LMX1B Research Articles

Nail-Patella Syndrome with an Emphasis on the Risk of Renal and Ocular Findings

We describe a 6-year-old girl presenting with nail dysplasia affecting all nails and hands for 2 years. Changes were seen on the ulnar side of the nails. She was assessed for limitation of elbow movements at 3 weeks of age and underwent physiotherapy for thickened biceps tendon. She subsequently developed laxity of knees and ankles, and x-ray revealed absent patellae at 32 weeks. She had behavioral abnormalities and sleep disturbances. X-ray of the pelvis revealed iliac horns, and urinalysis showed 3+ proteinuria. She had mixed hyperlipidemia. Her chromosomal analysis was normal but showed a mutation in the LMX1B gene. She was diagnosed to have Nail-patella syndrome or Hereditary osteo-onychodysplasia (HOOD Syndrome). Her renal imaging was normal, as were her ocular pressures. She is under regular surveillance by a multi-disciplinary team of genetic counselors, orthopedists, rheumatologists and ophthalmologists. She is currently prescribed enalapril, melatonin and simvastatin.

Dopaminergic cell differentiation from human embryonic nerve cells in vitro

Cell population enriched by determined cells, the precursors of dopaminergic neurons can be used for neurotransplantation. In order to obtain such cell population, we studied the potentialities of dopaminergic differentiation of human embryonic nervous tissue in a cell culture. A number of dopaminergic neurons during cultivation significantly increased from 5.6 ± 4.3% of cells within eyeshot to 23.5 ± 2.6% of cells; at the same time, a number of cells in control specimens reached 8.1 ± 6.5% of cells. The analysis of gene expression associated with this kind of neuronal differentiation confirmed its dopaminergic character. We showed that in the course of cultivation the expression of early transcriptional factors Nurr 1 and Lmx 1b disappears; at the same time, the level of TH expression increases. Thus, the use of special conditions of cultivation allows for obtaining a cell population enriched by the precursors of dopaminergic neurons.

Cardiogenetics, Neurogenetics, and Pathogenetics of Left Ventricular Hypertrabeculation/Noncompaction

Left ventricular hypertrabeculation (LVHT), also known as noncompaction or spongy myocardium, is a cardiac abnormality of unknown etiology and pathogenesis frequently associated with genetic cardiac and noncardiac disorders, particularly genetic neuromuscular disease. This study aimed to review the current knowledge about the genetic or pathogenetic background of LVHT. A literature review of all human studies dealing with the association of LVHT with genetic cardiac and noncardiac disorders, particularly neuromuscular disorders, was conducted. Most frequently, LVHT is associated with mitochondrial disorders (mtDNA, nDNA mutations), Barth syndrome (G4.5, TAZ mutations), hypertrophic cardiomyopathy (MYH7, ACTC mutations), zaspopathy (ZASP/LDB3 mutations), myotonic dystrophy 1 (DMPK mutations), and dystrobrevinopathy (DTNA mutations). More rarely, LVHT is associated with mutations in the DMD, SCNA5, MYBPC3, FNLA1, PTPN11, LMNA, ZNF9, AMPD1, PMP22, TNNT2, fibrillin2, SHP2, MMACHC, LMX1B, HCCS, or NR0B1 genes. Additionally, LVHT occurs with a number of chromosomal disorders, polymorphisms, and not yet identified genes, as well in a familial context. The broad heterogeneity of LVHT's genetic background suggests that the uniform morphology of LVHT not only is attributable to embryonic noncompaction but also may result from induction of hypertrabeculation as a compensatory reaction of an impaired myocardium. Most frequently, LVHT is associated with mutations in genes causing muscle or cardiac disease, or with chromosomal disorders. These associations require comprehensive cardiac, neurologic, and cytogenetic investigations.

Clinico-Genetic Study of Nail-Patella Syndrome

Nail-patella syndrome (NPS) is an autosomal dominant disease that typically involves the nails, knees, elbows and the presence of iliac horns. In addition, some patients develop glomerulopathy or adult-onset glaucoma. NPS is caused by loss-of-function mutations in the LMX1B gene. In this study, phenotype-genotype correlation was analyzed in 9 unrelated Korean children with NPS and their affected family members. The probands included 5 boy and 4 girls who were confirmed to have NPS, as well as 6 of their affected parents. All of the patients (100%) had dysplastic nails, while 13 patients (86.7%) had patellar anomalies, 8 (53.3%) had iliac horns, 6 (40.0%) had elbow contracture, and 4 (26.7%) had nephropathy including one patient who developed end-stage renal disease at age 4.2. The genetic study revealed 8 different LMX1B mutations (5 missense mutations, 1 frame-shifting deletion and 2 abnormal splicing mutations), 6 of which were novel. Genotype-phenotype correlation was not identified, but inter- and intrafamilial phenotypic variability was observed. Overall, these findings are similar to the results of previously conducted studies, and the mechanism underlying the phenotypic variations and predisposing factors of the development and progression of nephropathy in NPS patients are still unknown.

A Synonymous Genetic Alteration of LMX1B in a Family with Nail-Patella Syndrome

The gene responsible for nail-patella syndrome, LMX1B, has recently been identified on chromosome 9q. Here we present a patient with nail-patella syndrome and an autosomal dominant pattern of inheritance. A 17-year-old girl visited our clinic for the evaluation and treatment of proteinuria. She had dystrophic nails, palpable iliac horns, and hypoplastic patellae. Electron microscopy of a renal biopsy showed irregular thickening of the glomerular basement membrane. A family history over three generations revealed five affected family members. Genetic analysis found a change of TCG to TCC, resulting in a synonymous alteration at codon 219 in exon 4 of the LMX1B gene in two affected family members. The same alteration was not detected in an unaffected family member. This is the first report of familial nail-patella syndrome associated with an LMX1B in Korea mutation, However, we can not completely rule out the possibility that the G-to-C change may be a single nucleotide polymorphism as this genetic mutation cause no alteration in amino acid sequence of LMX1B.

Nail-patella syndrome, infantile nephrotic syndrome: complete remission with antiproteinuric treatment

A girl, second child of healthy parents, was referred to the Renal Unit at the age of 9 months with haematuria (230 RBC/microl) and proteinuria (2.4 g/l). Serum creatinine was normal (0.25 mg/dl), albumin low (34 g/l) and cholesterol elevated (223 mg/dl). Physical examination showed bilateral webbing of the elbows, equinovarus of both feet and absent patellae. The clinical diagnosis of nail-patella syndrome was confirmed by demonstrating a splice mutation in the intron 5 (750 + 1 G>A) of the LMX1B gene. Treatment with enalapril for 2 years (0.1-1 mg/kg per day) did not bring about any change in urinary protein excretion. However, enalapril (1 mg/kg per day) associated with losartan (1 mg/kg per day) resulted in complete remission (proteinuria 140 mg/24 h) at the age of 7 years.

The lmx1b gene is pivotal in glomus development in Xenopus laevis

We have previously shown that lmx1b, a LIM homeodomain protein, is expressed in the pronephric glomus. We now show temporal and spatial expression patterns of lmx1b and its potential binding partners in both dissected pronephric anlagen and in individual dissected components of stage 42 pronephroi. Morpholino oligonucleotide knock-down of lmx1b establishes a role for lmx1b in the development of the pronephric components. Depletion of lmx1b results in the formation of a glomus with reduced size. Pronephric tubules were also shown to be reduced in structure and/or coiling whereas more distal tubule structure was unaffected. Over-expression of lmx1b mRNA resulted in no significant phenotype. Given that lmx1b protein is known to function as a heterodimer, we have over-expressed lmx1b mRNA alone or in combination with potential interacting molecules and analysed the effects on kidney structures. Phenotypes observed by over-expression of lim1 and ldb1 are partially rescued by co-injection with lmx1b mRNA. Animal cap experiments confirm that co-injection of lmx1b with potential binding partners can up-regulate pronephric molecular markers suggesting that lmx1b lies upstream of wt1 in the gene network controlling glomus differentiation. This places lmx1b in a genetic hierarchy involved in pronephros development and suggests that it is the balance in levels of binding partners together with restricted expression domains of lmx1b and lim1 which influences differentiation into glomus or tubule derivatives in vivo.

A novel mutation in LMX1B gene causes nail-patella syndrome in a large Chinese family

We conducted clinical and genetic studies in a large Chinese family with nail-patella syndrome (NPS) involving multi-organ (such as limb, renal and eye) and investigated the functional consequences of a novel LMX1B mutation identified in the family. Twenty individuals at risk for inheriting NPS in the Chinese family participated in the study and a physical examination was performed and blood was drawn for DNA extraction. Linkage analysis and mutation screening of LMX1B gene were performed and the functional study in vitro for the mutation was conducted by luciferase assay. The disease phenotype of this family was linked to D9S290 with LOD Score=5.8 at θ=0; a novel mutation 742 A>G (R248G) within the homeodomain was found in a conserved site and co-segregated with the disease phenotype of the family. The functional study in vitro by luciferase assay indicated that the R248G mutation within the binding domain of the gene affected the transactivation. This is the first report that a mutation in the LMX1B gene causes NPS in a Chinese population, which will expand the spectrum of mutations in the LMX1B gene and provide insight into the underlining pathology of NPS.

Nail-patella syndrome and renal involvement: Description of three cases and literature review

Nail-patella syndrome (NPS) is a rare, autosomal dominant disorder reported in approximatively 1/50,000 individuals. It is characterized by hypoplastic or absent patellae, dystrophic nails, dysplasia of the elbows and iliac horns. Less frequently renal and ocular damages occur. The abnormal gene in NPS is located at the distal end of the long arm of Chromosome 9. Mutations in the human LMX1B gene have been demonstrated to be responsible for NPS. It encodes a LIM-homeodomain transcription factor which plays an important role in limb development in vertebrates. Extensive mutation analysis of different NPS families by different groups failed to demonstrate any genotype-phenotype correlation. Renal involvement occurs in 30-60% of patients and presents with proteinuria and/or microscopic hematuria, edema, hypertension. Progression to nephrotic syndrome occurs in less than 20% of patients, and renal failure in about 10% of NPS patients requiring dialysis and/or transplantation. We report three cases of NPS with different degrees of renal involvement and present a review of the literature on this rare hereditary condition.

Identification of entire LMX1B gene deletions in nail patella syndrome: evidence for haploinsufficiency as the main pathogenic mechanism underlying dominant inheritance in man

Heterozygous mutations in the LMX1B gene cause nail patella syndrome (NPS) that is associated with nail and skeletal malformations, nephropathy, and glaucoma. Previous phenotype studies of Lmx1b null mice revealed dorsal limb and renal anomalies similar to human NPS, which contributed to the identification of heterozygous mutations in this LIM-homeodomain protein LMX1B as the genetic defect responsible for NPS. Despite advanced insight into the role of the Lmx1b transcription factor in a broad range of animal developmental programs, the pathogenic mechanism underlying dominant inheritance of NPS in man remained unclear. Here, we describe for the first time the detection of two entire LMX1B gene deletions and one smaller exonic LMX1B deletion by multiplex ligation-dependent probe amplification (MLPA) in a series of eight unrelated families with classical features of NPS in whom no pathogenic LMX1B mutation was found by sequence analysis. The identification of entire LMX1B deletions strongly confirms that haploinsufficiency is the principal pathogenetic mechanism of NPS and suggests a difference in dosage sensitivity for this gene between mice and man.

Expression and function of nr4a2, lmx1b, and pitx3in zebrafish dopaminergic and noradrenergic neuronal development

Background:Dopaminergic neurons form in diverse areas of the vertebrate di- and mesencephalon to constitute several major neuromodulatory systems. While much is known about mammalian mesencephalic dopaminergic neuron development, little is known about the specification of the diencephalic dopaminergic groups. The transcription factors Pitx3 and Lmx1b play an important role in mammalian mesencephalic dopaminergic specification, and Nurr1/Nr4a2 has been shown to contribute to specification of the dopaminergic neurotransmitter phenotype. We use zebrafish to analyze potentially evolutionarily conserved roles of these transcription factors in a vertebrate brain that lacks a mesencephalic dopaminergic system, but has an ascending dopaminergic system in the ventral diencephalon.Results:We use a combination of fluorescent in situ hybridization and immunohistochemistry to determine whether nr4a2, lmx1b, and pitx3 genes are expressed in mature dopaminergic neurons or in potential precursor populations. We identify a second nr4a2 paralogue, nr4a2a, and find it co-expressed with Tyrosine hydroxylase in preoptic, pretectal and retinal amacrine dopaminergic neurons, while nr4a2b is only expressed in preoptic and retinal dopaminergic neurons. Both zebrafish nr4a2 paralogues are not expressed in ventral diencephalic dopaminergic neurons with ascending projections. Combined morpholino antisense oligo mediated knock-down of both nr4a2a and nr4a2b transcripts reveals that all zebrafish dopaminergic neurons expressing nr4a2a depend on Nr4a2 activity for tyrosine hydroxylase and dopamine transporter expression. Zebrafish lmx1b.1 is expressed in noradrenergic neurons of the locus coeruleus and medulla oblongata, but knock-down reveals that it is specifically required for tyrosine hydroxylase expression only in the medulla oblongata area postrema noradrenergic neurons. Both lmx1b genes and pitx3 are not expressed in dopaminergic neurons, but in a diencephalic territory that might contain precursor cells for ventral diencephalic dopaminergic neurons. Upon morpholino knock-down of both lmx1b paralogues, the number of neurons in diencephalic dopaminergic clusters with ascending projections appears specifically reduced. Thus lmx1b paralogues may contribute to the generation of diencephalic dopaminergic precursors. Conversely, knock-down of pitx3 does not specifically affect any diencephalic DA cluster.Conclusion:Our data indicate a conserved evolutionary role of Nr4a2 proteins in specification of the neurotransmitter phenotype, albeit it appears to be only one of several regulatory modules of dopaminergic differentiation, as most ventral diencephalic dopaminergic neurons do not express nr4a2 genes in zebrafish. For zebrafish lmx1b genes, which are not expressed in mature dopaminergic neurons, our data suggest a role in diencephalic precursor populations contributing to the ascending dopaminergic systems. A di-mesencephalic longitudinal domain of lmx1b expression may be the basis for the expansion and posterior shift of ventral di-/mesencephalic dopaminergic populations with ascending projections during evolution.

Complete heart block associated with noncompaction, nail-patella syndrome, and mitochondrial myopathy

Objectives Complete heart block has not been reported in association with left ventricular hypertrabeculation (LVHT)/noncompaction, nail-patella syndrome (NPS), and mitochondrial myopathy (MMP). Case Report A 47-year-old man with congenital NPS from a 17-bp deletion in exon 5 of the LMX1B gene, MMP from the A3243G mitochondrial DNA transition, and LVHT was acutely admitted after a syncope followed by dizziness. Cardiological examinations revealed bradycardia. Blood pressure was 70/30 mm Hg. Blood work revealed a creatine kinase of 389 U/L (normal, <175 U/L), renal insufficiency, anemia, and reduced calcium, phosphorus, and magnesium. Electrocardiogram showed complete heart block with an escape rhythm of 30/min. A temporary pacemaker was inserted. During the procedure, the patient became asystole but was successfully resuscitated. One day later, he received a permanent pacemaker. Echocardiography showed normal systolic function and LVHT. The patient recovered without sequelae. Conclusion In single cases, LVHT may be associated with complete heart block. Most likely, complete heart block was due to MMP. Patients with NPS and MMP require multidisciplinary surveillance, including regular electrocardiograms, for indicating anticipatory intervention in time.

The podocyte-specific inactivation of Lmx1b, Ldb1 and E2a yields new insight into a transcriptional network in podocytes

Patients with nail-patella syndrome, which among other symptoms also includes podocyte-associated renal failure, suffer from mutations in the LMX1B gene. The disease severity among patients is quite variable and has given rise to speculations on the presence of modifier genes. Promising candidates for modifier proteins are the proteins interacting with LMX1B, such as LDB1 and E47. Since human kidney samples from patients are difficult to obtain, conventional Lmx1b knock-out mice have been extremely valuable to study the role of Lmx1b in podocyte differentiation. In contrast to findings in these mice, however, in which a downregulation of the Col4a3, Col4a4 and Nphs2 genes has been described, no such changes have been detected in kidney biopsies from patients. We now report on our results on the characterization of constitutive podocyte-specific Lmx1b, Ldb1 and E2a knock-out mice. Constitutive podocyte-specific Lmx1b knock-out mice survive for approximately 2 weeks after birth and do not present with a downregulation of the Col4a3, Col4a4 and Nphs2 genes, therefore they mimic the human disease more closely. The podocyte-specific Ldb1 knock-out mice survive longer, but then also succumb to renal failure, whereas the E2a knock-out mice show no renal symptoms for at least 6 months after birth. We conclude that LDB1, but not E2A is a promising candidate as a modifier gene in patients with nail-patella syndrome.

Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences

Currently, polymerase chain reaction is the most used technique in many laboratories for either diagnostic or molecular biology purposes. Despite the large number of DNA sequences that can be easily analyzed, some GC-rich sequences are refractory to amplification due to the formation of secondary intramolecular structures. To overcome this problem, several molecules have been described to improve polymerization. Here we show that a combination of three additives--betaine, dimethyl sulfoxide, and 7-deaza-dGTP--was essential to achieve amplification of DNA sequences of three disease genes showing a GC content ranging from 67 to 79%.

Interaction of the LMX1B and PAX2 gene products suggests possible molecular basis of differential phenotypes in Nail–Patella syndrome

The LMX1B gene, encoding a protein involved in limb, kidney and eye development, is mutated in patients affected by Nail-Patella syndrome. Inter- and intrafamilial variability is common in this disorder for skeletal abnormalities, presence and severity of nephropathy and ocular anomalies. Phenotypic variability might depend on interactions of the LMX1B causative gene with other genes during development of both kidney and eye, which might act as modifier genes. Results are presented on the interaction between LMX1B and PAX2 proteins, obtained by both direct yeast two-hybrid assay and coimmunoprecipitation. Such interaction provides support to further studies on pathways underlying important developmental processes.

Functional Characterization of LMX1B Mutations Associated with Nail-Patella Syndrome

Nail-patella syndrome (NPS) is an autosomal dominant disease characterized by dysplastic nails, absent or hypoplastic patellae, elbow dysplasia, and nephropathy. Recently, it was shown that NPS is the result of heterozygous mutations in the LIM-homeodomain gene, LMX1B. Subsequently, many mutations of the LMX1B gene have been reported in NPS patients. However, functional analyses of the mutant proteins have been performed in only a few mutations. Furthermore, the mechanisms of dominant inheritance in humans have not been established. In the present study, we analyzed the LMX1B gene in three Japanese patients with NPS and identified two novel mutations, 6 nucleotide deletion (Delta246N 247Q) and V242L. These two mutations are located in the homeodomain of LMX1B. Functional analyses of the LMX1B mutants revealed that these mutants had diminished transcriptional activity and had lost DNA binding ability. Furthermore, we demonstrated that each mutant did not manifest a dominant-negative effect on the transcriptional activity of wild-type LMX1B. These results suggested that NPS is caused by loss-of-function mutations of LMX1B, and haploinsufficiency of LMX1B should be the predominant pathogenesis of NPS in humans.

Phenotype Severity and Genetic Variation at the Disease Locus: An Investigation of Nail Dysplasia in the Nail Patella Syndrome

The genetic bases underlying the range and severity of phenotypes in Mendelian disorders is poorly understood; however, improvements in this area have the potential to facilitate analysis of oligogenic disorders. The nail dysplasia observed in Nail Patella Syndrome (NPS) was selected as a quantifiable variable within a Mendelian disorder, for which data could be readily obtained, to allow investigation of the genetic basis of variation. Analysis of SNP haplotypes across the LMX1B gene demonstrated association between the haplotype of the mutant allele and the variability in the nail score (p = 0.024). These results are in contrast to those obtained previously, which supported a modifying role for the wild-type allele. Since there is no evidence that particular mutations, or classes of mutation, are associated with the variation (p > 0.5), further work is required to identify the elements associated with the LMX1B gene that mediate phenotypic severity.

Skeletal Integrity in Patients with Nail Patella Syndrome

Nail patella syndrome (NPS) is a rare autosomal dominant disorder resulting from a heterogeneous loss of function in the LMX1B gene. It is associated with multiple skeletal deformities, yet it is unknown whether this is associated with osteoporosis. To examine bone mass and the prevalence of fragility fractures, we assessed bone mineral density (BMD) of the spine and hip in 31 adults and 12 children with mutation-confirmed NPS and 60 healthy age- and gender-matched adult controls. For the adults with NPS, BMD was 11-20% lower at the hip sites (P < or = 0.001) and 8% lower at the spine (P < 0.05) than that of controls. Even when adjusted for body mass index, the BMD remained significantly lower in patients with NPS in all hip regions but not in the spine. Adults with NPS also had a significantly lower Z-score (sd values from normal) at all hip sites (all P < 0.05), compared with age- and gender-matched controls in the manufacturer's database. However, children had significantly lower Z-scores only at the femoral neck and trochanter. Participants with NPS also had a higher prevalence of fractures (odds ratio 30.9, 95% confidence interval 6.4-149.6, P < 0.001) and scoliosis (odds ratio 16.0, 95% confidence interval 3.3-78.2, P < 0.001). The majority of these fractures occurred in women before puberty and in long bones, especially the clavicle. We conclude that adults with NPS have a BMD that is 8-20% lower than controls, which is associated with an increase in the prevalence of fractures and scoliosis. Future studies are needed to determine whether bone quality, geometry, or turnover could account for these findings.

The human LMX1B gene: transcription unit, promoter, and pathogenic mutations

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.

Confirmation of CLIM2/LMX1B interaction by yeast two-hybrid screening and analysis of its involvement in nail-patella syndrome

Nail-patella syndrome (NPS), an autosomal dominant disorder characterized by nail dysplasia, absent or hypoplastic patellae, iliac horns, and often associated with nephropathy and, less frequently, with open angle glaucoma, is caused by mutations in the LMX1B gene. Inter-familial and intra-familial phenotypic variability raises the question whether modifier genes can be identified to explain differences in the expression and severity of clinical features of NPS. Genes encoding proteins that interact with the LMX1B protein are good candidates and, therefore, methods to search for interactions can be used to this purpose. By the yeast two-hybrid screening we detected the CLIM2 gene as a LMX1B interactor, confirming previous reports which described the same interaction by biochemical methods. Sequencing of the CLIM2 coding region in seven NPS cases in which no LMX1B mutation had been found, did not detect any molecular variant in these patients. Moreover, by genotyping a polymorphic dinucleotide repeat close to the CLIM2 gene in affected members of a large Dutch NPS family with high incidence of nephropathy, we were unable to find a correlation between the presence of a specific allele and the expression of nephropathy. In conclusion, although the results of this study could not provide any proof of CLIM2 involvement in the pathogenesis of NPS or in determination of the clinical phenotype, we suggest that the CLIM2 gene can be considered as a good candidate for further studies on normal and disturbed kidney development associated with NPS or other hereditary glomerulopathies.