Identification of limb-specific Lmx1b auto-regulatory modules with Nail-patella syndrome pathogenicity

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.

A Novel LMX1B Variant Identified in a Patient Presenting with Severe Renal Involvement and Thin Glomerular Basement Membrane

We report a case of nail-patella syndrome (NPS) with unusual thinning of the glomerular basement membrane (GBM) associated with a novel heterozygous variant in the <i>LMX1B</i> gene. A 43-year-old female patient with a previous diagnosis of NPS, referred to our hospital for persistent proteinuria, underwent a renal biopsy, which revealed minor glomerular abnormalities. She underwent a second renal biopsy at the age of 56 owing to the presence of persistent proteinuria and decline in serum albumin, meeting the diagnostic criteria for nephrotic syndrome. Light microscopy demonstrated glomerulosclerosis and cystic dilatation of the renal tubules. Notably, electron microscopy revealed unusual thinning of the GBM, which is quite different from typical biopsy findings observed in patients with NPS, characterized by thick GBM with fibrillary material and electron-lucent structures. Comprehensive genetic screening for 168 known genes responsible for inherited kidney diseases using a next-generation sequencing panel identified a novel heterozygous in-frame deletion-insertion (c.723_729delinsCAAC: p.[Ser242_Lys243delinsAsn]) in exon 4 of the <i>LMX1B</i> gene, which may account for the disrupted GBM structure. Further studies are warranted to elucidate the complex genotype-phenotype relationship between <i>LMX1B</i> and proper GBM morphogenesis.

Case Report: Corneal Leucoma as a Novel Clinical Presentation of Nail-Patella Syndrome in a 5-Year-Old Girl

Nail-patella syndrome (NPS) is a rare autosomal-dominant disorder characterized by the classic tetrad of absent or hypoplastic finger and toe nails, absent or hypoplastic patella, skeletal deformities involving the elbow joints, and iliac horns. This disease is caused by heterozygous pathogenic variations in the LMX1B gene, which encodes the LIM homeodomain transcription factor protein (LMX1B). We report a case of corneal leucoma and dysplasia prior to overt steroid-resistant nephrotic syndrome (SRNS) in a patient with NPS. At presentation, the parents of a 5-year-old female patient reported their daughter had corneal leucoma, psychomotor delay and speech defect. We also noted the presence of bilateral edema of the lower extremities, hypertension, nail dystrophy, and the bilateral absence of patella. She developed steroid-resistant nephrotic syndrome. Lowe oculocerebrorenal syndrome and NPS were the conditions considered in differential diagnosis. Trio-based whole genome sequencing indicated a heterozygous de novo likely pathogenic variation in the LMX1B gene (c.805A&gt;C [p.Asn269His]). Patients with NPS often develop nail, ocular, or orthopedic symptoms prior to nephrotic syndrome. Corneal leucoma may be a novel clinical presentation of NPS.

MO1027EFFICACY OF CALCINEURIN INHIBITORS IN CHILDREN WITH MONOGENIC STEROID-RESISTANT NEPHROTIC SYNDROME

Background and Aims Monogenic causes of steroid-resistant nephrotic syndrome (SRNS) have been reported for up to one-third of children depending on age of the disease onset. Immunosuppressive treatment of genetic SRNS with calcineurin inhibitors (CNIs) is still controversial. The aim of the study was to investigate the efficacy of CNIs with focus on inducing remission and long-term kidney function in children with monogenic SRNS. Method Retrospective analysis of efficacy CNIs in five children (2M/3F) with monogenic SRNS was performed. Kidney biopsy prior CNIs revealed FSGS (n=4) and MCD (n=1). The initial cyclosporine (CsA) dose was 5 mg/kg/24h to keep a target level of 80-150 ng/ml and tacrolimus (TAC) - 0.1 mg/kg/24h to achieve a target level of 5-10 ng/ml. CsA took all 5 patients with subsequent switching to TAC in 2 children due to cosmetic side effects. The median follow-up period was 165.0 (IQR: 59.0; 185.5) months. Next generation sequencing (NGS) was used for identification of pathogenic variants in all patients. Results The median age at onset of monogenic SRNS was 33.0 (IQR: 16.5; 63.0) months. 2/5 (40%) patients presented with acute SRNS, 1/5 (20%) child with infantile NS, 1/5 (20%) - with isolated nephrotic range proteinuria with hypoalbuminemia and 1/5 (20%) - with NS and extrarenal features of Nail-Patella syndrome. NGS identified previously described pathogenic variants in all 5 children, including NPHS2 homozygous c.28dup (p.Glu87Ter) (n=1), NPHS2 compound heterozygous c.868G&gt;A (p.Val290Met) in combination with c.686G&gt;A (p.Arg229Gln) (n=1), LMX1B heterozygous c.788T&gt;G (p.Val263Gly) (n=1), LMX1B heterozygous c.737G&gt;A (p.Arg246Gln) (n=1), and COL4A3 heterozygous c.2962G&gt;A (p.Gly988Arg) variant (n=1). The median time from diagnosis to initiation of CNIs treatment was 72.0 (IQR: 33.0; 93.0) months. CNIs induced complete remission in 2/5 (40%) patients, presented with acute SRNS, including one girl with MCD due to NPHS2 compound heterozygous variants with mutation-dependent pathogenicity of one (p.R229Q) of them and one boy with FSGS due to COL4A3 heterozygous variant (n=1). Partial remission was induced by CNIs in 2/5 (40%) girls with FSGS due to LMX1B heterozygous variants with isolated SRNS (n=1) and Nail-Patella syndrome (n=1). The median duration of CNIs treatment to obtain complete or partial remission was 13.5 (IQR: 6.8; 15.8) months. Acute CNIs-associated nephrotoxicity had 2 patients with LMX1B variants. At the last follow up full and partial responders to CNIs treatment aged of 16.5 (IQR: 11.8; 17.5) years had CKD-1 (n=3) and CKD-2 (n=1). 1/5 (20%) boy with NPHS2-associated infantile NS was CNI resistant and developed CKD-5 at the age of 6.5 years with subsequent living related kidney transplantation. Conclusion We found that 4/5 (80%) children with monogenic SRNS demonstrated partial or full response to CNIs treatment with stable long-term kidney function. We assume that CNIs might improve podocyte function by stabilization of their cytoskeleton disrupted in patients with monogenic SRNS.