Nail-patella syndrome (NPS) is a multi-system disorder characterized by hypoplastic nails, hypoplastic patella, skeletal deformities, and iliac horns, which is caused by heterozygous variants of LMX1B. Nephropathy ranging from mild urinary abnormality to end-stage renal disease occurs in some individuals with NPS. Because of the low prevalence of NPS and the lack of longitudinal studies of its kidney involvement, the clinical, pathological, and genetic features characterizing severe nephropathy remain unclear. We conducted a Japanese survey of NPS with nephropathy, and analyzed their clinical course, pathological features, and factors associated with severe renal phenotype. LMX1B gene analysis and luciferase reporter assay were also performed. Among 13 NPS nephropathy cases with genetic validation, 5 patients who had moderate-to-massive proteinuria progressed to advanced chronic kidney disease or end-stage renal disease. Pathological findings in the early phase did not necessarily correlate with renal prognosis. Variants associated with deteriorated renal function including a novel variants were confined to the N-terminal region of the LIM domain and a short sequence in the LMX1B homeodomain, which were distinct from reported variants found in isolated nephropathy without extrarenal manifestation (LMX1B-associated nephropathy). Luciferase reporter analysis demonstrated that variants in patients with severe renal phenotype caused haploinsufficiency, but no dominant-negative effects on promoter activation. A distinct proportion of NPS nephropathy patients progressed to end-stage renal disease in adolescence or young adulthood. Patients with moderate or severe proteinuria, especially those with variants in specific regions of LMX1B, should be monitored for potential deterioration of renal function.

LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Accordingly in mice, Lmx1b has been shown to play crucial roles in the development of the limb, kidney and eye. Although one functional allele of Lmx1b appears adequate for development, Lmx1b null mice display ventral-ventral distal limbs with abnormal kidney, eye and cerebellar development, more disruptive, but fully concordant with NPS. In Lmx1b functional knockouts (KOs), Lmx1b transcription in the limb is decreased nearly 6-fold, indicating autoregulation. Herein, we report on two conserved Lmx1b-associated cis-regulatory modules (LARM1 and LARM2) that are bound by Lmx1b, amplify Lmx1b expression with unique spatial modularity in the limb, and are necessary for Lmx1b-mediated limb dorsalization. These enhancers, being conserved across vertebrates (including coelacanth, but not other fish species), and required for normal locomotion, provide a unique opportunity to study the role of dorsalization in the fin to limb transition. We also report on two NPS patient families with normal LMX1B coding sequence, but with loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.

Nail-patella syndrome (NPS) is an autosomal dominant developmental disorder most commonly characterized by dyplasia of nail or patella, the radial head or the humeral head hypoplasia, and, frequently ocular abnormalities and renal disease. It is caused by heterozygous loss-of-function mutations in the LMX1B gene, which encodes LIM homeodomain transcription factor and is essential for regulating the dorsal limb fate.

In a Danish family, multiple individuals in five generations present with early-onset paroxysmal cranial dyskinesia, musculoskeletal abnormalities, and kidney dysfunction.

Nail patella syndrome (NPS) is an autosomal dominant disorder characterized by nail malformations, patellar apoplasia, or patellar hypoplasia. Mutations within the LMX1B gene are found in 85% of families with NPS; thus, this gene has been characterized as the causative gene of NPS. In this study, we identified a heterozygous microdeletion of the entire LMX1B gene using multiplex ligation-dependent probe amplification (MLPA) in a Chinese family with NPS. The determination of the deletion breakpoints by Illumina genome-wide DNA analysis beadchip showed that the deletion was located in chromosome 9q33.3 and spanned about 0.66 Mb in size. This heterozygous deletion provides strong evidence for haploinsufficiency as the pathogenic mechanism of NPS.

Nail-Patella syndrome (NPS) is an inherited disease characterized by nail and skeletal anomalies, nephropathy and glaucoma. The diagnosis of NPS is based on clinical findings, including hypoplastic or absent patella, dystrophic nails, dysplasia of the elbows and iliac horns. However, the main determinant of NPS prognosis is nephropathy, which may range from asymptomatic proteinuria to end-stage renal disease. NPS is caused by heterozygous loss-of-function mutations in the LMX1B gene, which encodes the LIM homeodomain transcription factor LMX1B. LMX1B serves an essential role in the physiological development of dorsal-ventral limb structures, morphogenesis and function of podocytes, as well as in development of the anterior segments of the eyes, and in certain types of neurons. The present study aimed to identify the disease-causing mutation in a 2-year old girl with nephrotic syndrome that evolved rapidly to end-stage renal disease. The patient showed classical symptoms of NPS including dystrophic nails and an absence of the patellae. DNA sequence analysis identified a novel missense variant in exon 4 of LMX1B (c.709T>C, p.S237P); this substitution affected a conserved serine residue in the homeodomain of LMX1B and was predicted to be pathogenic. In silico modeling of the homeodomain revealed that the p.S237P mutation converted the A236-S237-F238 segment of α-helix 1 into a strand. It was hypothesized that this mutation affected binding of the transcription factor to its target DNA, thus abrogating transcription activation, which would explain the phenotype that manifested in the patient.

Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.

Long noncoding RNAs (lncRNAs) represent a group of regulatory RNAs that play critical roles in numerous cellular events, but their functional importance in development remains largely unexplored. Here, we discovered a series of previously unidentified gene clusters harboring conserved lncRNAs at the nonimprinting regions in brain (CNIBs). Among the seven identified CNIBs, human CNIB1 locus is located at Chr 9q33.3 and conserved from Danio rerio to Homo sapiens. Chr 9q33.3-9q34.11 microdeletion has previously been linked to human nail-patella syndrome (NPS) which is frequently accompanied by developmental and visual deficiencies. By generating CNIB1 deletion alleles in zebrafish, we demonstrated the requirement of CNIB1 for proper growth and development, and visual activities. Furthermore, we found that the role of CNIB1 on visual activity is mediated through a regulator of ocular development-lmx1bb. Collectively, our study shows that CNIB1 lncRNAs are important for zebrafish development and provides an lncRNA cluster-mediated pathophysiological mechanism for human Chr 9q33.3-9q34.11 microdeletion syndrome.

End-stage renal disease (ESRD) presenting in a familial autosomal dominant pattern points to an underlying monogenic cause. Nail-patella syndrome (NPS) is an autosomal dominant disorder that may lead to ESRD caused by mutations in the transcription factor LMX1B. Renal-limited forms of this disease, termed nail-patella-like renal disease (NPLRD), and LMX1B nephropathy have recently been described. We report a large family, from the North East of England, with seven affected members with varying phenotypes of renal disease, ranging from ESRD at 28 years of age to microscopic haematuria and proteinuria and relatively preserved renal function. In this family, there were no extra-renal manifestations to suggest NPS. Genome-wide linkage studies and inheritance by descent (IBD) suggested disease loci on Chromosome 1 and 9. Whole exome sequencing (WES) analysis identified a novel sequence variant (p.R249Q) in the LMX1B gene in each of the three samples submitted, which was confirmed using Sanger sequencing. The variant segregated with the disease in all affected individuals. In silico modelling revealed that R249 is putatively located in close proximity to the DNA phosphoskeleton, supporting a role for this residue in the interaction between the LMX1B homeodomain and its target DNA. WES and analysis of potential target genes, including CD2AP, NPHS2, COL4A3, COL4A4 and COL4A5, did not reveal any co-inherited pathogenic variants. In conclusion, we confirm a novel LMX1B mutation in a large family with an autosomal dominant pattern of nephropathy. This report confirms that LMX1B mutations may cause a glomerulopathy without extra-renal manifestations. A molecular genetic diagnosis of LMX1B nephropathy thus provides a definitive diagnosis, prevents the need for renal biopsies and allows at risk family members to be screened.

Congenital talipes equinovarus (CTEV) is a rotational foot deformity that affects muscles, bones, connective tissue, and vascular or neurological tissues. The etiology of CTEV is complex and unclear, involving genetic and environmental factors. Nail-patella syndrome is an autosomal dominant disorder caused by variants of the LIM homeobox transcription factor 1 beta gene (LMX1B, OMIM:602575). LMX1B plays a key role in the development of dorsal limb structures, the kidneys, and the eyes, and variants in this gene may manifest as hypoplastic or absent patella, dystrophic nails, and elbow and iliac horn dysplasia; glomerulopathy; and adult-onset glaucoma, respectively. This study aimed to identify pathogenic variants in a fetus with isolated talipes equinovarus diagnosed by ultrasound in the second trimester, whose father exhibited dysplastic nails and congenital absence of bilateral patella.

LMX1B is a LIM-homeodomain transcription factor required for the normal development of dorsal limb structures, the glomerular basement membrane, the anterior segment of the eye, and dopaminergic and serotonergic neurons. Heterozygous loss-of-function mutations in LMX1B cause nail patella syndrome (NPS). To further understand LMX1B gene regulation and to identify pathogenic mutations within the coding region, a detailed analysis of LMX1B gene structure was undertaken. 5' -RACE and primer extension identified a long 5' -untranslated region of 1.3 kb that contains two upstream open-reading frames (uORFs). Transient transfection assays showed that sequences required for basal promoter activity extend no further than 112 bp upstream. An additional 47 mutations have been identified in the coding region, as well as nine deletions of large portions of the gene, but not in the promoter or highly conserved intronic sequences. The range of mutations and the identification of uORFs suggest further complexity in the regulation of LMX1B expression.

Mutations in the LIM homeobox transcription factor 1-beta (LMX1B) are a cause of nail patellar syndrome, a condition characterized by skeletal changes, glaucoma and focal segmental glomerulosclerosis. Recently, a missense mutation (R246Q) in LMX1B was reported as a cause of glomerular pathologies without extra-renal manifestations, otherwise known as nail patella-like renal disease (NPLRD). We have identified two additional NPLRD families with the R246Q mutation, though the mechanisms by which LMX1BR246Q causes a renal-specific phenotype is unknown. In this study, using human podocyte cell lines overexpressing either myc-LMX1BWT or myc-LMX1BR246Q, we observed dominant negative and haploinsufficiency effects of the mutation on the expression of podocyte genes such as NPHS1, GLEPP1, and WT1. Specifically, we observed a novel LMX1BR246Q-mediated downregulation of WT1(-KTS) isoforms in podocytes. In conclusion, we have shown that the renal-specific phenotype associated with the LMX1BR246Q mutation may be due to a dominant negative effect on WT1(-KTS) isoforms that may cause a disruption of the WT1 (-KTS):(+KTS) isoform ratio and a decrease in the expression of podocyte genes. Full delineation of the LMX1B gene regulon is needed to define its role in maintenance of glomerular filtration barrier integrity.

Understanding the genetic causes of kidney disease is essential for accurate diagnosis and could lead to improved therapeutic strategies and prognosis. To accurately and promptly identify the genetic background of kidney diseases, we applied a targeted next-generation sequencing gene panel including 203 genes associated with kidney disease, as well as diseases originating in other organs with mimicking symptoms of kidney disease, to analyze 51 patients with nonspecific nephrogenic symptoms, followed by validation of its efficacy as a diagnostic tool. We simultaneously screened for copy number variants (CNVs) in each patient to obtain a higher diagnostic yield (molecular diagnostic rate: 39.2%). Notably, one patient suspected of having Bartter syndrome presented with chloride-secreting diarrhea attributable to homozygous SLC26A3 variants. Additionally, in eight patients, NGS confirmed the genetic causes of undefined kidney diseases (8/20, 40%), and initial clinical impression and molecular diagnosis were matched in 11 patients (11/20, 55%). Moreover, we found seven novel pathogenic/likely pathogenic variants in PKD1, PKHD1, COL4A3, and SLC12A1 genes, with a possible pathogenic variant in COL4A3 (c.1229G>A) identified in two unrelated patients. These results suggest that targeted NGS-panel testing performed with CNV analysis might be advantageous for noninvasive and comprehensive diagnosis of suspected genetic kidney diseases.

Mutanlallemand (mtl) and Belly Spot and Deafness (bsd) are two new spontaneous alleles of the Lmx1a gene in mice. Homozygous mutants show head tossing and circling behaviour, indicative of vestibular defects, and they have short tails and white belly patches of variable size. The analysis of auditory brainstem responses (ABR) showed that mtl and bsd homozygotes are deaf, whereas heterozygous and wildtype littermates have normal hearing. Paint-filled inner ears at E16.5 revealed that mtl and bsd homozygotes lack endolymphatic ducts and semicircular canals and have short cochlear ducts. These new alleles show similarities with dreher (Lmx1a) mutants. Complementation tests between mtl and dreher and between mtl and bsd suggest that mtl and bsd are new mutant alleles of the Lmx1a gene. To determine the Lmx1a mutation in mtl and bsd mutant mice we performed PCR followed by sequencing of genomic DNA and cDNA. The mtl mutation is a single point mutation in the 3' splice site of exon 4 leading to an exon extension and the activation of a cryptic splice site 44 base pairs downstream, whereas the bsd mutation is a genomic deletion that includes exon 3. Both mutations lead to a truncated LMX1A protein affecting the homeodomain (mtl) or LIM2-domain (bsd), which is critical for LMX1A protein function. Moreover, the levels of Lmx1a transcript in mtl and bsd mutants are significantly down-regulated. Hmx2/3 and Pax2 expression are also down-regulated in mtl and bsd mutants, suggesting a role of Lmx1a upstream of these transcription factors in early inner ear morphogenesis. We have found that these mutants develop sensory patches although they are misshapen. The characterization of these two new Lmx1a alleles highlights the critical role of this gene in the development of the cochlea and vestibular system.