Mutations of the Uromodulin Gene in MCKD Type 2 Patients Cluster in Exon 4, Which Encodes Three EGF-Like Domains
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Kidney International, Vol. 64 (2003), pp. 1580–1587 Mutations of the Uromodulin gene in MCKD type 2 patients cluster in exon 4, which encodes three EGF-like domains MATTHIAS T.F. WOLF,BETTINA E. MUCHA,MASSIMO ATTANASIO,ISABELLA ZALEWSKI, STEPHANIE M. KARLE,HARTMUT P.H. NEUMANN,NAZNEEN RAHMAN,BIRGIT BADER, CONRAD A. BALDAMUS,EDGAR OTTO,RALPH WITZGALL,ARNO FUCHSHUBER, and FRIEDHELM HILDEBRANDT Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan; University Children’s Hospital, Freiburg University, Freiburg, Germany; Department of Internal Medicine, University Hospital, Freiburg University, Freiburg, Germany; Section of Cancer Genetics, Institute of Cancer Research, Sutton, United Kingdom; Department of Internal Medicine, University Hospital, Tuebingen University, Tuebingen, Germany; Department of Internal Medicine, University Hospital, Cologne University, Cologne, Germany; and Institute for Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany Mutations of the Uromodulin gene in MCKD type 2 patients Medullary cystic kidney disease type 2 (MCKD2) cluster in exon 4, which encodes three EGF-like domains. (OMIM 603860) includes clinical features of reduced Background. Autosomal-dominant medullary cystic kidney urinary concentration, salt wasting, and end-stage renal disease type 2 (MCKD2) is a tubulointerstitial nephropathy that causes renal salt wasting, hyperuricemia, gout, and end-stage re- failure. In contrast to MCKD1, in MCKD2, a more se- nal failure in the fifth decade of life. The chromosomal locus for vere phenotype concerning hyperuricemia and gout has MCKD2 was localized on chromosome 16p12. Within this chro- been described. Moreover, in MCKD2, an earlier median mosomal region, Uromodulin (UMOD) was located as a candi- age of onset has been reported (age of onset in MCKD2 date gene. UMOD encodes the Tamm-Horsfall protein. By se- is 32 years of age compared to 62 years in MCKD1) [1]. quence analysis, one group formerly excluded UMOD as the disease-causing gene. In contrast, recently, another group de- Otherwise, these two diseases are clinically undistinguish- scribed mutations in the UMOD gene as responsible for MCKD2 able. In MCKD, small corticomedullary cysts are com- and familial juvenile hyperuricemic nephropathy (FJHN). mon, but not always detected. Kidney size is normal or Methods. Haplotype analaysis for linkage to MCKD2 was slightly reduced. MCKD shows a renal histologic triad of performed in 25 MCKD families. In the kindreds showing (1) tubular basement disintegration, (2) tubular atrophy linkage to the MCKD2 locus on chromosome 16p12, muta- tional analysis of the UMOD gene was performed by exon with cyst development at the corticomedullary border, polymerase chain reaction (PCR) and direct sequencing. and (3) interstitial cell infiltration associated with fibrosis Results. In 19 families, haplotype analysis was compatible [2]. Neither imaging results, nor pathologic findings, are with linkage to the MCKD2 locus. All these kindreds were pathognomic for MCKD2. The condition shares clinical examined for mutations in the UMOD gene. In three different and morphologic similarities to recessive juvenile neph- families, three novel heterozygous mutations in the UMOD ronophthisis (NPH) [3]. In contrast to juvenile onset in gene were found and segregated with the phenotype in affected individuals. Mutations were found only in exon 4. NPH, end-stage renal failure in MCKD occurs in adult- Conclusion. We confirm the UMOD gene as the disease- hood. Moreover NPH is inherited in an autosomal-reces- causing gene for MCKD2. All three novel mutations were sive pattern, whereas MCKD is autosomal-dominant. found in the fourth exon of UMOD, in which all mutations Two loci for MCKD have been described: MCKD1 except one (this is located in the neighboring exon 5) published showed linkage to chromosome 1q21 in two Cypriot fam- so far are located. These data point to a specific role of exon 4 encoded sequence of UMOD in the generation of the MCKD2 ilies [4] and MCKD2 was localized on chromosome 16p12 renal phenotype. in an Italian family [1]. Two groups showed indepen- dently that loci for familial juvenile hyperuremic ne- phropathy (FJHN) and MCKD2 mapped to the same Key words: MCKD2, uromodulin, Tamm-Horsfall protein. chromosomal region on 16p12 [5, 6], assuming that FJHN and MCKD2 might be different facets of the same dis- Received for publication March 20, 2003 and in revised form May 28, 2003 ease caused by mutations in one and the same gene. Accepted for publication June 23, 2003 Later, an additional FJHN locus was mapped to a more 2003 by the International Society of Nephrology centromeric region [7] on chromosome 16, resulting in 1580 Wolf et al: Mutations in the UMOD gene in MCKD type 2 patients 1581 two different FJHN loci, which are overlapped by the 5 to 10 years, 4.1 Ϯ 1 mg/dL; female, 12 years, 4.5 Ϯ 0.9 MCKD2 locus [8]. Two years after publication of the mg/dL; 15 years, 4.5 Ϯ 0.9 mg/dL; Ն18 years, 4.0 Ϯ 0.7 chromosomal candidate region of MCKD2, Uromodulin mg/dL; male, 12 years, 4.4 Ϯ 1.1 mg/dL; 15 years, 5.5 Ϯ (UMOD) was published as excluded from representing 1.1 mg/dL; Ն18 years, 6.2 Ϯ 0.8) [18]. the disease-causing gene [9]. In contrast however, Hart The study was approved by the Ethics Committee of et al [10] later detected four different mutations in the the Albert-Ludwigs-University Freiburg and all partici- UMOD gene, indicating that this is the gene responsible pating family members provided informed consent. for FJHN and MCKD2. UMOD encodes the Tamm-Horsfall protein (THP), Haplotype analysis which is expressed primarily at the luminal side of renal Genomic DNA was isolated by standard methods di- epithelial cells of the thick ascending loop of Henle and rectly from blood samples using the QIAamp blood kit of early distal convoluted tubules. THP is the most abun- (Qiagen, Valencia, CA, USA) or from blood lymphocytes dant protein in the urine of humans [11]. Functional after Epstein-Barr virus (EBV) transformation. Haplo- roles of UMOD have been described in urinary tract type analysis was performed in 224 individuals (including infections, in binding to complement factors, in myeloma 84 affected individuals) and inferred in 18 additional kidney, and nephrolithiasis [12–15]. The UMOD protein individuals (nine additional affected individuals), using contains (1) a zona pellucida domain (ZP), which is 13 consecutive polymorphic microsatellite markers that necessary for polymerization into the supramolecular span the critical MCKD2 region in the following order: structure of a filament, (2) an elastase-sensitive fragment cen-D16S3116-D16S401-D16S3113-D16S420-D16S417- containing three calcium-binding epidermal growth fac- D16S412-D16-S3036-D16S3041-D16S3056-D16S501- tor (EGF)-like domains and a signal peptide, and (3) D16S499-D16S-3060-D16S764-tel. Fluorescently labeled a potential glycosyl-phosphatidylinositol (GPI)-anchor polymerase chain reaction (PCR) products were detected cleavage site. UMOD is a transmembrane-bound pro- by a Genetic Analyzer 3100 (Applied Biosystems, Foster tein, which can be secreted into the urine by cleavage City, CA, USA) and were analyzed by the GENOTYPER of the GPI-anchor [16]. software. We here report three novel mutations in UMOD and confirm UMOD as the disease-causing gene. So far, eight Mutational analysis of the UMOD gene mutations published are located in exon 4 and one muta- Mutational analysis was performed by exon PCR tion was found at the beginning of the neighboring exon of the Uromodulin gene. Primer sequences were deter- 5 of the UMOD gene that encodes three EGF-like do- mined using the UCSC sequence (November 2002 mains [10, 17]. freeze) encompassing the coding sequence of UMOD [19]. The following primers and conditions were em- ployed: exon 3, 5Ј-CAATCAAAGCACTCCTTCCAG- METHODS 3Ј and 5Ј-GACAGGTGCTACATTGCTTCC-3Ј; exon Patients 4 was divided into two different overlapping parts: exon We ascertained 25 MCKD families (224 individuals, 4a, 5Ј-CCTGGAGAATGAGGGAAGG-3Ј and 5Ј-CTG of which 84 were affected). Fourteen families were from GACGAGTACTGGCGC-3Ј; exon 4b, 5Ј-GGCAGCT Germany, three from the United States, two each from ACTTGTGCGTATG-3Ј and 5Ј-CTCTGCAGTGCCT the United Kingdom, Hungary, and Turkey, respec- TTCCAG-3Ј; exon 5, 5Ј-GTCTCCCCACAGTCCTC tively, one each from Belgium and China, respectively. ATC-3Ј and 5Ј-GGCAGTGACAGGTTTCTCAAC-3Ј; The age at diagnosis, the age at onset of end-stage renal exon 6, 5Ј-GGCCCCCAAGCTATAGACAC-3Ј and disease (ESRD), hyperuricemia, imaging data, and biopsy 5Ј-CCATGAATTGCTTTCTTTATTTG-3Ј; exon 7,5Ј- results were reviewed if available. Clinical criteria neces- TCATGCCCCTTTCTCTCATC-3Ј and 5Ј-GCTCATG sary for inclusion were (1) compatibility of pedigree GTTAAGGGGTTTG-3Ј; exon 8, 5Ј-TGCCTAGGA with autosomal-dominant inheritance, (2) chronic renal ATGCAAATCAG-3Ј and 5Ј-CCTTACCTGTCCTGG failure, (3) defective urine concentration (Ͻ800 mOsm/L ATGATG-3Ј; exon 9, 5Ј-TCAGCTCTGCACTGATGA after overnight dehydration) with polyuria (Ͼ3 L/day), CAG-3Ј and 5Ј-TGGCCAAATGTCTTTGGTTAC-3Ј; and (4) at least one pedigree member with chronic renal exon 10, 5Ј-AACCACATTCAGGCTCCTTC-3Ј and failure in whom renal biopsy showed tubulointerstitial 5Ј-AGGTGCCAGGCTCTGTTATC-3Ј; exon 11, 5Ј- fibrosis with infiltrates, tubular atrophy, and thickening GTCAGGATGCTTGCCAAATC-3Ј and 5Ј-CACTG of the tubular basement membrane. Optional clinical CAGAAAGGACCTGAAC-3Ј; and exon 12, 5Ј-GG criteria were normal or small-sized kidneys with occa- GTGTCCTCTTCTGATTGG-3Ј and 5Ј-CAGGTA sional small corticomedullary cysts. Hyperuricemia was CACCGTCACAAGTCC-3Ј using a “touch down” pro- defined as serum uric acid concentration Ͼ1 SD greater gram with a starting annealing temperature of 72ЊC (ex- than the normal values for age and gender (both genders ons 4a, 5, and 10), decreasing every step by 0.7ЊC for 24 1582 Wolf et al: Mutations in the UMOD gene in MCKD type 2 patients times and a next round of amplification with an annealing since there is age-related penetrance in MCKD. On this temperature of 55ЊC for 20 times.