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Refinement of the Gene Locus for Autosomal Dominant Medullary Cystic Kidney Disease Type 1 (MCKD1) and Construction of a Physical and Partial Transcriptional Map of the Region
Genomics 72, 278 –284 (2001) doi:10.1006/geno.2000.6486, available online at http://www.idealibrary.com on
Refinement of the Gene Locus for Autosomal Dominant Medullary Cystic Kidney Disease Type 1 (MCKD1) and Construction of a Physical and Partial Transcriptional Map of the Region A. Fuchshuber,* ,1 S. Kroiss,* S. Karle,* S. Berthold,* K. Huck,* C. Burton,† N. Rahman,‡ M. Koptides,§ C. Deltas,§ E. Otto,* F. Ru¨schendorf, ¶ T. Feest,† and F. Hildebrandt* *University Children’s Hospital, Mathildenstrasse 1, 79106 Freiburg, Germany; †The Richard Bright Renal Unit, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, United Kingdom; ‡Institute of Medical Genetics, University of Wales, Cardiff, United Kingdom; ¶ Max Delbru¨ck Centre for Molecular Medicine, Berlin, Germany; and §The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus Received October 6, 2000; accepted December 18, 2000
Autosomal dominant medullary cystic kidney disease (MCKD) is an adult onset tubulointerstitial nephropathy that leads to salt wasting and end-stage renal failure. A gene locus (MCKD1) has been mapped on chromosome 1q21. Here we report on a large MCKD1 family of British origin linked to the MCKD1 locus. Haplotype analysis performed with markers spanning the previously reported critical MCKD1 region allowed for the refinement of this interval to 4 cM by definition of D1S305 as a new proximal flanking marker. Furthermore, we constructed a yeast artificial chromosome, P1-related artificial chromosome, and bacterial artificial chromosome contig of this region, which is only sparsely covered by the Human Genome Sequencing Project. This enabled us to map numerous expressed sequence tags within the critical interval. This physical and partial transcriptional map of the MCKD1 region is a powerful tool for the identification of positional and functional candidate genes for MCKD1 and will help to identify the diseasecausing gene. © 2001 Academic Press
INTRODUCTION
Autosomal dominant medullary cystic kidney disease [MCKD1 (MIM 174000); MCKD2 (MIM 603860)] is a tubulointerstitial nephropathy of the so-called nephronophthisis/MCKD complex of diseases (Waldherr et al., 1982). It shares with nephronophthisis the characteristic histological hallmarks of tubular basement membrane disintegration, interstitial cell infiltration, tubular atrophy, and cyst formation at the cortico-medullary border of the kidneys (Hildebrandt and Otto, 2000). In contrast to juvenile nephronophthisis [NPH1 (MIM 256100)], extrarenal organ involve1 To whom correspondence should be addressed. Telephone: ⫹49 761 270 4301. Fax: ⫹49 761 270 4533. E-mail: fuchshub@ kkl200.ukl.uni-freiburg.de.
0888-7543/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
ment has not been reported. However, hyperuricemia and gout seem to be common facultative symptoms in MCKD1 (Stavrou et al., 1998) as well as in MCKD2 (Scolari et al., 1999). Up to now, two genes for MCKD have been mapped: MCKD1 was localized to chromosome 1q21 in a large Cypriot family and restricted to an 8-cM interval between flanking markers D1S498 and D1S2125 (Christodoulou et al., 1998). A second gene locus (MCKD2) was identified on chromosome 16p12 in an Italian pedigree (Scolari et al., 1999). However, the disorder was shown to be even more heterogeneous, since MCKD families linked neither to MCKD1 nor to MCKD2 have been described (Fuchshuber et al., 1998; Kroiss et al., 2000). Here we report on a family of British origin, with a total of 11 affected individuals, who meet the clinical criteria for MCKD. By haplotype analysis, we found linkage to MCKD1, and we refined the genetic MCKD1 interval to a critical region of 4 cM of sex-averaged genetic distance now flanked by proximal marker D1S305, thereby reducing the critical physical interval from 7.1 Mb to approximately 3.3 Mb. Furthermore, we constructed a highresolution physical map with yeast artificial chromosome (YAC), P1-related artificial chromosome (PAC), and bacterial artificial chromosome (BAC) clones between markers D1S498 and D1S2125. This contig enabled us to generate a partial transcriptional map of the MCKD1 region. SUBJECTS AND METHODS Patients. The pedigree of the four-generation MCKD1 family originating from the United Kingdom and including 11 affected individuals is depicted in Fig. 1. We have collected blood samples from 15 family members (6 affected and 9 unaffected individuals). The diagnosis was based on the following clinical criteria, compatible with MCKD (Fuchshuber et al., 1998; Kroiss et al., 2000): (i) all affected individuals progressed to end-stage renal failure, preceded by a typical history of polyuria, polydipsia, and/or anemia; (ii) renal histology of at least one affected individual was consistent with MCKD, characterized by disintegration of the tubular basement
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FIG. 1. Pedigree of the large five-generation MCKD1 family with segregation of chromosome 1 markers. Black symbols indicate affected individuals; white symbols indicate either unaffected or unknown status. Haplotypes of 12 consecutive microsatellite markers spanning the critical MCKD1 region on chromosome 1q21 are shown as differently shaded and colored bars. Black bars indicate the affected allele. Haplotypes were generated by minimizing recombinants; inferred alleles are shown in parentheses. Marker order from top to bottom (centromere to telomere) is given on the left for each generation (flanking markers are D1S305 and D1S2635). Paternal haplotypes are drawn to the left; maternal haplotypes are drawn to the right. Generations are numbered in roman numerals. membranes, tubulo-interstitial fibrosis, and eventually tubular cyst formation at the cortico-medullary border; and (iii) the pedigree was consistent with an autosomal dominant trait. For the definition of an unaffected status, the following criteria had to be met: (i) normal renal function (glomerular filtration rate ⬎80 ml/min/1.73 m 2) at age ⬎65 years and (ii) at the same time, no polyuria, polydipsia, and/or anemia should be observed. Haplotype and linkage analysis. Genomic DNA was isolated by standard methods directly from blood samples or after EBV transformation of peripheral blood lymphocytes as previously described (Vollmer et al., 1998). In total, 15 individuals including 6 affected patients were haplotyped by using 12 consecutive polymorphic microsatellite markers that span the MCKD1 region. The marker order was as follows: cen–D1S514 –D1S498 –D1S305–D1S2777–D1S2140 – D1S1595–D1S2624–D1S394–D1S1600–D1S1653–D1S2125–D1S2635– tel. Haplotype analysis was evaluated graphically (see Fig. 1) by use of CYRILLIC, version 2.1.3 (Cherwell Scientific). Because of the
age-dependent disease penetrance, linkage analysis was performed using an affecteds-only strategy (Vollmer et al., 1998). Full penetrance was assumed for affected individuals. Two-point linkage analysis was performed with 10 consecutive polymorphic markers in the MCKD1 region. For calculation of lod scores, the LINKAGE program (version 5.1) (Lathrop et al., 1984) was used. To conduct multipoint linkage analysis, the program VITESSE was used (O’Connell and Weeks, 1995). Intermarker distances were taken from Dib et al. (1996). Allele frequencies for the markers tested were assumed to be equally distributed. The disease frequency was set at 10 ⫺4 (Christodoulou et al., 1998). YAC contig construction. For construction of a YAC contig, YACs were identified from the CEPH mega-YAC library using a sequencetagged site (STS) content mapping strategy as described previously (Nothwang et al., 1998; Vollmer et al., 2000). Genetic marker information, as well as PCR conditions, was obtained from different public databases [The Whitehead Institute for Biomedical Research,
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TABLE 1 New STS Markers to BAC-End Sequences within the MCKD1 Critical Interval STS name
Primer sequence (5⬘-3⬘)
66D17 SP6-F 66D17 SP6-R 66D17 T7-F 66D17 T7-R 356J7 SP6-F 356J7 SP6-R 110J1 T7-F 110J1 T7-R 110J1 SP6-F 110J1 SP6-R 183E10 SP6-F 183E10 SP6-R
5⬘-ATA CTT TCC TCC TGC CAC AG-⬘3 5⬘-CTC TGT CAC TGT GAC GAT AG-⬘3 5⬘-TCA CCA GCA CTT AGA TGG AC-⬘3 5⬘-TTT CTG AGC GCT CTG CTC TG-⬘3 5⬘-AAG CAG GGC TCC AAG GTG AG-⬘3 5⬘-TAA AAG GTC AAC AGG GCC CC-⬘3 5⬘-ACT ATG TTG ATA GAG CCT GC-⬘3 5⬘-AGC TAA TAA ATG TCA GGG CC-⬘3 5⬘-ATT TCT GAC CCA TCT GTG CC-⬘3 5⬘-TTG GTT TCG AGC AGT AGG AG-⬘3 5⬘-ACC AGT TCA CAC TCA CTC AG-⬘3 5⬘-TTT CCA CCT TCA CTT TCA CC-⬘3
http://www-genome.wi.mit.edu/; The Genetic Epidemiology Research Group, http://cedar.genetics.soton.ac.uk/public_html/ (Collins et al., 1996); The Ge´ne´thon Human Genetic Linkage Map (Dib et al., 1996); GeneMap 1999, http//:www.ncbi.nlm.nih.gov/GeneMap/].
Product size (bp)
Annealing temperature (°C)
112
55
159
60
113
62
212
51
136
60
126
55
In the seven affected family members (individuals 10, 13, 17, 19, 20, 25, and 37) for whom data were available, the age at first renal replacement therapy for terminal renal failure ranged from 37 to 65 years with a median age of 50 years. Renal histology was available from patients 17, 25, and 37, showing the characteristics of MCKD with tubular basement membrane disintegration, tubulo-interstitial cell infiltration, tubular atrophy, and cyst development. Extrarenal organ involvement and hyperuricemia or episodes of gout were not observed.
fected individuals and also by two individuals in generation IV, who are too young for definition of the affected status (Fig. 1). Shared markers were as follows: cen–D1S2777, D1S2140, D1S1595, D1S2624, D1S394, D1S1600, D1S1653, and D1S2125–tel. To test the hypothesis of linkage of the family to MCKD1, we performed a two-point linkage analysis using 10 polymorphic markers located within the interval (D1S514, D1S498, D1S305, D1S2777, D1S2140, D1S1595, D1S2624, D1S394, D1S1653, and D1S2125). Since disease penetrance in MCKD is age dependent (Kroiss et al., 2000), linkage was calculated using an affectedsonly strategy. A maximum two-point lod score of Z max ⫽ 2.27 at a recombination fraction of ⫽ 0 was calculated with marker D1S2777. Multipoint linkage analysis yielded Z max ⫽ 2.4 for a region delimited by the flanking markers D1S305 and D1S2635 (Fig. 2). In linkage studies where only a few polymorphic markers are tested to confirm linkage to a known candidate gene locus with a small number of markers as well as in diseases with X-chromosomal inheritance, the criterion for significance for linkage can be released to ⬎2 from ⬎3 as postulated in a total genome search for linkage, where multiple testing is performed with approximately 400 polymorphic markers (Ott, 1985, p. 71). A lod score of 2.27 therefore clearly demonstrates linkage of the disease locus in this family to the MCKD1 locus. Recombination events are observed with marker D1S305 at the centromeric border and marker D1S2635 at the telomeric border of chromosome 1q21, thus refining the MCKD1 gene locus from 8 cM described previously (Christodoulou et al., 1998) to a critical interval of 4 cM. These results are supported by a previous report, where we could exclude linkage to the MCKD2 locus on chromosome 16p12 in this family (family MCD3) (Kroiss et al., 2000).
Linkage and Haplotype Analysis
Physical Map
Haplotype analysis with 12 polymorphic markers from the critical MCKD1 region revealed a common haplotype of 8 consecutive markers shared by all af-
In total the contig contains 47 YAC clones defined by 50 genetic markers (15 polymorphic microsatellite markers, 16 STSs, and 19 ESTs). Seven overlapping
PAC and BAC contig construction. Using a hybridization-based library screening of a human genomic PAC library [RPCI1.3–5 Human PAC, Resource Center, Max-Planck Institute, Berlin (RZPD): http://www.rzpd.de/] provided by Pieter de Jong (Ioannou et al., 1994), PAC clones were mapped within the YAC contig. Inter-Alu PCR was performed to derive probes from the YACs 790H6, 753B9, 874D5, 955E11, 950E2, 713B11, 887H8, 873E5, 726F3, and 748F9. STS markers known to map to the MCKD1 critical region according to the radiation hybrid maps from different public databases (for addresses see above) were used to screen the PAC library. Positive clones were obtained from the RZPD and isolated as described previously (Vollmer et al., 2000). Furthermore, 10 BAC clones provided by RZPD (http://www.rzpd.de) were also positioned within the MCKD1 interval. BAC-end sequencing. BAC ends were directly sequenced using BigDye chemistry on an ABI 377 automated sequencer by using vector-based T7 and SP6 primers. Resulting sequences were analyzed using the BLASTN 2.0.8 program (Altschul et al., 1997). Primers were designed to amplify unique end-sequence markers, which then were mapped back onto the YAC contig (Table 1).
RESULTS
Patients
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FIG. 2. Multipoint linkage analysis graph: The horizontal axis represents the relative genetic positions of the markers in centimorgan sex-averaged genetic distance (Dib et al., 1996). The position of marker D1S514 is arbitrarily set at 0 cM. The respective lod score values are indicated on the vertical axis. A horizontal dashed line is drawn at a lod score of 0. Note that for the markers D1S2777, D1S2140, D1S1595, D1S2624, D1S394, D1S1600, and D1S2125, lod score values above 2 in this candidate region indicate linkage to the MCKD1 locus.
YACs (“minimal contig”) between the previously reported flanking markers D1S489 and D1S2125 (Christodoulou et al., 1998) span an approximate physical distance of 7.1 Mb, defined by the combined length of clones 790H6 (760 kb), 753B9 (1740 kb), 955E11 (1310 kb), 950E2 (1290 kb), 713B11 (212 kb), 873E5 (210 kb), and 748F9 (1600 kb) (Fig. 3). According to the marker information of the Center for Medical Genetics (http://www.marshmed.org/genetics/), this physical distance corresponds to a total genetic sex-averaged distance of approximately 8 cM. The identification of marker D1S305 as a new flanking marker allowed for refinement of the critical MCKD1 region to a physical distance of approximately 3.3 Mb. Three YACs [YACs 790H6 (760 kb), 753B9 (1740 kb) and 955E11 (1310 kb)] spanning 3.8 Mb at the centromeric border of the contig can thereby be excluded from the critical MCKD1 interval. To generate a high-resolution physical map of the critical MCKD1 interval, we isolated 31 partially overlapping PAC clones by library hybridization using inter-Alu PCR from the RPCI PAC library to this interval, and the PAC clones were subsequently screened with STS markers previously mapped to the YAC contig. Additionally, by using the same approach, 10 BAC clones were mapped within the MCKD1 interval. Furthermore, BAC ends were sequenced with SP6 and T7 universal primers. Six novel BAC-end sequence markers were generated from the resulting sequences (Fig. 3, Table 1). Nineteen ESTs representing known genes were localized to the MCKD1 region. Among them are HAX-1, a nuclear gene encoding a mitochondrial protein, a human skeletal muscle 1.3-kb mRNA for tropomyosin, a fibroblast growth factor receptor 2, a human
small proline-rich protein, a RNA-specific adenosine deaminase (ADAR), an interleucin 6 receptor (IL6R), and a human transcriptional activator mRNA. DISCUSSION
In a large British pedigree of 11 affected individuals with the classic clinical criteria for MCKD, we found linkage to MCKD1. Renal histology showed tubulointerstitial abnormalities consistent with MCKD. No extrarenal organ involvement was observed. End-stage renal failure occurred with a median age of 50 years, similar to the MCKD1 family reported by Christodoulou et al. (1998). By haplotype analysis we refined the genetic interval initially reported to span 8 cM (Christodoulou et al., 1998) to a critical region of 4 cM according to the genetic map of the Marshfield Center of Medical Genetics (http://www.marshmed.org/genetics). Furthermore we constructed a high-resolution YAC, PAC, and BAC contig between markers D1S498 and D1S2125, which facilitates assignment of positional candidate genes in the refined region. This contig allowed for narrowing of the critical interval by 3.8 Mb on the centromeric side and for generation of a partial transcriptional map of the region. Although the genes responsible for MCKD1 and MCKD2 are not yet known, the gene causing NPH1 (NPHP1) was identified recently (Hildebrandt et al., 1997; Saunier et al., 1997). It codes for nephrocystin and includes a Src homology 3 (SH3) domain (Hildebrandt, 1998; Otto et al., 2000; Hildebrandt and Otto, 2000). It has been shown that nephrocystin interacts through this SH3 domain with Crk-associated sub-
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FIG. 3. YAC, PAC, and BAC contig of the MCKD1 region on chromosome 1q21 with partial transcriptional map. (A) STS and EST markers examined by STS content mapping in this study. Flanking markers D1S498 and D1S2125 reported initially (Christodoulou et al., 1998) are shown in boldface type. The newly defined flanking marker D1S305 from this study is shown in a box. (B) YAC clones of the CEPH-Mega-YAC library with their respective names, which have been mapped by STS content mapping with the respective markers. YAC sizes are given in parentheses. A vertical line indicates a positive PCR amplification with the respective marker. The YACs of the “minimal contig” spanning the entire interval are depicted in boldface type. The previously reported maximum physical size of 7.1 Mb is now restricted to approximately 3.3 Mb by definition of the new flanking marker D1S305. (C) BAC clones of the RPCI-11.1 library provided by the RZPD (http://www.rzpd.de). (D) PAC clones from a human genomic PAC library (RPCI1,3–5, Human PAC, Resource Center, RZPD) provided by Pieter de Jong. (E) Expressed sequences, which have been assigned to known genes.
strate (Cas), which is implicated in signal transduction and organization of the actin cytoskeleton at sites of cell adhesion (Donaldson et al., 2000). Considering the similarities of the clinico-pathological presentation of MCKD and NPH1, it is reasonable to assume that the two diseases may be the result of defects in the same developmental or metabolic pathway. Therefore, genes containing SH3 or SH2 domains represent strong candidates for MCKD. One of the genes found to map within the critical MCKD1 region is HAX-1 (HS1-associated protein X-1), an SH3-domain-containing protein associated with the actin cytoskeleton. It has been demonstrated that HAX-1 interacts with PKD2, the gene causing autosomal-dominant polycystic kidney disease type 2 (Gallagher et al., 2000). Since the pathogenesis of cyst formation in ADPKD is still unclear, assuming a common pathway of cystogenesis in both diseases makes HAX-1 an interesting positional and functional candidate gene for MCKD. With definition of D1S305 as a new proximal flanking marker, this gene is, however,
excluded as a positional candidate gene. Mutational analysis in one MCKD1 family revealed no mutations in the seven exons of HAX-1 (data not shown). Furthermore, there are several positional candidate genes that we mapped within the critical MCKD1 region. Some of them are not considered to be good functional candidate genes: Trichohyalin and S100A9 (synonyme: calgranulin B) are genes coding for proteins that are expressed during terminal differentiation in the epidermis (Volz et al., 1993); the small proline-rich protein 1B, called cornifin, appears to function as a component of the crosslinked envelope in squamous differentiating cells (Marvin et al., 1992); tropomyosin 3 is involved in a somatic rearrangement that creates the chimeric TRK onkogene and was found to be associated with autosomal dominant nemaline myopathy (Laing et al., 1995); the RNA-specific adenosine deaminase converts adenosine to iosine in dsRNA with various functions including site-specific RNA editing of transcripts of the glutamate receptors, probably resulting in malfunction of the glutamate-gated ion
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channels and modification of viral RNA genomes (Weier et al., 1995); the alpha subunit of erythroid spectrin is a predominant component of the membrane skeleton of the red cell and has been found to be associated with a subset of hereditary elliptocytosis (Lawler et al., 1984). KIAA0144, KIAA0080, and KIAA0907 are unidentified human genes whose sequences have been deduced by analysis of cDNA clones from human cell line KG-1 (Nomura et al., 1994). IL6R, the interleukin 6 receptor, stimulates the expression of hepatocyte growth factor, HGF (Matsumoto and Nakamura, 1992), which is postulated to be involved in tubular damage (Yorioka et al., 1996) or even postulated to be a tubular cyst-inducing mutagen (Horie et al., 1994) and therefore represents an interesting functional and positional candidate gene for MCKD1. The physical and partial transcriptional map of the MCKD1 region allows for identification of further candidate genes suited to mutational analysis in individuals affected by MCKD1 and thus represents a powerful tool for the identification of the disease-causing gene. ACKNOWLEDGMENTS A.F. was supported by a grant from the German Research Foundation (DFG FU 202/3-1) and from the Fritz-Thyssen-Stiftung, Germany. F.H. was supported by the Zentrum klinische Forschung (ZKF-A1). A.F. and F.H. were supported by the State of BadenWu¨rttemberg (FSP “Zystogenese”). N.R. was supported by the National Kidney Research Fond (NKRF). We thank R. Witzgall for information on HAX-1 as a candidate gene. We thank Barbara Scho¨nfeld, Anita Imm, and Cornelia Klein for their technical assistance. We thank the patients and their family for their collaboration.
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fibronectin glomerulopathy maps to 1q32, in the region of the regulation of complement activation gene cluster. Am. J. Hum. Genet. 63: 1724 –1731. Volz, A., Korge, B. P., Compton, J. G., Ziegler, A., Steinert, P. M., and Mischke, D. (1993). Physical mapping of a functional cluster of epidermal differentiation genes on chromosome 1q21. Genomics 18: 92–99. Waldherr, R., Lennert, T., Weber, H. P., Fodisch, H. J., and Scharer, K. (1982). The nephronophthisis complex. A clinicopathologic study in children. Virchows Arch. 394: 235–254. Weier, H. U., George, C. X., Greulich, K. M., and Samuel, C. E. (1995). The interferon-inducible, double-stranded RNA-specific adenosine deaminase gene (DSRAD) maps to human chromosome 1q21.1– q21.2. Genomics 30: 372–375. Yorioka, N., Taniguchi, Y., Yamashita, K., Usui, K., Shigemoto, K., Harada, S., Taguchi, T., and Yamakido, M. (1996). Hepatocyte growth factor in nephronophthisis–medullary cystic disease complex. Pediatr. Nephrol. 10: 515–516.
Refinement of the Gene Locus for Autosomal Dominant Medullary Cystic Kidney Disease Type 1 (MCKD1) and Construction of a Physical and Partial Transcriptional Map of the Region A. Fuchshuber,* ,1 S. Kroiss,* S. Karle,* S. Berthold,* K. Huck,* C. Burton,† N. Rahman,‡ M. Koptides,§ C. Deltas,§ E. Otto,* F. Ru¨schendorf, ¶ T. Feest,† and F. Hildebrandt* *University Children’s Hospital, Mathildenstrasse 1, 79106 Freiburg, Germany; †The Richard Bright Renal Unit, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, United Kingdom; ‡Institute of Medical Genetics, University of Wales, Cardiff, United Kingdom; ¶ Max Delbru¨ck Centre for Molecular Medicine, Berlin, Germany; and §The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus Received October 6, 2000; accepted December 18, 2000
Autosomal dominant medullary cystic kidney disease (MCKD) is an adult onset tubulointerstitial nephropathy that leads to salt wasting and end-stage renal failure. A gene locus (MCKD1) has been mapped on chromosome 1q21. Here we report on a large MCKD1 family of British origin linked to the MCKD1 locus. Haplotype analysis performed with markers spanning the previously reported critical MCKD1 region allowed for the refinement of this interval to 4 cM by definition of D1S305 as a new proximal flanking marker. Furthermore, we constructed a yeast artificial chromosome, P1-related artificial chromosome, and bacterial artificial chromosome contig of this region, which is only sparsely covered by the Human Genome Sequencing Project. This enabled us to map numerous expressed sequence tags within the critical interval. This physical and partial transcriptional map of the MCKD1 region is a powerful tool for the identification of positional and functional candidate genes for MCKD1 and will help to identify the diseasecausing gene. © 2001 Academic Press
INTRODUCTION
Autosomal dominant medullary cystic kidney disease [MCKD1 (MIM 174000); MCKD2 (MIM 603860)] is a tubulointerstitial nephropathy of the so-called nephronophthisis/MCKD complex of diseases (Waldherr et al., 1982). It shares with nephronophthisis the characteristic histological hallmarks of tubular basement membrane disintegration, interstitial cell infiltration, tubular atrophy, and cyst formation at the cortico-medullary border of the kidneys (Hildebrandt and Otto, 2000). In contrast to juvenile nephronophthisis [NPH1 (MIM 256100)], extrarenal organ involve1 To whom correspondence should be addressed. Telephone: ⫹49 761 270 4301. Fax: ⫹49 761 270 4533. E-mail: fuchshub@ kkl200.ukl.uni-freiburg.de.
0888-7543/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
ment has not been reported. However, hyperuricemia and gout seem to be common facultative symptoms in MCKD1 (Stavrou et al., 1998) as well as in MCKD2 (Scolari et al., 1999). Up to now, two genes for MCKD have been mapped: MCKD1 was localized to chromosome 1q21 in a large Cypriot family and restricted to an 8-cM interval between flanking markers D1S498 and D1S2125 (Christodoulou et al., 1998). A second gene locus (MCKD2) was identified on chromosome 16p12 in an Italian pedigree (Scolari et al., 1999). However, the disorder was shown to be even more heterogeneous, since MCKD families linked neither to MCKD1 nor to MCKD2 have been described (Fuchshuber et al., 1998; Kroiss et al., 2000). Here we report on a family of British origin, with a total of 11 affected individuals, who meet the clinical criteria for MCKD. By haplotype analysis, we found linkage to MCKD1, and we refined the genetic MCKD1 interval to a critical region of 4 cM of sex-averaged genetic distance now flanked by proximal marker D1S305, thereby reducing the critical physical interval from 7.1 Mb to approximately 3.3 Mb. Furthermore, we constructed a highresolution physical map with yeast artificial chromosome (YAC), P1-related artificial chromosome (PAC), and bacterial artificial chromosome (BAC) clones between markers D1S498 and D1S2125. This contig enabled us to generate a partial transcriptional map of the MCKD1 region. SUBJECTS AND METHODS Patients. The pedigree of the four-generation MCKD1 family originating from the United Kingdom and including 11 affected individuals is depicted in Fig. 1. We have collected blood samples from 15 family members (6 affected and 9 unaffected individuals). The diagnosis was based on the following clinical criteria, compatible with MCKD (Fuchshuber et al., 1998; Kroiss et al., 2000): (i) all affected individuals progressed to end-stage renal failure, preceded by a typical history of polyuria, polydipsia, and/or anemia; (ii) renal histology of at least one affected individual was consistent with MCKD, characterized by disintegration of the tubular basement
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FIG. 1. Pedigree of the large five-generation MCKD1 family with segregation of chromosome 1 markers. Black symbols indicate affected individuals; white symbols indicate either unaffected or unknown status. Haplotypes of 12 consecutive microsatellite markers spanning the critical MCKD1 region on chromosome 1q21 are shown as differently shaded and colored bars. Black bars indicate the affected allele. Haplotypes were generated by minimizing recombinants; inferred alleles are shown in parentheses. Marker order from top to bottom (centromere to telomere) is given on the left for each generation (flanking markers are D1S305 and D1S2635). Paternal haplotypes are drawn to the left; maternal haplotypes are drawn to the right. Generations are numbered in roman numerals. membranes, tubulo-interstitial fibrosis, and eventually tubular cyst formation at the cortico-medullary border; and (iii) the pedigree was consistent with an autosomal dominant trait. For the definition of an unaffected status, the following criteria had to be met: (i) normal renal function (glomerular filtration rate ⬎80 ml/min/1.73 m 2) at age ⬎65 years and (ii) at the same time, no polyuria, polydipsia, and/or anemia should be observed. Haplotype and linkage analysis. Genomic DNA was isolated by standard methods directly from blood samples or after EBV transformation of peripheral blood lymphocytes as previously described (Vollmer et al., 1998). In total, 15 individuals including 6 affected patients were haplotyped by using 12 consecutive polymorphic microsatellite markers that span the MCKD1 region. The marker order was as follows: cen–D1S514 –D1S498 –D1S305–D1S2777–D1S2140 – D1S1595–D1S2624–D1S394–D1S1600–D1S1653–D1S2125–D1S2635– tel. Haplotype analysis was evaluated graphically (see Fig. 1) by use of CYRILLIC, version 2.1.3 (Cherwell Scientific). Because of the
age-dependent disease penetrance, linkage analysis was performed using an affecteds-only strategy (Vollmer et al., 1998). Full penetrance was assumed for affected individuals. Two-point linkage analysis was performed with 10 consecutive polymorphic markers in the MCKD1 region. For calculation of lod scores, the LINKAGE program (version 5.1) (Lathrop et al., 1984) was used. To conduct multipoint linkage analysis, the program VITESSE was used (O’Connell and Weeks, 1995). Intermarker distances were taken from Dib et al. (1996). Allele frequencies for the markers tested were assumed to be equally distributed. The disease frequency was set at 10 ⫺4 (Christodoulou et al., 1998). YAC contig construction. For construction of a YAC contig, YACs were identified from the CEPH mega-YAC library using a sequencetagged site (STS) content mapping strategy as described previously (Nothwang et al., 1998; Vollmer et al., 2000). Genetic marker information, as well as PCR conditions, was obtained from different public databases [The Whitehead Institute for Biomedical Research,
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TABLE 1 New STS Markers to BAC-End Sequences within the MCKD1 Critical Interval STS name
Primer sequence (5⬘-3⬘)
66D17 SP6-F 66D17 SP6-R 66D17 T7-F 66D17 T7-R 356J7 SP6-F 356J7 SP6-R 110J1 T7-F 110J1 T7-R 110J1 SP6-F 110J1 SP6-R 183E10 SP6-F 183E10 SP6-R
5⬘-ATA CTT TCC TCC TGC CAC AG-⬘3 5⬘-CTC TGT CAC TGT GAC GAT AG-⬘3 5⬘-TCA CCA GCA CTT AGA TGG AC-⬘3 5⬘-TTT CTG AGC GCT CTG CTC TG-⬘3 5⬘-AAG CAG GGC TCC AAG GTG AG-⬘3 5⬘-TAA AAG GTC AAC AGG GCC CC-⬘3 5⬘-ACT ATG TTG ATA GAG CCT GC-⬘3 5⬘-AGC TAA TAA ATG TCA GGG CC-⬘3 5⬘-ATT TCT GAC CCA TCT GTG CC-⬘3 5⬘-TTG GTT TCG AGC AGT AGG AG-⬘3 5⬘-ACC AGT TCA CAC TCA CTC AG-⬘3 5⬘-TTT CCA CCT TCA CTT TCA CC-⬘3
http://www-genome.wi.mit.edu/; The Genetic Epidemiology Research Group, http://cedar.genetics.soton.ac.uk/public_html/ (Collins et al., 1996); The Ge´ne´thon Human Genetic Linkage Map (Dib et al., 1996); GeneMap 1999, http//:www.ncbi.nlm.nih.gov/GeneMap/].
Product size (bp)
Annealing temperature (°C)
112
55
159
60
113
62
212
51
136
60
126
55
In the seven affected family members (individuals 10, 13, 17, 19, 20, 25, and 37) for whom data were available, the age at first renal replacement therapy for terminal renal failure ranged from 37 to 65 years with a median age of 50 years. Renal histology was available from patients 17, 25, and 37, showing the characteristics of MCKD with tubular basement membrane disintegration, tubulo-interstitial cell infiltration, tubular atrophy, and cyst development. Extrarenal organ involvement and hyperuricemia or episodes of gout were not observed.
fected individuals and also by two individuals in generation IV, who are too young for definition of the affected status (Fig. 1). Shared markers were as follows: cen–D1S2777, D1S2140, D1S1595, D1S2624, D1S394, D1S1600, D1S1653, and D1S2125–tel. To test the hypothesis of linkage of the family to MCKD1, we performed a two-point linkage analysis using 10 polymorphic markers located within the interval (D1S514, D1S498, D1S305, D1S2777, D1S2140, D1S1595, D1S2624, D1S394, D1S1653, and D1S2125). Since disease penetrance in MCKD is age dependent (Kroiss et al., 2000), linkage was calculated using an affectedsonly strategy. A maximum two-point lod score of Z max ⫽ 2.27 at a recombination fraction of ⫽ 0 was calculated with marker D1S2777. Multipoint linkage analysis yielded Z max ⫽ 2.4 for a region delimited by the flanking markers D1S305 and D1S2635 (Fig. 2). In linkage studies where only a few polymorphic markers are tested to confirm linkage to a known candidate gene locus with a small number of markers as well as in diseases with X-chromosomal inheritance, the criterion for significance for linkage can be released to ⬎2 from ⬎3 as postulated in a total genome search for linkage, where multiple testing is performed with approximately 400 polymorphic markers (Ott, 1985, p. 71). A lod score of 2.27 therefore clearly demonstrates linkage of the disease locus in this family to the MCKD1 locus. Recombination events are observed with marker D1S305 at the centromeric border and marker D1S2635 at the telomeric border of chromosome 1q21, thus refining the MCKD1 gene locus from 8 cM described previously (Christodoulou et al., 1998) to a critical interval of 4 cM. These results are supported by a previous report, where we could exclude linkage to the MCKD2 locus on chromosome 16p12 in this family (family MCD3) (Kroiss et al., 2000).
Linkage and Haplotype Analysis
Physical Map
Haplotype analysis with 12 polymorphic markers from the critical MCKD1 region revealed a common haplotype of 8 consecutive markers shared by all af-
In total the contig contains 47 YAC clones defined by 50 genetic markers (15 polymorphic microsatellite markers, 16 STSs, and 19 ESTs). Seven overlapping
PAC and BAC contig construction. Using a hybridization-based library screening of a human genomic PAC library [RPCI1.3–5 Human PAC, Resource Center, Max-Planck Institute, Berlin (RZPD): http://www.rzpd.de/] provided by Pieter de Jong (Ioannou et al., 1994), PAC clones were mapped within the YAC contig. Inter-Alu PCR was performed to derive probes from the YACs 790H6, 753B9, 874D5, 955E11, 950E2, 713B11, 887H8, 873E5, 726F3, and 748F9. STS markers known to map to the MCKD1 critical region according to the radiation hybrid maps from different public databases (for addresses see above) were used to screen the PAC library. Positive clones were obtained from the RZPD and isolated as described previously (Vollmer et al., 2000). Furthermore, 10 BAC clones provided by RZPD (http://www.rzpd.de) were also positioned within the MCKD1 interval. BAC-end sequencing. BAC ends were directly sequenced using BigDye chemistry on an ABI 377 automated sequencer by using vector-based T7 and SP6 primers. Resulting sequences were analyzed using the BLASTN 2.0.8 program (Altschul et al., 1997). Primers were designed to amplify unique end-sequence markers, which then were mapped back onto the YAC contig (Table 1).
RESULTS
Patients
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FIG. 2. Multipoint linkage analysis graph: The horizontal axis represents the relative genetic positions of the markers in centimorgan sex-averaged genetic distance (Dib et al., 1996). The position of marker D1S514 is arbitrarily set at 0 cM. The respective lod score values are indicated on the vertical axis. A horizontal dashed line is drawn at a lod score of 0. Note that for the markers D1S2777, D1S2140, D1S1595, D1S2624, D1S394, D1S1600, and D1S2125, lod score values above 2 in this candidate region indicate linkage to the MCKD1 locus.
YACs (“minimal contig”) between the previously reported flanking markers D1S489 and D1S2125 (Christodoulou et al., 1998) span an approximate physical distance of 7.1 Mb, defined by the combined length of clones 790H6 (760 kb), 753B9 (1740 kb), 955E11 (1310 kb), 950E2 (1290 kb), 713B11 (212 kb), 873E5 (210 kb), and 748F9 (1600 kb) (Fig. 3). According to the marker information of the Center for Medical Genetics (http://www.marshmed.org/genetics/), this physical distance corresponds to a total genetic sex-averaged distance of approximately 8 cM. The identification of marker D1S305 as a new flanking marker allowed for refinement of the critical MCKD1 region to a physical distance of approximately 3.3 Mb. Three YACs [YACs 790H6 (760 kb), 753B9 (1740 kb) and 955E11 (1310 kb)] spanning 3.8 Mb at the centromeric border of the contig can thereby be excluded from the critical MCKD1 interval. To generate a high-resolution physical map of the critical MCKD1 interval, we isolated 31 partially overlapping PAC clones by library hybridization using inter-Alu PCR from the RPCI PAC library to this interval, and the PAC clones were subsequently screened with STS markers previously mapped to the YAC contig. Additionally, by using the same approach, 10 BAC clones were mapped within the MCKD1 interval. Furthermore, BAC ends were sequenced with SP6 and T7 universal primers. Six novel BAC-end sequence markers were generated from the resulting sequences (Fig. 3, Table 1). Nineteen ESTs representing known genes were localized to the MCKD1 region. Among them are HAX-1, a nuclear gene encoding a mitochondrial protein, a human skeletal muscle 1.3-kb mRNA for tropomyosin, a fibroblast growth factor receptor 2, a human
small proline-rich protein, a RNA-specific adenosine deaminase (ADAR), an interleucin 6 receptor (IL6R), and a human transcriptional activator mRNA. DISCUSSION
In a large British pedigree of 11 affected individuals with the classic clinical criteria for MCKD, we found linkage to MCKD1. Renal histology showed tubulointerstitial abnormalities consistent with MCKD. No extrarenal organ involvement was observed. End-stage renal failure occurred with a median age of 50 years, similar to the MCKD1 family reported by Christodoulou et al. (1998). By haplotype analysis we refined the genetic interval initially reported to span 8 cM (Christodoulou et al., 1998) to a critical region of 4 cM according to the genetic map of the Marshfield Center of Medical Genetics (http://www.marshmed.org/genetics). Furthermore we constructed a high-resolution YAC, PAC, and BAC contig between markers D1S498 and D1S2125, which facilitates assignment of positional candidate genes in the refined region. This contig allowed for narrowing of the critical interval by 3.8 Mb on the centromeric side and for generation of a partial transcriptional map of the region. Although the genes responsible for MCKD1 and MCKD2 are not yet known, the gene causing NPH1 (NPHP1) was identified recently (Hildebrandt et al., 1997; Saunier et al., 1997). It codes for nephrocystin and includes a Src homology 3 (SH3) domain (Hildebrandt, 1998; Otto et al., 2000; Hildebrandt and Otto, 2000). It has been shown that nephrocystin interacts through this SH3 domain with Crk-associated sub-
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FIG. 3. YAC, PAC, and BAC contig of the MCKD1 region on chromosome 1q21 with partial transcriptional map. (A) STS and EST markers examined by STS content mapping in this study. Flanking markers D1S498 and D1S2125 reported initially (Christodoulou et al., 1998) are shown in boldface type. The newly defined flanking marker D1S305 from this study is shown in a box. (B) YAC clones of the CEPH-Mega-YAC library with their respective names, which have been mapped by STS content mapping with the respective markers. YAC sizes are given in parentheses. A vertical line indicates a positive PCR amplification with the respective marker. The YACs of the “minimal contig” spanning the entire interval are depicted in boldface type. The previously reported maximum physical size of 7.1 Mb is now restricted to approximately 3.3 Mb by definition of the new flanking marker D1S305. (C) BAC clones of the RPCI-11.1 library provided by the RZPD (http://www.rzpd.de). (D) PAC clones from a human genomic PAC library (RPCI1,3–5, Human PAC, Resource Center, RZPD) provided by Pieter de Jong. (E) Expressed sequences, which have been assigned to known genes.
strate (Cas), which is implicated in signal transduction and organization of the actin cytoskeleton at sites of cell adhesion (Donaldson et al., 2000). Considering the similarities of the clinico-pathological presentation of MCKD and NPH1, it is reasonable to assume that the two diseases may be the result of defects in the same developmental or metabolic pathway. Therefore, genes containing SH3 or SH2 domains represent strong candidates for MCKD. One of the genes found to map within the critical MCKD1 region is HAX-1 (HS1-associated protein X-1), an SH3-domain-containing protein associated with the actin cytoskeleton. It has been demonstrated that HAX-1 interacts with PKD2, the gene causing autosomal-dominant polycystic kidney disease type 2 (Gallagher et al., 2000). Since the pathogenesis of cyst formation in ADPKD is still unclear, assuming a common pathway of cystogenesis in both diseases makes HAX-1 an interesting positional and functional candidate gene for MCKD. With definition of D1S305 as a new proximal flanking marker, this gene is, however,
excluded as a positional candidate gene. Mutational analysis in one MCKD1 family revealed no mutations in the seven exons of HAX-1 (data not shown). Furthermore, there are several positional candidate genes that we mapped within the critical MCKD1 region. Some of them are not considered to be good functional candidate genes: Trichohyalin and S100A9 (synonyme: calgranulin B) are genes coding for proteins that are expressed during terminal differentiation in the epidermis (Volz et al., 1993); the small proline-rich protein 1B, called cornifin, appears to function as a component of the crosslinked envelope in squamous differentiating cells (Marvin et al., 1992); tropomyosin 3 is involved in a somatic rearrangement that creates the chimeric TRK onkogene and was found to be associated with autosomal dominant nemaline myopathy (Laing et al., 1995); the RNA-specific adenosine deaminase converts adenosine to iosine in dsRNA with various functions including site-specific RNA editing of transcripts of the glutamate receptors, probably resulting in malfunction of the glutamate-gated ion
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channels and modification of viral RNA genomes (Weier et al., 1995); the alpha subunit of erythroid spectrin is a predominant component of the membrane skeleton of the red cell and has been found to be associated with a subset of hereditary elliptocytosis (Lawler et al., 1984). KIAA0144, KIAA0080, and KIAA0907 are unidentified human genes whose sequences have been deduced by analysis of cDNA clones from human cell line KG-1 (Nomura et al., 1994). IL6R, the interleukin 6 receptor, stimulates the expression of hepatocyte growth factor, HGF (Matsumoto and Nakamura, 1992), which is postulated to be involved in tubular damage (Yorioka et al., 1996) or even postulated to be a tubular cyst-inducing mutagen (Horie et al., 1994) and therefore represents an interesting functional and positional candidate gene for MCKD1. The physical and partial transcriptional map of the MCKD1 region allows for identification of further candidate genes suited to mutational analysis in individuals affected by MCKD1 and thus represents a powerful tool for the identification of the disease-causing gene. ACKNOWLEDGMENTS A.F. was supported by a grant from the German Research Foundation (DFG FU 202/3-1) and from the Fritz-Thyssen-Stiftung, Germany. F.H. was supported by the Zentrum klinische Forschung (ZKF-A1). A.F. and F.H. were supported by the State of BadenWu¨rttemberg (FSP “Zystogenese”). N.R. was supported by the National Kidney Research Fond (NKRF). We thank R. Witzgall for information on HAX-1 as a candidate gene. We thank Barbara Scho¨nfeld, Anita Imm, and Cornelia Klein for their technical assistance. We thank the patients and their family for their collaboration.
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