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Nail-Patella Syndrome

Identification of Mutations in the LMX1B Gene in Dutch Families

NINE V.A.M. KNOERS^*, ERNIE M.H.F. BONGERS^*, SYLVIA E.C. VAN BEERSUM^*, ED J.P. LOMMEN, HANS VAN BOKHOVEN^* and FRANS A. HOL^*

^* Department of Human Genetics, University Hospital Nijmegen, Nijmegen, The Netherlands
Department of Pediatrics, St. Joseph Hospital, Veldhoven, The Netherlands.

Correspondence to Dr. Nine Knoers, Department of Human Genetics, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: 31-24-3613946; Fax: 31-24-3565026; E-mail: n.knoers{at}antrg.azn.nl

	Abstract

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Abstract. Nail-patella syndrome is an autosomal dominant disorder characterized by dyplasia of finger nails, skeletal anomalies, and, frequently, renal disease. It has recently been shown that this disorder is caused by putative loss-of-function mutations in a transcription factor (LMX1B) belonging to the LIM-homeodomain family, members of which are known to be important for pattern formation during development. A cohort of eight Dutch NPS families were screened for mutations in the LMX1B gene; seven different mutations, including one novel variant, were identified. Three of the mutations are very likely to result in truncated LMX1B proteins, three are predicted to influence sequence-specific DNA binding, and one is presumed to prevent the formation of a stable protein by abolishing the Zn(II) binding site of the protein. Although there was a remarkable high incidence of renal disease in one of the families, the nephropathy was not seen in all affected family members and the severity of renal impairment varied significantly among the patients. This indicates that the incidence and severity of nephropathy within this family cannot be attributed to the LMX1B genotype. In addition, evidence of a correlation between other characteristics of the NPS phenotype and specific mutations has not been found.

	Introduction

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Nail-patella syndrome (NPS) (MIM 161200), also known as hereditary osteo-onychodysplasia, is a rare autosomal dominant disorder that is characterized by nail and bone abnormalities and, frequently, renal disease. Well more than 500 cases have been described after the first description of the disorder by Little in 1837 (1). Nail hypoplasia or dysplasia and absent or hypoplastic patellae are essential features for the diagnosis. Other diagnostic signs are deformation or luxation of the head of the radius, resulting in impaired mobility of the elbow, and iliac horns, which are pathognomonic but reported to be present in only 70% of cases. Various other skeletal anomalies, such as pes equinovarus, dislocated hips, and contractures of major joints, have been described frequently in NPS but do not contribute to the diagnosis. Approximately 50% of affected individuals exhibit cloverleaf pigmentation of the inner margin of the iris. Its significance is unclear. One report described two families in which NPS cosegregated with open-angle glaucoma (2), suggesting that glaucoma may be another variable feature of the syndrome.

The most serious component of NPS is the nephropathy, but this does not occur in all patients with the syndrome. In a retrospective study analyzing renal features of 123 observations in the literature, Meyrier et al. (3) discovered renal involvement in more than 60% of cases. In most cases, the nephropathy manifests only by chronic benign proteinuria. In approximately 15% of cases, however, the disease developed toward end-stage renal disease. It is unknown which factors are responsible for progression to renal insufficiency, and therefore renal prognosis is unpredictable. The light microscopy findings of renal biopsies in NPS are nonspecific. At the ultrastructural level, however, the glomeruli show a uniform and characteristic picture: irregular thickening of the glomerular basement membrane (GBM) with electron-lucent areas giving it a so-called "moth-eaten appearance." Within the GBM and the mesangium, fibrillar collagen-like material is found, and finally there is fusion of epithelial foot processes. Remarkably, these ultrastructural changes have been found in all biopsied cases, independent of clinical involvement, and therefore do not seem to correlate with impaired renal function.

In the late 1960s, the gene for NPS was assigned to the distal end of the long arm of chromosome 9 by the establishment of linkage of NPS to the ABO blood group locus and the adenylate kinase gene (4,5). It lasted a few decades, however, before the localization of the NPS gene could be significantly refined to a genetic interval of 1 cM in region 9q34.1 (6,7,8,9).

Recently, Dreyer et al. (10) showed that NPS is caused by mutations in the LMX1B gene. The involvement of this gene in NPS was subsequently confirmed by others (11,12). Mutations in LMX1B were also found in families with NPS and glaucoma (11). LMX1B belongs to a family of highly related LIM-homeodomain transcription factors that are involved in pattern formation during development. These proteins contain two tandem LIM domains, which are cysteine-rich, zinc-binding domains that facilitate the interaction with other transcription factors: a homeodomain that has DNA-binding activity and a transcriptional activation domain (13). The identification of the LMX1B gene as being responsible for NPS was anticipated by investigations of the role of LMX1B homologous genes in limb development. In the chicken, it had been shown that lmx1 specifies the dorsal cell fate of the limb. Thus, absence of lmx1 expression leads to the development of biventral limbs (14). Mice with a homozygous deletion of exon 3 to 7 in the mouse lmx1 homologue, LmX1b, exhibit nail dysplasia and skeletal defects similar to those observed in NPS, such as iliac hypoplasia, aplasia of the patellae, and joint abnormalities marked by osseous misarticulations. In addition, ultrastructural examination of the kidneys showed prominent irregular thickening of the GBM with occasional regions of membrane discontinuity, reminiscent of the renal findings in NPS (15).

In this report, we present the identification of seven different mutations, including one novel variant, in the LMX1B gene in eight Dutch families with NPS and discuss the presumptive effect of these mutations on the function of the LMX1B protein, as well as the absence of convincing genotype—phenotype correlations.

	Materials and Methods

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Patients
We studied three large families with NPS, two smaller families with NPS, and three families with sporadic cases of NPS. The clinical phenotype of affected individuals in the eight families is summarized in Table 1. The two largest families (families 3 and 8) were studied more then 10 yr ago, at that time with the aim of determining the presence or absence of nephropathy (16). Recently, most of the patients of family 8 were seen again for molecular genetic analysis and confirmation of the absence/presence of other major NPS signs. Some patients that were included in the study of 10 yr ago had died, and three additional patients had received diagnoses. We discovered that one recently identified patient who was a presumed sporadic patient belongs to family 3. We were not able to examine all of the affected individuals of this family again; therefore, we have the data only on nail, patellar, and iliac signs from three patients. The nephropathy data from families 3 and 8 were taken from the original paper (16). We also were not able to examine clinically all patients from family 4.

Mutation Detection Analysis
Genomic DNA was extracted from whole blood according to the procedure described by Miller et al. (17). To find mutations in the LMX1B gene, we performed single-strand conformation polymorphism (SSCP) analysis. The different LMX1B exons were amplified by PCR using standard PCR buffer in a total volume of 25 ul, containing 50 ng of genomic DNA, 15 pmol of each appropriate primer (Table 2), 1 U of Taq polymerase (Life Technologies, Breda, The Netherlands) and 0.5 mM of each dNTP. Amplifications were performed in a Thermal Cycler (Perkin Elmer, Norwalk, CT). Samples were denatured at 92°C for 5 min and then subjected to 35 cycles of amplification: 1 min at 95°C, 1 min at annealing temperature (Table 2), 1 min at 72°C. SSCP analysis was performed using the Genephor Gelsystem (Amersham Pharmacia Biotech, Roosendaal, the Netherlands). PCR products were loaded on precast, ready-to-use GeneGel Excel 12.5/24 polyacrylamide gels (Pharmacia). After electrophoresis, gels were silver stained in an automated gel stainer using the Plus-one DNA Silverstaining Kit (Pharmacia), according to the protocol of the manufacturer. Samples were run in duple at 5°C and 15°C, respectively, to increase sensitivity. When a band shift was detected, the corresponding fragment was bidirectionally sequenced to reveal the nature of the mutation. In case no mutations were found through SSCP analysis, all exons were sequenced. Automated DNA sequencing was performed on an ABI PRISM 377 (PE Biosystems, Nieuwerkerk ald Yssel, The Netherlands) using dye terminator chemistry.

	Results

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Human genomic DNA from the probands of five families and of three sporadic patients was screened for mutations in the LMX1B gene by PCR-SSCP of exons 2 to 8, followed by sequencing of amplified DNA fragments that had resulted in SSCP band shifts. We identified mutations in all of them (Table 3). Two patients from different families (families 3 and 4) share the same R198X mutation. The seven different mutations were all single base-pair substitutions, consisting of four missense mutations, two nonsense mutations, and 1 splice-site mutation. One of the identified mutations (A249P) has not been described previously. Of the remaining six mutations, three (Q64X, C95Y, splice-site mutation) have been found in one other family, and the other three (R198X, R200Q, A213P) are more frequently occurring mutations and have been found in five, six, and four families, respectively (10,11,12,18). Both nonsense mutations (R198X and Q64X) are predicted to cause premature termination of translation, resulting in truncation of the LMX1B protein. The G to C transversion and the G to A transition in exon 4, leading to an A213P and an R200Q missense mutation, respectively, in the homeodomain of the LMX1B protein, have been shown to have a negative effect on DNA binding (11). The G to A transition in exon 3, resulting in a C95Y missense mutation in the second LIM domain, may abolish the Zn(II) binding site and prevent the formation of a stable protein. The splice-site mutation (672 +1GA) will probably lead to a shorter LMX1B mRNA as a result of exon skipping. Because LMX1B-expressing tissue, such as kidney, was not available, we were not able to confirm this presumed effect of this mutation. In families 2, 3, 4, 7, and 8, the mutations cosegregate with the NPS phenotype. The seven mutations were not found in a panel of 100 control chromosomes. In addition to these putative loss-of-function mutations, five common polymorphisms, two silent mutations (Ser219Ser; Glu124Glu), and three intron polymorphisms (IVS 2, +7 GC; IVS 4, -49 CT; IVS 8, -49 CA) were found in our series.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In our group of Dutch families with NPS, we identified seven different mutations in the LMX1B gene, including one novel variant, which brings the total number of different mutations identified to 65 (10,11,12,18,19). Approximately 43% of these 65 mutations are localized in the homeodomain, 40% in the LIM1 domain, and the rest in the LIM2 domain. Remarkably, mutations in the transcriptional activation domain in the C-terminus of the LMX1B protein have not yet been found.

Three of the mutations identified in our study are frequently occurring mutations. R198X has been found in 4%, R200Q in 5%, and A213P in 3% of all families tested. Although they did not show their data, McIntosh et al. (12) concluded, on the basis of haplotype analysis with polymorphic microsatellite markers from the 9q34 region, that these mutations recur in unrelated individuals. Haplotype analysis in our Dutch cohort has been performed only in the larger families, at the time when the gene responsible for NPS was not yet identified, to narrow the chromosomal region encompassing the disease gene. The patients carrying the R198X, R200Q, and A213P mutations did not belong to these previously analyzed families, either because they were sporadic patients or because the family was too small or only a few family members were available for study. Therefore, although unlikely, we cannot exclude the possibility that our patients are related to any of the other families carrying a similar mutation. The high rate of recurrence of the R198X and R200Q mutations might be explained by the fact that these mutations are the result of a transition at a CpG dinucleotide, which is a mutational hot spot because of the tendency for the cytosine to be methylated and subsequently deaminated to thymine. The recurrence of the A213P mutation, however, remains unexplained.

On the basis of the nature of the mutations and/or the mutations' localization in functionally important domains of the LMX1B protein, together with the consegregation of the mutations with the NPS phenotype within the families and the absence of these mutations in 100 control chromosomes, it is very likely that these seven mutations are indeed harmful mutations and not innocuous polymorphisms. Thus, the two nonsense mutations and the splice-site mutation are predicted to result in truncated LMX1B proteins. The four remaining mutations are missense mutations, and three of these amino-acid substitutions are localized in the homeodomain of the protein, where they are likely to have an effect on DNA binding. R200Q and A213P were shown to reduce and abolish DNA binding, respectively, in in vitro DNA binding studies (12). The homeodomain-DNA interactions of homeobox genes have been intensively studied in many species (for review, see reference (20). In the course of the evolution, the amino-acid sequence of the homeodomain has been conserved to a high degree, reflecting the importance of this sequence for establishing DNA binding. Because it is known which amino acids in the homeodomain are important for DNA-binding specificity, direct DNA-backbone contacts, and secondary structure, the putative effects of the identified mutations in the homeodomain of LMX1B on DNA binding may be predicted. The R200Q substitution affects a highly conserved amino acid located in the flexible N-terminal arm of the homeodomain, which is important in establishing contact to the minor groove of the DNA and as such significantly contributes to the high DNA-binding affinity of the homeodomain. The alanine substitution in the first helix of the homeodomain (A213P) is likely to disturb the formation of a three-dimensional structure that is determined by the folding of the helices I, II, and III into a tight globular structure (21). Because this three-dimensional structure of the homeodomain ensures the positioning of the third (recognition) helix in the major groove of the DNA, where it establishes contact with both strands of the DNA, it can be predicted that disturbance of the three-dimensional structure by the A213P mutation will indirectly disrupt direct DNA contact with the third helix and therefore DNA binding. The substitution of an alanine in the third helix of the homeodomain (A249P) is likely to hinder the base-specific contact with DNA in the major groove and as such the specificity of DNA binding.

From the function and expression studies of lmx-1 in chicken and Lmx1b in mice, respectively, it can be deduced that the skeletal defects seen in NPS reflect a disruption of normal dorsoventral patterning of the limb during development (14,15). Although it is known from these animal studies that Lmx1b is expressed in fetal and adult kidney tissue, the exact role of this gene in the development of the kidney is much less clear. On the basis of the renal manifestations in NPS, it is likely that Lmx1b has an important role in patterning and cell differentiations in the kidney also. Recent studies by Morello et al. (22) showed that Lmx1b localizes to the glomeruli of E15.5 and newborn wild-type mice. In addition, these investigators demonstrated absence of 3 (IV) and 4 (IV) collagen expression in Lmx1b —/— mutant kidneys. It is widely known that during development, there is a switch from an 1/2 to an 3/4 network of collagen IV expression in the GBM (23). Therefore, on the basis of the findings in Lmx1b mutant kidneys, it was postulated that Lmx1b might play an integral role in this coordinated transcriptional switch (22). For humans with a heterozygous mutation in LMX1B, however, the situation may be much less clear. Thus, previous immunologic studies of the GBM using MCAP1 antibody, which recognizes the NC1 domain of 3 (IV), gave conflicting results: normal labeling in three patients and no GBM labeling in two others (24,25).

Consistent with the findings of other investigators (11,12,18), we have not found convincing evidence of a correlation between the presence and severity of certain features of NPS and the type or location of the LMX1B mutation. The absence of a genotype—phenotype correlation is not surprising given that there is not only interfamilial but also intrafamilial variability in the expression and severity of symptoms. Family 8 is a very good example of intrafamilial phenotypic variability. It is intriguing that a large percentage of the patients within this family have nephropathy. Nevertheless, renal abnormalities are not present in all affected individuals of this family; in addition, the severity of the nephropathy varies among the patients, ranging from very mild proteinuria in nine patients to end-stage renal disease in four of them. Patients from a family reported by McIntosh et al. (12), who carry the same splice-site mutation (672 +1GA) as patients in family 8, do not have nephropathy. Therefore, the high incidence and the significant difference in severity of nephropathy within family 8 cannot be attributed to the LMX1B genotype. Recently, Farley et al. (26), on the basis of genetic studies in a very small family with NPS and quantified variable expression of orthopaedic symptoms, found support for the old hypothesis of Renwick (27), who proposed that the severity of NPS symptoms is modulated by the allele contributed by the unaffected parent. We believe that more genetic studies in clinically well characterized large families with NPS are necessary to evaluate the possible influence of modifying alleles on phenotypic expression and variability.

GBM abnormalities similar to those seen in NPS have been observed in several families without bone or nail abnormalities (28). It will be of interest to determine, by LMX1B mutation analysis, whether these cases represent partial expression of NPS or an independent genetic form of GBM disease. In case this NPS-like nephropathy turns out not to be due to LMX1B mutations, the possibility that genes encoding proteins that interact with LMX1B are involved should be investigated. We are performing yeast-two-hybrid analysis with the ultimate aim of identifying these genes.

	Acknowledgments

 
This study is supported by the Dutch Kidney foundation (Grant No. C97-1698).

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Little EM: Congenital absence or delayed development of the patella. Lancet 2:781 -784, 1897
2. Lichter PR, Richards JE, Downs CA, Stringham HM, Boehnke M, Farley FA: Cosegregation of open-angle glaucoma and the nail-patella syndrome. Am J Ophthalmol 124:506 -515, 1997[Medline]
3. Meyrier A, Rizzo R, Gubler MC: The nail-patella syndrome. A review. J Nephrol 2:133 -140, 1990
4. Renwick JH, Lawler SD: Genetic linkage between the ABO and nail-patella loci. Ann Hum Genet19 : 312-331,1955[Medline]
5. Schleutermann DA, Bias BW, Murdoch JL, McKusick VA: Linkage of the loci for the nail-patella syndrome and adenylate kinase. Am J Hum Genet 21:606 -630, 1969[Medline]
6. Campeau E, Watkins D, Rouleau GA, Babul R, Buchanan JA, Mechino W, Der Kaloustian VM: Linkage analysis of the nail-patella syndrome. Am J Hum Genet 56:243 -247, 1995[Medline]
7. McIntosh I, Clough MV, Schaffer AA, Puffenberger EG, Horton VK, Peters K, Abbott MH, Roig CM, Cutine S, Ozelius L, Kwiatkowski DJ, Pyeritz RE, Brown LJ, Pauli RM, McCormick MK, Francomano CA: Fine mapping of the nail-patella syndrome locus at 9q34. Am J Hum Genet60 : 133-142,1997[Medline]
8. Hol FA, Geurds MPA, Mariman ECM, Knoers N: Refinement of the locus for the nail-patella syndrome at 9q34. Am J Hum Genet61S : 1627,1997
9. Silahtaroglu A, Hol FA, Jensen PKA, Erdel M, Duba H-C, Geurds MPA, Knoers NVAM, Mariman ECM, Tumer Z, Utermann G, Wirth J, Bugge M, Tommerup N: Molecular cytogenetic detection of 9q34-breakpoints associated with nail-patella syndrome. Eur J Hum Genet7 : 68-76,1999[Medline]
10. Dreyer SD, Zhou G, Baldini A, Winterpacht A, Zabel B, Cole W, Johnson RL, Lee B: Mutations in LMX1B cause abnormal skeletal patterning and renal dysplasia in nail-patella syndrome. Nat Genet 19: 47-50,1998[Medline]
11. Vollrath D, Jaramillo-Babb VL, Clough MV, McIntosh I, Scott KM, Lichter PR, Richards JE: Loss-of-function mutations in the LIM-homeodomain gene LMX1B, in nail-patella syndrome. Hum Mol Genet 7:1091 -1098, 1998[Abstract/Free Full Text]
12. McIntosh I, Dreyer SD, Clough MV, Dunston JA, Eyaid W, Roig CM, Montgomery T, Ala-Mello S, Kaitila I, Winterpacht A, Zabel B, Frydman M, Cole WG, Francomano CA, Lee B: Mutation analysis of LMX1B gene in nail-patella syndrome patients. Am J Hum Genet63 : 1651-1658,1998[Medline]
13. Sanchez-Garcia I, Rabbitts TH: The LIM domain: A structural motif found in zinc-finger-like proteins. Trends Genet10 : 315-320,1994[Medline]
14. Vogel A, Rodriguez G, Warnken W, Belmonte JCI: Dorsal cell fate specified by chick lmx1 during vertebrate limb development. Nature 378:715 -720, 1995
15. Chen H, Ovchinnikov D, Kokubo H, Oberg KC, Pepicelli CV, Gan L, Lee B, Johnson RL: Limb and kidney defects in Lmx1b mutant mice suggest the involvement of Lmx1b in human nail patella syndrome. Nat Genet 19:51 -55, 1998[Medline]
16. Looij BJ, te Slaa RL, Hogewind BL, van de Kamp JJP: Genetic counseling in hereditary osteo-onychodysplasia (HOOD, nail-patella syndrome) with nephropathy. J Med Genet25 : 682-686,1988[Abstract/Free Full Text]
17. Miller SA, Dykes DD, Polesky HF: A simple staining out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16: 1215,1988[Free Full Text]
18. Clough MV, Hamlington JD, McIntosh I: Restricted distribution of loss-of-function mutations within the LMX1B genes of nail-patella syndrome patients. Hum Mutat14 : 459-465,1999[Medline]
19. Seri M, Melchionda S, Dreyer S, Marini M, Carella M, Cusano R, Piemontese MR, Caroli F, Silengo M, Zelante L, Romeo G, Ravazzolo R, Gasparini P, Lee B: Identification of LMX1B gene mutations in Italian patients affected with nail-patella syndrome. Int J Mol Med4 : 285-2901999[Medline]
20. Gehring WJ, Qian YQ, Billeter M, Furukubo-Tokunaga K, Schief AF, Resendez-perez D, Affolter M, Otting G, Wüthrich K: Homeodomain-DNA recognition. Cell78 : 211-223,1994[Medline]
21. Qian YQ, Billeter M, Otting G, Müller M, Gehring WJ, Wüthrich K: The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: Comparison with prokaryotic repressors. Cell59 : 573-580,1989[Medline]
22. Morello R, Dreyer SD, Oberg K, Lee B: Localization of Lmx1b during kidney and limb development and pathogenesis of nail-patella syndrome. Am J Hum Genet65S : A77,1999
23. Miner JH, Sanes JR: Collagen IV 3, 4, and 5 chains in rodent basal laminae: Sequence, distribution, association with laminins, and developmental switches. J Cell Biol127 : 879-891,1994[Abstract/Free Full Text]
24. Noel LH, Gubler MC, Bobrie G, Savage CO, Lockwood CM, Grünfeld JP: Inherited defects of renal basement membranes. In: Advances in Nephrology, edited by Grünfeld JP, Maxwell MH, Bach JF, St. Louis, Year Book Medical Publishers, 1989, pp77 -94
25. Sutcliffe NP, Cashman SJ, Savage CO, Fox JG, Boulton-Jones JM: Variability of the antigenicity of glomerular basement membrane in nail-patella syndrome. Nephrol Dial Transplant4 : 262-2651989[Abstract/Free Full Text]
26. Farley FA, Lichter PR, Downs CA, McIntosh I, Vollrath D, Richards J: An orthopaedic scoring system for nail-patella syndrome and application to a kindred with variable expressivity and glaucoma. J Pediatr Orthopaedics 19:624 -631, 1999
27. Renwick JH: Nail-patella syndrome: Evidence for modification by alleles at the main locus. Ann Hum Genet21 : 159-169,1956[Medline]
28. Dombros N, Katz A: Nail-patella-like lesions in the absence of skeletal abnormalities. Am J Kidney Dis1 : 237-240,1982[Medline]

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