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

© The Author(s) 2021.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.This work was supported in part by grants from the Spanish Ministerio de Ciencia, Innovación y Universidades (M.A.R) (BFU2017-88265-P); the National Organization for Rare Disorders (K.C.O.), and the Loma Linda University Pathology Research Endowment Fund (K.C.O.)

Two Lmx1b-associated cis-regulatory modules (LARM1/2) mediate Lmx1b auto-amplification during limb dorsalization and their disruption can cause a limb-specific form of Nail-Patella syndrome

Trabajo presentado en Limb Development and Regeneration: New Tools for a Classic Model System EMBO Workshop, celebrado en Barcelona (España) del 2 al 5 de julio de 2019.During limb development, the Lmx1b homeodomain transcription factor is restricted to dorsal mesenchyme and is required for distal dorsalization. Lmx1b also contributes to kidney, eye and cerebellar development. Haploinsufficiency of the human LMX1B genes causes Nail-Patella Syndrome (NPS) characterized by nail dysplasia, absent or hypoplastic patellae, eye abnormalities, and progressive kidney disease. In the functional Lmx1b knockout mouse, transcription of the mutant Lmx1b mRNA in the limb is reduced, indicating a role for Lmx1b in an auto-amplifying feedback loop. Previous Lmx1b-targeted ChIP-seq data identified two Lmx1b-associated regulatory modules (LARM1/2) bound by Lmx1b. Herein, we demonstrate that LARM1/2 activity overlaps Lmx1b expression in the dorsal limb mesoderm and is dependent on conserved Lmx1b binding sites for activity. We also demonstrate interaction between the LARM enhancers and the Lmx1b promoter. In addition, we performed a targeted CRISPR-Cas excision of the LARM1/2 region in mice; loss of LARM1/2 reduced the expression of Lmx1b in normal mice and generated a phenotype with nearly symmetrical ventral-ventral distal limbs. LARM1/2 loss was limb-specific and did not affect any other Lmx1b-related organs. We extended our investigation to humans with an NPS phenotype that lacked a variation within the LMX1B coding sequence. In two families with a limb-restricted phenotype, loss-of-function variations in the LARM region were identified. These data indicate that the limb-specific expression of Lmx1b is amplified through an auto-regulatory loop involving LARM1/2. Furthermore, disruption of LARM1/2 function can cause isolated limb malformations in mice and human NPS