Nail-Patella syndrome (NPS) or hereditary onycho-osteodysplasia (OMIM entry no. 161200) is a rare autosomal dominant disease that affects multiple organs including the limbs, skeleton, kidneys and eyes (1). The classical clinical manifestations consist of nail dysplasia, hypoplasia or absence of the patella, elbow dysplasia and iliac horns (1). Renal involvement occurs in 30-50% of patients with NPS and is the primary determinant of prognosis (2,3). Renal manifestations typically include proteinuria with or without hematuria, and nephrotic syndrome (5-10% of cases), which progresses to end-stage renal disease (ESRD) over varying periods of time (3). Additional relevant characteristics observed in patients with NPS include open angle glaucoma and ocular hypertension, which usually develop during adulthood and occur in ~10 and 7% of patients, respectively (2,4). The gene associated with NPS, LMX1B, maps on to chromosome 9q33.3 and contains 8 exons that encode a LIM-homeodomain transcription factor called LMXB1 (5-7). This protein belongs to the LIM-homeodomain protein family, and serves an essential role in the normal development of dorsal-ventral limb structures, morphogenesis, and in the functions of podocytes and the glomerular basal membrane, anterior segment of the eye, and some types of neurons (1,5).

LMX1B contains two cysteine-rich zinc-binding motifs (LIM-A and LIM-B domains) at the N-terminus, which are all involved in protein-protein interactions; a homeodomain in the middle responsible for the interaction with specific DNA sequences in target genes, and a C-terminal glutamine-rich domain of unknown function (1,7). Most loss-of-function mutations of LMX1B are clustered in the LIM domains and the homeodomain, encoded by exons 2-3 and 4-6, respectively (8-11). Several studies have reported entire deletions of the LMX1B gene in patients with NPS, supporting haploinsufficiency as the major pathogenic mechanism underlying the disease (8,9,12,13). However, a clear genotype-phenotype correlation among NPS patients is still lacking (8). Even patients within the same family present a varying range of symptoms and symptom severity, emphasizing the potential contribution of modifier genes in the manifestation of the phenotype (11).

In the present case report, an unusual case of a 2-year old girl from Syria who presented with nephrotic syndrome that evolved rapidly to ESRD is described. She exhibited classical NPS symptoms including dystrophic nails and absence of patellae. Using DNA sequencing analysis, a novel heterozygous missense mutation in LMX1B (c.709T>C, p.S237P), which affected the homeodomain of the LMX1B protein was identified, which was predicted to be pathogenic.

Based on the orthopedic manifestations and the renal course of the patient, mutational analysis of the LMX1B gene was performed. The results of the DNA sequencing analysis showed a novel heterozygous variant in exon 4 of LMX1B, c.709T>C (g.78849T>C, genomic location Chr9; 129455570>C GRCh37; Fig. 2A). This nucleotide substitution was predicted change a serine residue to proline at position 237 (p.S237P) in the homeodomain of the LMX1B protein (Ensembl transcript ID, ENST00000355497). Since blood samples from the patient's parents were not available, it was not possible to determine if the mutation was inherited from one of parents or if it originated de novo. Variant c.709T>C, p.S237P was absent from several databases, including gnomAD, 1000 Genomes Project, HGMD and ClinVar. Homology analysis showed that serine 237 of LMX1B is conserved amongst different species (Fig. 2B). Analysis of p.S237P with five different bioinformatics tools predicted that this variant was pathogenic (Table I). VarSome classified this substitution as likely pathogenic as it met 2 moderate (PM2 and PM3) and 3 supporting (PP2, PP3 and PP5) pathogenicity criteria established by the American College of Medical Genetics and Genomics (17). The novel LMX1B mutation described here was submitted to ClinVar (accession no. VCV000694525; variant, NM_002316.4; ncbi.nlm.nih.gov/clinvar/variation/694525/). As shown in Fig. 2C, the missense mutation p.S237P is located in α-helix 1 of the LMX1B homeodomain. To evaluate the structural impact of this mutation, 3D models of the wild-type and mutant homeodomains were constructed using SWISS-MODEL. The results suggested that the substitution of serine for proline at position 237 transformed the segment A236-S237-F238 of the α-helix 1 in the homeodomain into a strand (Fig. 2D).

NPS is an autosomal dominant disorder characterized by dysplastic nails, a hypoplastic or absent patella, and elbow dysplasia (1,2). In some cases, patients may also exhibit differing levels of proteinuria that can progress to ESRD, and less frequently open-angle glaucoma. The skeletal disorders can be detected at birth, but in some cases they are less significant, making the diagnosis problematic (1,3). In the present case report, NPS was challenging to diagnose immediately as nephrotic syndrome developed before obvious orthopedic symptoms, and there was no family history of NPS. This case was quite uncommon as the patient presented persistent nephrotic syndrome very early, at the age of 2 years, and developed rapidly to ESRD. The majority of patients with NPS exhibit a slow gradual loss of renal function, and only a small number of cases, where a rapid progression to ESRD at a young age, have been reported (15,18). The factors responsible for progression to ESRD in NPS are unknown, but it is possible that potential modifying genes may contribute to it. The nephropathy in the patient reported on in the present case report was treated with corticosteroids and diuretics, and the corticoresistance supported the association of the nephrotic syndrome with NPS. Moreover, renal biopsy of the patient revealed a characteristic irregular thickening of the glomerular basement membrane with progression to fibrosis and glomerulosclerosis observed in other patients with NPS (3). Additionally, the nail and skeletal phenotype of the patient was consistent with previously published data of NPS (2).

NPS is caused by loss-of-function mutations in the LMX1B gene, which is expressed in several different tissues implicated in the clinical phenotype (5-7). In the kidney, LMX1B is expressed in podocytes and regulates the expression of several genes required for correct podocyte differentiation and function (1). Studies using inducible podocyte specific Lmx1b knockout mice have established the importance of LMX1B in fully differentiated podocytes, and suggest that this transcription factor is essential for the maintenance of a properly structured actin cytoskeleton (19). Additionally, LMX1B has been shown to bind enhancer sequences (FLAT elements) in the promoter regions of ABRA and ARL4C, two genes encoding actin cytoskeleton-associated proteins (19). These studies concluded that podocyte pathogenesis in NPS likely results from a dysregulation of the actin cytoskeleton.

During embryogenesis, LMX1B expression is high in the dorsal mesenchyme of developing limbs and is involved in dorso-ventral patterning (20). Following the formation of the dorsal-ventral axis, LMX1B activates the expression of several dorsalizing genes, represses the expression of ventralizing genes, and regulates the development of dorsal distal limb structures, such as the nails and patellas (20). Therefore, the skeletal phenotype of patients with NPS is the result of a defect in dorsoventral patterning caused by LMX1B mutations.

LMX1B is also expressed during ocular development in the anterior portions of the murine eye (21); ~10% of NPS patients develop open-angle glaucoma and an additional 7% exhibit ocular hypertension (2,8). These ocular symptoms usually develop during adulthood. The patient reported in the present case report is now 14 years old and she has not yet developed glaucoma. She undergoes ophthalmological reviews every 6 months. The pathogenesis of glaucoma in NPS is still unknown, but homozygous lmx1b knockout mice display iris and ciliary body hypoplasia and corneal collagen fibril defects, suggesting a role for LMX1B in collagen regulation during development (21).

Several of the mutations identified in patients with NPS are localized in the homeodomain of LMX1B (8,9,11). This highly conserved DNA-binding motif forms part of several transcription factors present in numerous organisms, from yeast to humans (22). These transcription factors serve essential roles during development and adult homeostasis, and mutations in their genes are known to cause human diseases. These genes include HOXA13 and HOXD13, which are associated with skeletal defects in distal extremities; LHX3 and POU1F1, which are associated with pituitary hormone deficiency; and PAX6 which is associated with eye-related disorders (22). The homeodomain is comprised of ~60 amino acids and includes three α-helices, of which helices 2 and 3 adopt a helix-turn-helix motif, and a flexible N-terminal arm. Helix 3 and the N-terminal arm interact directly with the major groove and the adjacent minor groove of target DNAs, respectively, whereas helices 1 and 2 maintain the tertiary structure important in the helix-turn-helix configuration. Mutational analysis of LMX1B in the present case report identified a novel heterozygous variant, c.709T>C, p.S237P, that explained the phenotype observed. This variant, which was not found in any of the reference databases, resulted in a change in a conserved serine residue to a helix-disrupting proline residue, and was predicted to be pathogenic by several different bioinformatics tools. The evolutionary conservation of serine 237 indicates that this residue is essential for the structure and function of the LMX1B homeodomain. Although mutational analysis of the patient's parents was not possible, it is hypothesized that p.S237P is a de novo mutation as there was no history of nephropathy, skeletal defects or nail dystrophy in either parent, sister, grandparents or uncles. The identified mutation is located next to the amino acid residue affected by recurrent mutation c.706G>C, p.A236P in α-helix 1 of the homeodomain and appears as p.A213P in (6). p.A236P has been shown to abolish both DNA binding of the LMX1B homeodomain to the FLAT element of the insulin promoter and transactivation of a reporter gene (6,12). Knoers et al (23) indicated that p.A236P disrupted the 3D structure of the homeodomain and, therefore, disrupted DNA binding. Similarly, p.S237P also introduces a proline residue that may destabilize the helix conformation and affect the overall tertiary structure of the homeodomain. Analysis of the predicted structural model of the mutant homeodomain in the present study indicated that p.S237P indeed disturbed the carboxy-terminal segment of α-helix 1. Thus, together, these results suggests that p.S237P destabilizes α-helix 1, abolishing DNA binding and the subsequent transcriptional activity of LMX1B. This hypothesis requires confirmation using functional studies.

In conclusion, a novel missense mutation (c.709T>C, p.S237P) in the LMX1B gene of a child presenting with nephrotic syndrome and rapid progression to ESRD was identified. Based on in silico structural analysis of this mutation, the α-helix 1 was altered, which abrogated binding of the LMX1B homeodomain to the target DNA. This report further expands on the mutational spectrum of LMX1B, and provides insight into the molecular mechanisms of NPS pathology.