Identification of limb-specific Lmx1b auto-regulatory modules with Nail-Patella Syndrome pathogenicity

Resumen del trabajo presentado en el 17th Spanish Society for Developmental Biology Meeting Virtual Meeting, celebrado en modalidad virtual del 18 al 20 de noviembre de 2020.Lmx1b is a LIM homeodomain transcription factor that plays essential roles in a wide range of developmental processes including the development of the kidney, the eye, the dopaminergic and serotonergic neurons and the limb. In the limb, Lmx1b expression is restricted to the dorsal mesenchyme and is responsible for limb dorsalization. Mice lacking functional Lmx1b die at birth because of multisystemic malformations. Homozygous Lmx1b mutants develop a double-ventral limb phenotype with loss of dorsal structures such as the patella, whereas the ventral ones such as the sesamoid bones are duplicated and dorsal tendons and muscles are transformed into mirror images of ventral ones. At superficial level, hair and nails are absent while typical ventral features such as pads are also duplicated. Herein, we report on two conserved Lmx1bassociated cis-regulatory modules (LARM1 and LARM2) located upstream of Lmx1b that are bound by Lmx1b and mediate an autoregulatory loop required to provide adequate levels of Lmx1b expression necessary for limb dorsalization. Combined removal of both enhancer lead to a limb restricted phenotype identical to that of the Lmx1b Knockout mice with no other Lmx1b-related systemic defects, indicating that they are limb specific enhancers. Unexpectedly, the CRISPR/Cas9 deletion of each individual enhancer produced a double ventral phenotype clearly restricted to the posterior or anterior half of the autopod in LARM1 and LARM2 respectively, unveiling spatial modularity in the transcriptional control of Lmx1b in the limb. In humans, LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Of most interest, we report on two NPS patient families with normal LMX1B coding sequence and limb-restricted phenotype, but with loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis