NCHGR/NIH, and YAC DNA was extracted as described (32). PCR reactions included total yeast DNA (Ç50 ng) or YAC DNA (0.2 ng), 10 mM Tris – HCl buffer, pH 8.3, 200 mM dNTPs, 1 mM of each primer (Table 1), and 0.25 u Taq DNA polymerase (Boehringer-Mannheim) in a total volume of 10 ml. Amplification consisted of denaturing at 947C for 2 min followed by 35 cycles of 947C for 10 s, 567C for 10 s, and 727C for 40 s, followed by 727C for 7 min. For PCR of marker D17S1583, 40 cycles were used, and the annealing temperature was changed to 557C. Agarose gel electrophoresis was performed as previously described, using 3% NuSieve GTG agarose and 1.5% agarose ME (FMC Bioproducts) (31).
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TABLE 1 Reagents Used for Fine Mapping of the Cystinosis Gene Region Reagent
Source/Ref
YACs 767F9 898A10 416F9 770F3 Markers (polymorphic) D17S1584
NCHGR/NIH NCHGR/NIH NCHGR/NIH NCHGR/NIH 20, 25
D17S1828
20, 25
D17S1583
20, 25
D17S2167
This paper
D17S2169
This paper
Markers (nonpolymorphic) ASPA
22, 27
OLFR1
26
D17S126
27
D17S379 (A34)
27
Primers for SSR search Alu-5* Alu-3* Universal vectorette primer Universal vectorette linker
33 33 34 34
Cloning of New Polymorphic Markers Polymorphic simple sequence repeats (SSRs) were sought on YAC clone 898A10 (28). First, Alu PCR was performed using the Expand Long Template PCR System (Boehringer-Mannheim) and Alu-5* and Alu-3* primers (33). All three combinations of these two primers (Table 1) were used. Each reaction included 500 mM dNTPs, 1 mM of each primer, 0.75 ml enzyme mix (Taq and Pwo DNA polymerase), and 50 mM Tris–HCl buffer, pH 9.2, containing 16 mM ammonium sulfate and 2.25 mM magnesium chloride in a total volume of 50 ml. Cycling conditions were 947C for 2 min followed by 30 cycles of 947C for 10 s, 607C for 30 s, and 687C for 20 min, with 7 min at 687C for extension. Alu PCR products were separated on a 1% agarose gel (1 1 TAE), transferred and cross-linked to nylon filters, and hybridized with the 32P-labeled probes
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5*-AGC TGC TTC TGC AAA GAT G-3* 5*-TAC AAG TCC TGG GCC AC-3* 5*-TGC ACT CAC AGA TTT GCC-3* 5*-TTA AGC CAC TTC GGA TTT G-3* 5*-TGC CCA TGC TGA CAT A-3* 5*-GAC CTG ACT AAA ANA CTC CA-3* 5*-TTC CTA AGT CTC AAA TGA GAC C-3* (forward) 5*-ATC CTA GAA AGA AGC AGT TTG C-3* (reverse) 5*-GAT CAG ATG GAC AAG AGA G-3* (forward) 5*-TCA TGG ACA ACA GCC CAA G-3* (reverse) 5*-CTC TTG ATG GGA AGA CGA TC-3* 5*-ACA CCG TGT AAG ATG TAA GC-3* 5*-TGT GTG TTA TAC AGC AGT GAT TGG-3* 5*-CTG TAC TTT GGA TGT GTG CTA GAT G-3* 5*-GGT CAC ATT AAC CTA TGT CTT TG-3* 5*-CAT ATT GGC ACA CCA ACA CAT CAG-3* 5*-CCT AAC TGA ATG ACA TGG AGG AC-3* 5*-GTT GGA ACA GAA CTA TGA ATA AC-3* 5*-GGA TTA CAG GCG TGA GCC AC-3* 5*-GAT CGC GCC ACT GCA CTC C-3* 5*-C GAA TCG TAA CCG TTC GTA CGA GAA TCG CT-3* 5*-G ATC AAG GAG AGG ACG CTG TCT GTC GAA GGT AAG GAA CGG ACG AGA GAA GGG AGA G-3* 5*-CTC TCC CTT CTC GAA TCG TAA CCG TTC GTA CGA GAA TCG CTG TCC TCT CCT T-3*
(CA)22 , (AAAG)10 , (AAAT)10 , and (AGC)10 . The probes were 5*-end-labeled using g-[32P]ATP and T4 polynucleotide kinase. Hybridization was performed with 51 SSPE, 51 Denhart’s solution, 0.5% SDS, and 100 mg/ml sheared salmon sperm DNA at 657C overnight, followed by washing for 30 min at 657C twice in 21 SSC, 0.1% SDS. Only the (CA)22 probe revealed positive bands, of size 7.2, 5.3, and 3.9 kb. The Alu PCR products were digested with Sau3AI. A universal vectorette linker (Table 1) was ligated to the digested products, and these ligation products served as templates for PCR amplification. Since the primers used included the universal vectorette primer (34) and either G4(GT)12 or G4(CA)12 , amplification occurred in both directions from any GT/CA repeat. PCR was carried out as described for YAC DNA, except that annealing occurred at 657C for 30 s. The PCR products representing both-strand fragments were size fractionated on an agarose gel (3%
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NuSieve, 1.5% agarose ME) in 11 TAE and the small fragments (70–900 bp) cloned into TA cloning vector pCRII (In Vitrogen). Twelve GT-directed clones (70– 800 bp) and 9 CA-directed clones (70–300 bp) were sequenced using Sequenase Version 2.0 (U.S. Biochemicals). Primers were made which corresponded to sequences in 4 CA-direction clones and 6 GT-direction clones. Amplification was carried out as described for YAC DNA using all combinations of CAdirection and GT-direction primers. If a CA-direction and GT-direction pair of primers yielded a PCR product of the size predicted by their clones’ sequences, then the primer pair matched and flanked a CA repeat. Such was the case for the primer pairs which defined the SSRs D17S2167 and D17S2169 (Table 1). Radiation Hybrid Analysis Genotyping was performed for the 83 radiation hybrids in the Stanford G3 panel (Research Genetics, Birmingham, AL) as well as a positive human DNA control and a negative rodent DNA control. Each PCR reaction contained 25 ng hybrid DNA, 1.5 mM MgCl2 , 10 mM Tris, 50 mM KCl, 0.01% (w/v) gelatin, 0.1% Triton X-100, 200 mM each of dATP, dGTP, and dTTP, 2.4 mM dCTP, 2.5 mCi a[32P]dCTP (3000 Ci/mmol), 60 ng of each primer, and 0.45 u Taq DNA polymerase (Boehringer-Mannheim) in a total volume of 15 ml. Amplification consisted of 30 cycles of 947C for 1.5 min, 577C for 1 min, and 727C for 1 min. PCR products were fractionated on 6% acrylamide gels and each hybrid was scored by visual inspection of the resulting autoradiograph. The data were analyzed by multipoint maximum likelihood analysis using the RHMAP statistical package (35). A branch and bound ordering strategy was employed assuming equal retention probabilities for all fragments. Distances between markers were expressed in cR8000 , which correspond to 1% breakage frequency. RESULTS Construction of a Yeast Artificial Chromosome (YAC) Contig Several YAC clones from the CEPH YAC libraries were previously mapped to chromosome 17p13 (A.T.). These YACs were screened by PCR with primers for polymorphic markers D17S1584, D17S1828, D17S1583, and D17S1798 (25), which flank the cystinosis gene. The YACs were also screened for the nonpolymorphic markers ASPA (22), OLFR1 (26),
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D17S379(A34), and D17S126 (27), which are located in the area surrounding the cystinosis gene. A total of four YAC clones (767F9, 898A10, 416F9, and 770F3), forming a contig between markers D17S1584 and D17S379 (Fig. 1), were identified. Because the YAC clone 898A10 was completely contained within the cystinosis region as defined by markers D17S1828 and D17S1798, it was used as a template to identify new SSRs which could be used to further narrow the cystinosis gene region. Recombination Mapping and Identification of New SSRs The placement of polymorphic markers from 17p, including D17S1584, D17S1828, D17S1583, and D17S1798, on a genetic map with an odds ratio of 1000:1 allowed for accurate recombination mapping of the cystinosis gene (25). Haplotypes with six markers from 17p were constructed for 31 cystinosis families assuming the most parsimonious linkage phase. In family 30, recombination occurred in affected individuals between markers D17S1828 and D17S1583 on the paternal chromosome (Fig. 2). This recombination event indicated that the cystinosis gene was telomeric to D17S1828, confirming the findings of others (21). The narrowed region of interest, however, was still 3.1 cM in size, according to the most recent linkage map (25). Since this was too large for the identification of candidate genes, we sought new polymorphic markers in this interval by hybridizing YAC Alu PCR products with a poly(CA/GT) probe under conditions of high stringency (28). Two new SSRs, D17S2167 and D17S2169, were identified which were polymorphic in our cystinosis families. D17S2167 had the polymorphic sequence (TG)8TTTA(TG)6CG(TG)10 , with allele sizes of 161 and 169 bp in our families. D17S2169 consisted of an imperfect AG repeat followed by (AGG)8 ; its alleles were 122 and 131 bp in our families. PCR amplification verified that both D17S2167 and D17S2169 were located on YAC clones 767F9 and 898A10. However, the precise location of these new markers in relation to the previously known markers remained to be determined. Radiation Hybrid Mapping The 10 polymorphic and nonpolymorphic markers used to identify our YAC contig were examined by PCR amplification for their presence or absence in 83 radiation hybrids of the Stanford G3 panel (29). The order of the markers and the distances separating them were determined using the computer pro-
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FIG. 2. Haplotype analysis of cystinosis patients with critical recombination events. Genotypes are indicated for 6 polymorphic microsatellite loci in two cystinosis families. Affected individuals are shaded. Half-shaded individuals are obligate heterozygotes. Haplotypes associated with the cystinosis gene are boxed. The affected individuals in both families 30 and 20 share the region of 17p between markers D17S1828 and D17S2167.
gram RHMAP (Fig. 1). The two best orders, supported by odds of at least 100:1, differed only in the positions of OLFR1 and D17S126. We show in Fig. 1 the order which conforms to previous physical mapping of these markers (30), although inversion of this order and placement of OLFR1 telomeric to D17S126 would be the most parsimonious solution with respect to YAC 767F9. This alternative placement of these markers would not require an interstitial deletion of YAC 767F9. Markers D17S1583 and D17S1798 had identical retention patterns and, therefore, their order could not be determined. Both D17S2167 and D17S2169, as well as ASPA and OLFR1, were located within the cystinosis region flanked by D17S1828 and D17S1583 (Fig. 1). The total distance between D17S1583 and D17S1584 was 85.9 cR8000 , and the distance between D17S1828 and D17S2167 was 10.2 cR8000 . Recombination Mapping Using New SSRs Haplotype analysis, including the new polymorphic markers D17S2167 and D17S2169, was performed in five families previously shown to have obligate recombination between the cystinosis gene and other 17p loci. The genotypes for D17S2169 were uninformative in these families. However, in family 20, recombination occurred in an affected individual between markers D17S1828 and D17S2167 on the paternal chromosome (Fig. 2). This placed the cystinosis gene centromeric to D17S2167 and, together with the recombination event in family 30, defined a 10.2 cR8000 candidate region between D17S1828 and D17S2167.
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DISCUSSION The search for the cystinosis gene was advanced significantly by the Cystinosis Collaborative Research Group’s recent demonstration of linkage to chromosome 17p13 (20). In that report, and in our subsequent studies, there has been no evidence for locus heterogeneity for the cystinosis gene. Apparent inconsistencies in the previously reported family 12 resulted from misidentification of patient samples. Furthermore, two families with juvenile/adolescent cystinosis have now contributed positive lod scores for linkage to chromosome 17p (data not shown), consistent with allelism of this cystinosis variant with classical nephropathic cystinosis. The area of the cystinosis gene was narrowed to a region of approximately 3.1 cM according to a recently published genetic map of the region (21,25). The crucial recombination event defining this interval involved an individual determined to be a cystinosis carrier on the basis of leucocyte cystine levels (21). The authors called for confirmation of this finding, and the present paper provides such verification. A recombination event in an affected individual in our family 30 indicates that the gene lies telomeric to D17S1828 (Fig. 2). We previously demonstrated that the telomeric border of the cystinosis region was D17S1583 (20). Although D17S1583 could not be distinguished from D17S1798 by our radiation hybrid analysis, it appears to lie centromeric to D17S1798, according to the published genetic map generated by genotyping 134 individuals in 8 CEPH families (25), as well as our own analysis of 478 individuals in 37
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CEPH families (data not shown). In addition, two affected Pakistani children of a first-cousin marriage were homozygous for D17S1583, as well as more centromeric markers, but not for D17S1798 (data not shown). However, the frequencies of these different alleles in the Pakistani population are unknown. Our focused search for markers in the cystinosis gene region used the stable YAC clone 898A10 as a template and identified several candidate SSRs. One of these, D17S2167, was only 10.2 cR8000 from D17S1828. A fortuitous recombination event in family 20 indicated that the cystinosis gene lies centromeric to D17S2167 (Fig. 2). Since the gene was shown to be telomeric to D17S1828 through recombination in family 30, these data together place the gene between D17S1828 and D17S2167. Our radiation hybrid mapping estimated the length of the recently reported cystinosis interval (D17S1828–D17S1798) to be 66.5 cR8000 . The new interval reported here (D17S1828–D17S2167) has a length of 10.2 cR8000 , which is 15% of the previous size. Data from chromosome 21 suggest that one cR8000 is equivalent to approximately 50 kb (29). Therefore, the current cystinosis gene region may be 510 kb in size. An alternative size analysis would examine only the specific area of chromosome 17p surrounding the cystinosis gene. For example, the estimated distance between markers OLFR1 and D17S126 is 73 kb (30), and we found that these markers are 4.0 cR8000 apart, providing a conversion factor of 18.3 kb/cR8000 . Using these data, the new cystinosis interval would span only 187 kb. A segment of DNA this size should be suitable for the identification of candidate genes. We report here the physical mapping of four YAC clones which form a contig across the original cystinosis interval of D17S1584 and D17S1583. These data alone should prove useful in the future orientation of various genes in the area. In addition, markers D17S1828 and D17S2167, which flank the cystinosis gene, can be used to screen a chromosome 17-specific cosmid library, as well as P1 clones and BAC clones. These studies, currently underway, will allow creation of a smaller contig across the cystinosis region and the identification of expressed sequences. The ultimate description of the cystinosis gene should provide new insights into the synthesis, targeting, processing, and integration of functional transporters in the lysosomal membrane. ACKNOWLEDGMENTS We thank Drs. Frances L. Harley and Paul Roy for obtaining certain patient DNA samples and Dr. Settara Chandrasekhara-
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ppa for providing helpful YAC clones. Dr. Michael Boehnke graciously offered advice regarding the radiation hybrid analysis. M.M.T. and W.v.H. were supported in part by the Medical Research Council (UK). U.L.-K. was supported by the Deutche Forschungsgemeinschaft (D.F.G.) Grants Li 375/5-1, -2 (D.F.G. Schwerpunktprogramm ‘‘Analyse des menschen Genoms mit molekularbiologischen Methoden’’), Li 375/6.1, 6.2 (Habilitations stipendium).
REFERENCES 1. Gahl WA, Bashan N, Tietze F, Bernardini I, Schulman JD. Cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis. Science 217:1263– 1265, 1982. 2. Gahl WA, Tietze F, Bashan N, Steinherz R, Schulman JD. Defective cystine exodus from isolated lysosome-rich fractions of cystinotic leucocytes. J Biol Chem 257:9570–9575, 1982. 3. Jonas AJ, Smith ML, Schneider JA. ATP-dependent lysosomal cystine efflux is defective in cystinosis. J Biol Chem 257:13185–13188, 1982. 4. Gahl WA. Cystinosis coming of age. Adv Pediatr 33:95–126, 1986. 5. Gahl WA, Schneider JA, Aula P. Lysosomal transport disorders: Cystinosis and sialic acid storage disorders. In The Metabolic and Molecular Bases of Inherited Disease (Scriver CR, Beaudet AL, Sly WS Valle D, Eds.). New York: McGraw–Hill, 1995, pp. 3763–3797. 6. Theodoropoulos DS, Krasnewich D, Kaiser-Kupfer MI, Gahl, WA. Classical nephropathic cystinosis as an adult disease. JAMA 270:2200–2204, 1993. 7. Kaiser-Kupfer M, Caruso RC, Minckler DS, Gahl WA. Longterm ocular manifestations in nephropathic cystinosis postrenal transplantation. Arch Ophthalmol 104:706–711, 1986. 8. Charnas LR, Luciano CA, Dalakas M, Gilliatt RW, Bernardini I, Ishak K, Cwik VA, Fraker D, Brushart TA, Gahl WA. Distal vacuolar myopathy in nephropathic cystinosis. Ann Neurol 35: 181–188, 1994. 9. Sonies BC, Ekman EF, Andersson HC, Adamson M, Kaler S, Markello T, Gahl WA. Swallowing dysfunction in nephropathic cystinosis. N Engl J Med 323:565–570, 1990. 10. Fivush B, Flick JA, Gahl WA. Pancreatic exocrine insufficiency in a patient with nephropathic cystinosis. J Pediatr 112:49–51, 1988. 11. Fivush B, Green OC, Porter CC, Balfe JW, O’Regan S, Gahl WA. Pancreatic endocrine insufficiency in post-transplant cystinosis. Am J Dis Child 141:1087–1089, 1987. 12. Thoene JG, Oshima RG, Crawhall JC, Olson DL, Schneider JA. Cystinosis: Intracellular cystine depletion by aminothiols in vitro and in vivo. J Clin Invest 58:180–189, 1976. 13. Gahl WA, Reed GF, Thoene JG, Schulman JD, Rizzo WB, Jonas AJ, Denman DW, Schlesselman JJ, Corden BJ, Schneider JA. Cysteamine therapy for children with nephropathic cystinosis. N Engl J Med 316:971–977, 1987. 14. Markello TC, Bernardini IM, Gahl WA. Improved renal function in children with cystinosis treated with cysteamine. N Engl J. Med 328:1157–1162, 1993. 15. Kimonis VE, Troendle J, Yang ML, Rose SR, Markello TC, Gahl WA. Effects of early cysteamine therapy on thyroid
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FINE MAPPING OF THE CYSTINOSIS GENE function and growth in nephropathic cystinosis. J. Clin Endocrinol Metab 80:3257–3261, 1995. 16. Gahl WA, Charnas L, Markello T, Bernardini I, Ishak KG, Dalakas M. Parenchymal organ cystine depletion with longterm cysteamine therapy. Biochem Med Metab Biol 48:275– 285, 1992. 17. Kaiser-Kupfer MI, Fujikawa L, Kuwabara T, Gahl WA. Removal of corneal crystals by topical cysteamine in nephropathic cystinosis. N Engl J Med 316:775–779, 1987. 18. Kaiser-Kupfer MI, Gazzo MA, Datiles MB, Caruso RC, Kuehl EM, Gahl WA. A randomized placebo-controlled trial of cysteamine eyedrops in nephropathic cystinosis. Arch Ophthalmol 108:689–693, 1990. 19. Gahl WA, Tietze F, Bashan N, Bernardini I, Schulman JD. Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions. Biochem J 216:393–400, 1983. 20. The Cystinosis Collaborative Research Group. Linkage of the gene for cystinosis to markers on the short arm of chromosome 17. Nature Genet 10:246–248, 1995. 21. Jean G, Fuchshuber A, Town MM, Gribouval O, Schneider JA, Broyer M, van’t Hoff W, Niaudet P, Antignac C. Highresolution mapping of the gene for cystinosis, using combined biochemical and linkage analysis. Am J Hum Genet 58:535–543, 1996. 22. Kaul R, Gao GP, Balamurugan K, Matalon R. Cloning of the human aspartoacylase cDNA and a common missense mutation in Canavan disease. Nature Genet 5:118–123, 1993. 23. Kojis TL, Heinzmann C, Flodman P, Ngo JT, Sparkes RS, Spence MA, Bateman JB, Heckenlively JR. Map refinement of locus RP13 to human chromosome 17p13.3 in a second family with autosomal dominant retinitis pigmentosa. Am J Hum Genet 58:347–355, 1996. 24. Stratton RF, Dobyns WB, Airhart SD, Ledbetter DH. New chromosomal syndrome: Miller–Dieker syndrome and monosomy 17p13. Hum Genet 67:193–200, 1984. 25. Dib C, Faure S, Fizames C, Samson D, Drouot N, Vignal A, Millasseau P, Marc S, Hazan J, Seboun E, Lathrop M, Gyapay G, Morissette J, Weissenbach J. A comprehensive ge-
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26.
27.
28.
29.
30.
31.
32. 33.
34.
35.
141
netic map of the human genome based on 5,264 microsatellites. Nature 380:152–154, 1996. Schurmans S, Muscatelli F, Miot F, Mattei M-G, Vassart G, Parmentier M. The OLFR1 gene encoding the HGMP07E putative olfactory receptor maps to the 17p13–p12 region of the human genome and reveals an MspI restriction fragment length polymorphism. Cytogenet Cell Genet 63:200–204, 1993. Genome Data Base, GDB (TM). The Human Genome Data Base Project, Johns Hopkins University, Baltimore, MD. World Wide Web »URL: http://gdbwww.gdb.org/gdbdoc/ topq.html…, 1995. Rotman G, Vanagaite L, Collins FS, Shiloh Y. Rapid identification of polymorphic CA-repeats in YAC clones. Mol Biotech 3:85–92, 1995. Cox DR, Burmeister M, Price ER, Kim S, Myers RM. Radiation hybrid mapping: A somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes. Science 250:245–250, 1990. Ben-Arie N, Lancet D, Taylor C, Khen M, Walker N, Ledbetter DH, Carrozzo R, Patel K, Sheer D, Lehrach H, North MA. Olfactory receptor gene cluster on human chromosome 17: Possible duplication of an ancestral receptor repertoire. Hum Mol Genet 3: 229–235, 1994. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982. Philippsen P, Stotz A, Scherf C. DNA of Saccharomyces cerevisiae. Meth Enzymol 194:169–182, 1991. Tagle DA, Collins FS. An optimized Alu-PCR primer pair for human-specific amplification of YACs and somatic cell hybrids. Hum Mol Genet 1:121–122, 1992. Riley J, Butler R, Oglivie D, Finniear R, Jenner D, Powell S, Anand R, Smith JC, Markham AF. A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucl Acids Res 18:2887–2890, 1990. Boehnke M, Lange K, Cox DR. Statistical methods for multipoint radiation hybrid mapping. Am J Hum Genet 49:1174–1188, 1991.
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