Expanding the clinical phenotype associated with NIPAL4 mutation: Study of a Tunisian consanguineous family with erythrokeratodermia variabilis-Like Autosomal Recessive Congenital Ichthyosis

Abstract

Erythrokeratodermia variabilis (EKV) is a rare disorder of cornification usually associated with dominant mutations in the GJB3 and GJB4 genes encoding connexins (Cx)31 and 30.3. Genetic heterogeneity of EKV has already been suggested. We investigated at the clinical and genetic level a consanguineous Tunisian family with 2 sisters presenting an autosomal recessive form of EKV to better characterize this disease. Mutational analysis initially screened the connexin genes and Whole-exome sequencing (WES) was performed to identify the molecular aetiology of the particular EKV phenotype in the proband. Migratory shaped erythematous areas are the initial presenting sign followed by relatively stable hyperkeratotic plaques are the two predominates characteristics in both patients. However, remarkable variability of morphological and dominating features of the disease were observed between patients. In particular, the younger sister (proband) exhibited ichthyosiform-like appearance suggesting Autosomal Recessive Congenital Ichthyosis (ARCI) condition. No causative mutations were detected in the GJB3 and GJB4 genes. WES results revealed a novel missense homozygous mutation in NIPAL4 gene (c.835C>G, p.Pro279Ala) in both patients. This variant is predicted to be likely pathogenic. In addition, in silico analysis of the mutated 3D domain structure predicted that this variant would result in NIPA4 protein destabilization and Mg2+ transport perturbation, pointing out the potential role of NIPAL4 gene in the development and maintenance of the barrier function of the epidermis. Taken togheter, these results expand the clinical phenotype associated with NIPAL4 mutation and reinforce our hypothesis of NIPAL4 as the main candidate gene for the EKV-like ARCI phenotype.

Fig 1. Pedigree, clinical and histological features of Erythrokeratodermia variabilis (EKV) in family EKV-ICH1.

(a) Pedigree of the family segregating with autosomal recessive inheritance of EKV phenotype. The proband (EKV-ICH1.2) is indicated with a blue triangle. The sequence chromatograms illustrate the genotypes of tested family members for GJB4 and NIPAL4 genes. The likely polymorphism c.507C>G, (C169W) within the GBJ4 gene (RefSeq NM_001005752) is identified at heterozygous state in both affected sisters (EKV-ICH1.1, 2) and the unaffected brother (EKV-ICH1.3). The c.835C>G (p.Pro279Ala) mutation within the NIPAL4 gene (RefSeq NM_001099287) is identified at homozygous state in both affected sisters (EKV-ICH1.1, 2). Pedigree shows the cosegregation of the c.835C>G (p.Pro279Ala) mutation with EKV phenotype in a recessive mode. (b) The proband showed symmetric, well demarcated, brownish hyperkeratotic plaques localized on the knees, the feet (b1) and the dorsa of the hands (b2, b3) at the age of 18 months (first physical examination). Note the circinate borders of the erythematous plaques and the slight erythematous hyperkeratotic palmar keratoderma (b3). At the second visit, two weeks later, diffuse, migratory, figurative, erythematous spots and patches on the trunk and extremities (b4). Note the progression of the hyperkeratotic plaques and the variability of the distribution of hyperkeratotic plaques between the different clinical examination one month later (b5, b6). Widespread, large, adherent scale on extremities and trunk suggestive of ichtyosiform-lesions (b7). Two years later, fixed hyperkeratotic lesions on the forearms, knees, and dorsa of the hands (b7, b8). Histological findings from skin biopsy taken from the proband EKV-ICH1.2 showed hyperkeratosis associated with a compact, thickened stratum corneum (SC), hypogranulosis with a mild thinning stratum granulosum (SG) layer, papillomatosis, dilated follicles containing large keratin plugs, and a mild perivascular lymphocytic infiltrate (b9) (HE, x100). (c) The patient (EKV-ICH1.1) showed symmetric thick hyperkeratotic psoriasiform plaques over the knees at the age of 11 years. Two years later, symmetric hyperkeratotic squamous plaques with erythematous circinate borders on the elbows and erythematous and squamous plaques on the dorsa of the hands.

Fig 2. Multiple alignment of the NIPA4 protein sequences in different species showing the conserved nature of the proline residue at position 279 throughout species.

Fig 3. Structural representation of the wild-type and mutated p.(Pro279Ala) NIPA4 protein.

Left: top and bottom; Two perpendicular views (a and b) are shown. Top (a): Side view in ribbon cartoon of the wild-type. The helical transmembrane region colored in red, the N-terminal region colored in magenta, the C-terminal region colored in blue, proline 279 in CPK and colored in orange. The central transmembrane core of NIPA4 protein (amino acid: 116–411) is formed by nine transmembrane α-helices noted H1 to H9 (H1 (116–140); H2 (162–186); H3 (190–211); H4 (217–237); H5 (251–277); H6 (285–313); H7 (322–349); H8 (353–374); H9 (380–411) and a random coiled N- (1–115) and C-terminal (412–466) regions. This allows flexibility to the N-terminus and C-terminus regions independently of the transmembrane region. The model of NIPA4 protein was built based on herpesvirus fusion regulator complex gH-gL (PDB: 3m1c) and the structure of the membrane transporter YddG (PDB: 5i20) belonging to the large superfamily of the ubiquitous drug/metabolite transporter, export drugs and metabolites (DMT) [43]. The NIPA4 model presents a similar fold and topology of the template: the nine α-helices are organized like the predicted transmembrane helices, with a large substrate-binding cavity at the center of the receptor. The bottom view (b) highlights the intracellular gate and the cavity that accommodates the Mg²⁺ ion channel. Right: top and bottom (c and d); Two perpendicular views are shown. Top (c): Side view in ribbon cartoon of the p.Pro279Ala mutation. The helical transmembrane region colored in magenta, the N-terminal region colored in blue, the C-terminal region colored in red, Ala 279 in CPK and colored in yellow. The bottom view (d) highlights the intracellular gate and the cavity that accommodates the Mg²⁺ ion channel.

Fig 4. Docking analysis of the wild-type and the mutated p.(Pro279Ala) NIPA4 proteins against Mg²⁺.

Fig 5. Analysis of simulation trajectory of the wild-type and mutated (p. Pro279Ala) NIPA4 protein for 140 ps.

(a) Time evolution of backbone RMSD as a function of time, (b) RMSF of the Cα atoms as a function of residue. A lesser value was found in RMSD for the wild-type (blue) than that the mutant protein (grey indicating a more stability for the wild-type than the altered protein. These results indicate that the flexibility of NIPA4 was improved due to the mutation on Pro279Ala. The superimposition of the RMSF of all amino acids of the wild-type with the p.Pro279Ala mutation indicates a strong modification in the region containing the mutation. This region was surrounded by a rectangle in (b). The fluctuation of the loop (274–287) clearly demonstrates the mobility of the mutated region compared to the wild-type, probably leading to the dysfunction of the Mg²⁺ ion channel.