A Gene Locus for Steroid-Resistant Nephrotic Syndrome with Deafness Maps to Chromosome 14q24.2
Rainer G. Ruf*,
Matthias T. F. Wolf*,
Hans C. Hennies,
Barbara Lucke,
Christina Zinn,
Verena Varnholt,
Anne Lichtenberger*,
Andreas Pasch,
Anita Imm,
Sonia Briese,
Thomas Lennert¶,
Arno Fuchshuber,
Peter Nurnberg,# and
Friedhelm Hildebrandt*
*Departments of Pediatrics and Human Genetics, University of Michigan, Ann Arbor, Michigan; Gene Mapping Center and Department of Molecular Genetics, Max-Delbrueck Center for Molecular Medicine, Berlin-Buch, Germany; Department of Pediatrics, University Hospital Charite, Campus Virchow Klinikum, Humboldt-University, Berlin, Germany; Children’s University Hospital, Freiburg, Germany; ¶Department of Pediatrics, University Hospital Benjamin Franklin, Free University, Berlin, Germany; and #Institute of Medical Genetics, Charité University Hospital, Humboldt University, Berlin, Germany.
Correspondence to Dr. Friedhelm Hildebrandt, University of Michigan Health System, 8220C MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0646; Phone: 734-615-7285; Fax: 734-615-1386;
ABSTRACT. Steroid-resistant nephrotic syndrome (SRNS) leadsto end-stage renal disease (ESRD) in childhood or young adulthood.Positional cloning for genes causing SRNS has opened the firstinsights into the understanding of its pathogenesis. This studyreports a genome-wide search for linkage in a consanguineousPalestinian kindred with SRNS and deafness and detection ofa region of homozygosity on chromosome 14q24.2. Multipoint analysisof 12 markers used for further fine mapping resulted in a LODscore Zmax of 4.12 ( = 0) for marker D14S1025 and a two-pointLOD score of Zmax = 3.46 ( = 0) for marker D14S77. Lack of homozygositydefined D14S1065 and D14S273 as flanking markers to a 10.7 cMinterval. The identification of the responsible gene will providenew insights into the molecular basis of nephrotic syndromeand sensorineural deafness. E-mail: fhilde@umich.edu
Nephrotic syndrome (NS) is defined as the association of proteinuria,hypoalbuminemia, edema, and hyperlipidemia. Autosomal recessivesteroid-resistant nephrotic syndrome SRNS has been describedas childhood onset of proteinuria, rapid progression to ESRD,resistance to steroid therapy, and absence of recurrence afterrenal transplantation (1). It remains the most intractable causefor ESRD in the first two decades of life. Whereas renal biopsymostly shows minimal change nephrotic syndrome (MCNS) in steroid-sensitivenephrotic syndrome (SSNS), renal biopsy in SRNS reveals differentmorphologic changes, such as MCNS, mesangial sclerosis, or focalsegmental glomerulosclerosis (FSGS) (2). Treatment ranges fromearly bilateral nephrectomy and transplantation in the congenitalnephrotic syndrome of the Finnish type (NPHS1) (3) to the moreconservative therapy with steroids in SSNS. Two genes have beenidentified as autosomal recessive causes of SRNS: mutationsin NPHS1, encoding nephrin (OMIM 602716), cause congenital nephroticsyndrome of the Finnish type (3), and mutations in NPHS2, encodingpodocin (OMIM 604766), cause SRNS type 1 (4). Mutations in ACTN4,encoding -actinin 4 (OMIM 604638) have been identified as anautosomal dominant cause of SRNS (5). An additional locus hasbeen mapped to chromosome 11q21-q22 for an autosomal dominantform of nephrotic syndrome (OMIM 603965) (6). Gene identificationunderlined the importance of genetic factors in the pathogenesisof nephrotic syndrome. Through identification of these threegenes as causative for SRNS, their gene products, nephrin, podocin,and -actinin 4, were identified as important for the functionof the glomerular podocyte (7). Specifically, these gene productswere shown to represent integral components of the glomerularslit membrane localized between podocyte foot processes, whichrepresents the primary glomerular filter of the kidney (7).We here performed a total genome search for linkage, applyingthe paradigm of homozygosity mapping to a Palestinian kindredwith the association of SRNS and sensorineural deafness (SND),and identified a new gene locus (SRN2) for SRNS with SND withina 10.7 cM interval on chromosome 14q24.2.
Blood samples and clinical data of the family with SRNS andSND were obtained after informed consent was obtained from patientsand their parents and siblings. Ethnic origin of the familywas Palestinian. Affected individuals all showed symptoms ofnephrotic syndrome (gross proteinuria, hypoalbuminemia, edema,and hyperlipidemia). Diagnosis was established by a pediatricnephrologist. Onset of disease ranged from 0.3 to 6.4 yr (Table 1).The histopathologic diagnosis was FSGS. All affected individualsentered ESRD within the range of 1.25 to 9.3 yr (Table 1). Inthree patients, kidney transplantation was performed and norecurrence of FSGS was described. Congenital SND was diagnosedin all four patients. Patient IV:2 died at the age of 15 yrfrom acute heart failure when restarting dialysis after chronicrejection of the kidney transplant. Patient V:1 died at theage of 1.7 yr from peritonitis and cerebral candida infectionwhile on peritoneal dialysis (Table 1). Genomic DNA was isolated,by standard methods (8), either directly from blood samplesor after Epstein-Barr virus transformation of peripheral bloodlymphocytes (9). For the genome-wide search for linkage, DNAwas available in two affected individuals, four unaffected siblings,and both parents who were first cousins and for two parentsand their children, one affected, who were more distantly relatedto the first pedigree (Figure 1). All affected individuals wereoffspring of consanguineous marriages; therefore, an approachof homozygosity mapping was employed (10). A total of 380 microsatellitemarkers from the Genethon final linkage map (11), with an averagedistance of 11 cM, were used. For further fine mapping on chromosome14q24.2, ten additional markers, with an average spacing of1 cM, were used. The order and distance between markers wereobtained from the Genethon database (11), and they are, in orderfrom centromeric to telomeric: D14S1069 (0.8 cM), D14S1065 (0.1cM), D14S1029 (1.6 cM), D14S588 (1.1 cM), D14S258 (1.3 cM),D14S1002 (2.7 cM), D14S77 (1.7 cM), D14S71 (0.1 cM), D14S1025(1.6 cM), D14S1047 (0.5 cM), D14S273 (1.6 cM), and D14S270.Semi-automated genotyping was performed with a MegaBACE-1000analysis system. Data were analyzed by Genetic Profiler Software,version 1.1. Two-point LOD score calculations were performedby the LINKAGE program package (12), with the help of the LINKRUNcomputer program (Wienker TF, unpublished observations; furtherinformation can be obtained from the authors), using an autosomalrecessive model with 100% penetrance, a gene frequency for SRNS,with SND of 0.0001. There is a certain dependence on age-relatedpenetrance; therefore, an affecteds-only strategy was appliedfor LOD score analysis. The "LODmax - 1 support interval" wasdefined as the genetic map positions intersecting the LOD scorecurve at Zmax - 1 (13). For haplotyping and computation of multipointLOD scores, the program SIMWALK (14) was used, allele frequencieswere calculated according to the database of the Gene MappingCenter and Department of Molecular Genetics, Max-Delbrueck Centerfor Molecular Medicine, Berlin-Buch, Germany (unpublished data).The comprehensive data collected during hundreds of total genomesearches representing a wide range of different ethnicitiesare included in the database, which represents an appropriatepopulation to estimate allele frequencies. In addition, theLOD Score of 4.1 obtained in these data is robust enough fora wide range of allele frequencies (15).
Figure 1. Haplotypes on chromosome 14q24.2 of the Palestinian kindred with steroid-resistant nephrotic syndrome (SRNS) and sensorineural deafness (SND). Twelve microsatellites are shown from cen to q-ter (top to bottom). Haploptyes were generated by minimizing recombinants. Circles denote females, squares males; filled symbols indicate affected status. A slash through a symbol marks a deceased individual. Haplotypes are interpreted as differently shaded bars. Paternal haplotypes are drawn to the left, maternal ones to the right. Double lines indicate consanguineous marriages. Regions of contiguous homozygosity are denoted in boxes. The Proband is indicated with an arrow. Note that markers D14S1065 and D14S273 (underlined) are flanking the SRN2 locus.
By using haplotype analysis data of the genome-wide search,the known loci for SRNS on chromosomes 1q25–31 (NPHS2),11q21-q22 (6), and 19q13 (NPHS1 and ACTN4) were excluded. Ofall 380 markers employed in the genome-wide search, only markerD14S588 showed homozygosity for the same allele in all affectedindividuals. No homozygosity was found in the unaffected familymembers. The maximum two-point LOD score at D14S588 was 2.94( = 0). Further fine mapping with additional ten markers confirmedthis region as contiguously homozygous by descent extendingfrom marker D14S1029 to D14S1047. Homozygosity was present inall affected individuals and was absent in unaffected individuals.Lack of homozygosity in individual V:1 defined marker D14S1065as the centromeric flanking marker, and marker D14S273 as flankingon the q-telomeric side (Figure 1), thus restricting the criticalgenetic region for SRNS2 to a 10.7 cM interval on chromosome14q24.2. Multipoint analysis of the 12 markers resulted in aZmax of 4.12 for marker D14S1025 at relative position 71.6 (Figure 2).The 95% CI at Zmax - 1 (13) extends over a 10.1 cM intervalflanked by marker D14S1029 and D14S1047. Marker D14S77 showedthe highest two-point LOD score with Zmax = 3.46 ( = 0) (Table 1).
Figure 2. Multipoint LOD scores for the SRN2 locus versus the twelve markers shown in Figure 1 were calculated for the Palestinian SRNS family with SND. Relative positions are given in cM according to the Genethon map (10). The two markers, D14S1065 and D14S273, flanking the SRNS region (see Figure 1) are underlined. q-ter, q-terminal orientation; cen, centromeric orientation.
Employing a homozygosity mapping strategy, we thus identifiedby total genome search a new gene locus (SRN2) for a new diseaseentity of SRNS occurring in association with SND. Accordingto the UCSC Genome Browser, the interval between flanking markersD14S1065 and D14S273 spans a physical distance of approximately7 Mb (relative marker positions are 66.310.876 bp and 73.183.615bp, respectively) and contains approximately 50 known genes.Among these genes, the -actinin 1 (ACTN1) gene is located inthe region suitable for linkage in this family at 66.846.267to 66.951.188 Mb according to the UCSC Genome Browser. ACTN1shows over 70% amino acid homology to ACTN4 (16), which hasbeen identified as an autosomal dominant cause of SRNS (5).ACTN1 was found to be expressed in Corti’s organ of theadult rat and more specifically in the cuticularplate supportingthe stereocilia as well as in the stereocilila of the outerand inner hair cells. In addition, it is expressed in the pillarcells and supporting cells (17). No expression of ACTN1 in thekidney has been described so far (5,18). No mutation was foundwhen directly sequencing the 21 exons of ACTN1 for individualsIII:10 and IV:6 of the Palestinian pedigree (data not shown).
In the search for the gene responsible for the disease in thisfamily, genes that encode components of the podocyte foot processcytoskeleton and genes that encode proteins interacting withproteins already described to be impaired in NS should be excellentcandidate genes. In addition, a role in the function of theinner ear will have to be postulated for candidate genes. Identificationof the gene responsible for SRN2 will provide new insights intodisease mechanisms of SRNS as well as into the function of theinner ear.
Table 2. Two-point LOD scores at various recombination fractions for 12 microsatellite markers versus SRN2 on chromosome 14q24.2
Acknowledgments
We thank all family members and their physicians for the participationin this study.
URL for data in this article are as follows: Genethon map, http://www.cephb.fr/ceph-genethon-map.html;Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim;UCSC Genome Browser, http://genome.ucsc.edu./
Footnotes
Rainer G. Ruf and Matthias T. F. Wolf contributed equally tothis work.
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Received for publication January 14, 2003.
Accepted for publication February 20, 2003.
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Abstract
Introduction
= 0) for marker D14S1025 and a two-point LOD score of Zmax = 3.46 (
-actinin 4 (OMIM 604638) have been identified as an autosomal dominant cause of SRNS (5). An additional locus has been mapped to chromosome 11q21-q22 for an autosomal dominant form of nephrotic syndrome (OMIM 603965) (6). Gene identification underlined the importance of genetic factors in the pathogenesis of nephrotic syndrome. Through identification of these three genes as causative for SRNS, their gene products, nephrin, podocin, and 
