Degenerated hair follicle cells and partial loss of sebaceous and eccrine glands in a familial case of axenfeld-rieger syndrome: An emerging role for the FOXC1/NFATC1 genetic axis

Highlights

• A novel frame-shift mutation in FOXC1 in a Lebanese family with ARS presenting a cutaneous phenotype.

• The cutaneous phenotype includes hair follicle cell degeneration, and partial loss of sebaceous and eccrine glands.

• This is the first documented cutaneous role for both NFATC1 and FOXC1 in humans that recapitulates their function in mouse models with inactivation of either gene.

Abstract

Background

Cutaneous malformations are at times associated with some forms of congenital heart defects. Many a times subtle cutaneous phenotypes maybe overlooked as their significance on the lives of individuals is minimal. Lebanon represents an area of high consanguinity, where the rates can go beyond 70% in some districts. For the past 6 years, we have been studying several genodermatoses in Lebanon including those with cardiac malformations.

Objectives

The main aim of this study is to document the genetic basis of a familial case of Axenfeld-Rieger Syndrome (ARS) with a mild cutaneous phenotype represented histologically with degeneration/ absence of hair follicles and incomplete formation of sebaceous and eccrine glands, in addition to the cardiac and ocular phenotypes.

Methods

Whole exome sequencing was performed on two identical-twins with ARS along with their affected father and non-affected mother. Sanger sequencing was used to confirm the mutation, and the effects of the mutations on protein function was assessed in vitro using transient transfections.

Results

A novel mutation inFOXC1 designated p.L240Rfs*75 was found in both twins and their father. The affected individuals share also a rare documented variant in NFATC1 designated p.V197 M. Both were absent from 200 Lebanese exomes. Our in vitro results suggested a gain of function activity of the FOXC1/NFATC1 complex, confirming its documented role in controlling murine hair follicle stem cells quiescence and regeneration.

Conclusion

This is the first documented human case with a mutation inFOXC1 regulating multi-organ developmental pathways that reflect a conserved mechanism in cell differentiation and proliferation.

Introduction

Congenital heart diseases (CHD) are the leading cause of death during the first year of life in humans worldwide, with an incidence of 1 in 100 [1,2]. Despite the tremendous progress in the clinical care of patients, and the technological progress in diagnosis and surgery, the genetic insight of such diseases is still scarce [[3], [4], [5], [6], [7]]. One of the main challenges is to properly analyze all the variants in the genes using a network approach that would take into account the functional and evolutionary interactions of the proteins in the cellular context. In all cases, an a priori knowledge of the molecular and developmental events that shape up the cellular defect inside the given organ is crucial. Lineage-specific transcription factors not only can control cell fate, but also structurally affect the development of a given organ when expressed at particular doses and appropriate timing. Amongst such transcription factors, the forkhead family of helix-turn-helix have been shown to be involved in organogenesis at different levels: cellular proliferation, differentiation, migration, and programmed cell death [8]. The murine Foxc1 protein was shown to be expressed particularly in the mesoderm as well as in derivatives of the neural crest cells in different organs including the eye, the heart, the brain, and recently the skin [[9], [10], [11]]. Foxc1 -/- mice die directly after birth or during the late stages of development mainly from cerebovascular defects [[12], [13], [14], [15]]. In humans, deleterious mutations in FOXC1 lead to the Axenfeld-Rieger Syndrome (OMIM#602482), that manifests amongst others with anterior segment dysgenesis at the level of the eye, leading to glaucoma, with subtle cardiac defects, mainly atrial septal defects (ASD) [16,17]. Despite extensive in vitro studies to assess the underlying strcutrual and functional defects in the mutant FOXC1 proteins, a genotype-phenotype correlation could not be established to explain the variable expressivity of the phenotype [18,19]. This might be due to FOXC1 being expressed in various cellular contexts at different timings, and that members for the same family might partially compensate for its expression and/or function. One close member is FOXC2 whose expression often overlaps with that of FOXC1 in numerous cells mainly in neural crest derivative cells [12,13,20,21].

Only recently, we started to gain more knowledge about the function of Foxc1 using mouse models [[22], [23], [24]]. The murine Foxc1 gene was shown to be expressed both in hair follicle stem cells and sebaceous glands. Loss of function of this gene resulted in the premature decay of the hair follicle cells by affecting the NFATC1/calcineurin pathway. Interestingly, the deletion of Foxc1 in activated, but not quiescent cells allow their premature activation, while its targeted inactivation in the bulge leads to loss of the old hair without impairing quiescence [23,24]. Finally, the targeted deletion of Foxc1 from the skin, lead to hypohidrotic mice, a phenotype that resembles the human sweat retention disorder, miliaria [22].

In this report, using whole exome sequencing we describe the first human cutaneous phenotype associated with a potential gain of function mutation in FOXC1 in a familial case of ARS with a highly penetrant ocular and cardiac phenotype. The degeneration of the hair follicle and the miniaturization/absence of sebaceous glands as well as incomplete development of the eccrine glands in affected individuals, were directly linked to the novel frameshift mutation in FOXC1. Our extensive genetic analyses identified a second rare variant in the NFATC1 which in our in vitro functional studies showed an indirect interaction with the FOXC1 mutant.