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

Abstract

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.

Developmental disorders caused by haploinsufficiency of transcriptional regulators: a perspective based on cell fate determination

“…As outlined above, master TFs whose HI leads to DDs are generally involved in positive feedback ( and ). Crucially, for many of these TFs, disruption of positive feedback has been identified as the cause of human disease, including LMX1B in nail–patella syndrome ( Haro et al, 2021 ), PAX6 in aniridia ( Bhatia et al, 2013 ), SOX10 in Waardenburg syndrome ( Lecerf et al, 2014 ), SOX9 in sex reversal ( Croft et al, 2018 ), FOXG1 in FOXG1 syndrome ( Ye et al, 2022 ), FOXP2 in speech–language disorder ( Becker et al, 2018 ), NFIA in a brain malformation syndrome ( Trevino et al, 2021 ), GATA2 in immunodeficiency ( Johnson et al, 2012 ), and HNF1A and HNF4A in MODY ( Hansen et al, 2002 ). If disruption of positive feedback is due to loss-of-function mutations in CREs (regulatory loss of function; Box 2 ), this frequently results in the isolated, rather than the syndromic, form of the disorder.…”

“…If disruption of positive feedback is due to loss-of-function mutations in CREs (regulatory loss of function; Box 2 ), this frequently results in the isolated, rather than the syndromic, form of the disorder. For example, LMX1B regulatory loss of function results in limb anomalies rather than full nail–patella syndrome ( Haro et al, 2021 ), SOX10 regulatory loss of function results in isolated Hirschsprung's disease rather than Waardenburg syndrome ( Lecerf et al, 2014 ), and FOXG1 regulatory loss of function results in isolated strabismus rather than FOXG1 syndrome ( Ye et al, 2022 ). The reason is that most CREs are tissue-specific and will, when mutated, only affect that tissue.…”

Failure of digit tip regeneration in the absence of Lmx1b suggests Lmx1b functions disparate from dorsoventral polarity

“…We have recently characterized two limb-specific Lmx1b enhancers, termed LARM1 and LARM2 , that mediate Lmx1b limb dorsalization (Haro et al 2021). Mice homozygous for the removal of these two enhancers ( ΔLARM1/2 ) replicate the Lmx1b-null limb phenotype consisting of double-ventral distal limbs with sole pads in both surfaces and no nails (Haro et al 2021; Fig 1A-A’).…”

“…We have recently characterized two limb-specific Lmx1b enhancers, termed LARM1 and LARM2 , that mediate Lmx1b limb dorsalization (Haro et al 2021). Mice homozygous for the removal of these two enhancers ( ΔLARM1/2 ) replicate the Lmx1b-null limb phenotype consisting of double-ventral distal limbs with sole pads in both surfaces and no nails (Haro et al 2021; Fig 1A-A’). In contrast to Lmx1b-null mutants that die perinatally, ΔLARM1/2 homozygous mutants survive, as they show no other Lmx1b-related systemic defects, allowing the analysis of fully differentiated double-ventral limbs.…”

“…Lmx1b plays essential roles in a wide range of developmental processes including the development of the kidney, the anterior segment of the eye, and central nervous system (specifically dopaminergic and serotonergic neurons) (Sweeney et al 2003). Recently, we characterized two limb-specific Lmx1b enhancers, termed LARM1 and LARM2 ( L mx1b a ssociated r egulatory m odule 1 and 2) that are key regulators of Lmx1b expression in the limb (Haro et al 2017; 2021). Accordingly, deletion of both LARM enhancers results in mice ( ΔLARM1/2 ) that replicate the Lmx1b-null phenotype exclusively in the limb and are, therefore, viable.…”