A Recurrent Nonsense Mutation in NECTIN4 Underlying Ectodermal Dysplasia-Syndactyly Syndrome with a Novel Phenotype in a Consanguineous Kashmiri Family

EDSS1, a syndrome characterized by ectodermal dysplasia-syndactyly, is inherited in an autosomal recessive manner due to mutations in the NECTIN4/PVRL4 gene. Clinical manifestations of the syndrome include defective nail plate, sparse to absent scalp and body hair, spaced teeth with enamel hypoplasia, and bilateral cutaneous syndactyly in the fingers and toes. Here, we report a consanguineous family of Kashmiri origin presenting features of EDSS1. Using whole exome sequencing, we found a recurrent nonsense mutation (NM_030916: c.181C > T, p.(Gln61 ∗)) in the NECTIN4 gene. The variant segregated perfectly with the disorder within the family. The candidate variant was absent in 50 in-house exomes pertaining to other disorders from the same population. In addition to the previously reported clinical phenotype, an upper lip cleft was found in one of the affected members as a novel phenotype that is not reported by previous studies in EDSS1 patients. Therefore, the study presented here, which was conducted on the Kashmiri population, is the first to document a NECTIN4 mutation associated with the upper lip cleft as a novel phenotype. This finding broadens the molecular and phenotypic spectrum of EDSS1.


Background
EDs are a genetically heterogeneous group of disorders characterized by developmental malformations of ectodermal structures, including nails, teeth, hair, or sweat glands.Te prevalence of EDs varies depending on the subtype, with an approximate incidence of 7/10,000 cases live births [1].To date, nearly, more than 200 forms have been described under the term ED [2].EDs are further divided into two forms syndromic and nonsyndromic: nonsyndromic is pure; however, syndromic is associated with other defects including intellectual disability, eye diseases, skeletal defects, facial dysmorphism, and other systemic phenotypes [3,4].Te identifcation of the molecular basis and pathogenesis of the increasing number of EDs has eased the development of a new classifcation system which combines both clinical and molecular data [2].ED-syndactyly syndrome 1 (EDSS1; OMIM#613573) is a sporadic form of ED characterized by extremely thin or absent scalp hair, thin eyebrows, and eyelashes, palmoplantar hyperkeratosis, distant and conically shaped teeth, and enamel hypoplasia, along with partial cutaneous syndactyly in both fngers and toes.Te etiology of ED-syndactyly syndrome 1 (EDSS1; OMIM#613573) has been attributed to genetic variations within the poliovirus receptor related 4 (PVRL4) gene, which has been more recently designated as NECTIN4 located on chromosome 1q23.1.NECTIN4 encodes a member of the nectin family, and its enhanced expression has been seen in adhering junctions of the suprabasal epidermis and the hair follicle, cultured keratinocytes, and in separating digits of the murine embryo [5].Te encoded protein contains one Ig-like V-type domain and two immunoglobulin-like (Ig-like) C2type domains.Te protein is involved in cell adhesion through trans-homophilic and heterophilic interactions.In multicellular organisms, cell-cell adhesion is crucial for the regeneration, ontogenesis, and maintenance of tissues and organs.All these functions are performed by diferent proteins.Dysregulation of genes associated with cell-cell adhesion proteins contribute signifcantly to the pathogenesis of many disorders such as, cancers, neuropsychiatric illnesses, and reproductive and sensory organs disorders along with other syndromic entities [6].
Tese cell-cell adhesion gene mutations have also been notably associated with a specifc form of syndromic ectodermal dysplasias.For instance, the CDH3 gene mutation underlies the emergence of human juvenile macular dystrophy (HJMD; OMIM#601553), characterized by progressive central retinal degeneration and genetic hair loss [7,8].Similarly, mutations in the NECTIN1/PVRL1 gene located on 11q23.3 are linked with cleft lip/palate ectodermal dysplasia (CLPED1; OMIM#225060) [9].Afected individuals from both EDSS1 and CLPED1 showed overlapping clinical features including thin scalp hair, eyebrows and eyelashes, tooth enamel hypoplasia, conical-shaped teeth, hypoplastic nails, palmoplantar keratoderma, and partial cutaneous syndactyly, whereas facial anomalies and cleft lip/palate are only seen in CLPED1 patients [10][11][12].Till now, a limited number of mutations in the NECTIN4 gene have been reported in patients with EDSS1, and there is a lack of comprehensive genotype-phenotype associations.However, despite the diverse phenotypes of NECTIN4 mutations in EDSS1 patients and the lack of comprehensive genotype-phenotype associations, three clinical features, including cutaneous syndactyly, hair abnormalities, and dental anomalies, have been consistently observed in individuals with EDSS1, regardless of the ethnic background of patients [5,[11][12][13][14][15]. were followed in the handling of human subjects.Family elders were informed in detail about the purpose of the study in their local language, and all individuals taking part in this research gave informed written consent.A comprehensive medical report was obtained from the Department of Dermatology, Combined Military Hospital (CMH), Azad Jammu and Kashmir, Muzafarabad, Pakistan.

DNA Extraction.
Venous blood stored in EDTAcontaining tubes was used to isolate genomic DNA using the phenol-chloroform method [16].DNA quality was verifed by using a photo spectrometer at 260 nm (UV-VIS SPECTROMETER/T60UV) and gel electrophoresis, respectively.

Whole Exome Sequencing and Variant Annotation.
Whole exome sequencing was performed on the DNA sample obtained from the afected proband (III-3) (Figure 1).Te SeqCap EZ Exome v3 kit from Roche NimbleGen was used to enrich DNA libraries for whole exome sequencing, and the Illumina HiSeq 4000 (Illumina, San Diego, CA, USA) platform was used for whole exome sequencing.Te mean depth of sequencing reads was maintained at 36x, ensuring adequate coverage of the target region, with each read covering approximately 94% of the region.
Low-quality reads were excluded by using Picard (https://broadinstitute.github.io/picard/),and then, the reads were mapped to the human reference genome (UCSC GRCh37/hg19) by using Burrows-Wheeler Aligner (http:// biobwa.sourceforge.net/).Variants were called using Genome Analysis Tool Kit (https://software.broadinstitute.org/gatk/).Subsequently, the KGGSeq tool was utilized to perform a comprehensive set of annotations against reference sequences (Hg19).Tis included assessing frequency in publicly available databases, making conservative predictions, and predicting pathogenicity using available website-based tools for the detected variants [17].

Verifcation of the Candidate Region by Sanger
Sequencing.Sanger sequencing was performed to validate the segregation of the candidate variant screened by WES among all the family members.A set of primer (forward: 5′TAATGGTGGCTGTCCCTCTCT 3′; reverse 3′ CACTCG TACTCGCCCTCATC 5′) was used for the amplifcation of the target region.

Protein Modeling and Functional Interactions.
Utilizing the NCBI database (https://www.ncbi.nlm.nih.gov/ ), the amino acid sequence of NECTIN4 was obtained and used to create a 3D model of the protein through the application of I-Tasser software (Iterative Treading ASSEmbly Refnement) [19].Te produced structural models were subsequently visualized with PyMOL (https://www.pymol.org/), a renowned molecular graphics system.Te functional interactions of NECTIN were evaluated using the string database (https://stringdb.org/)(Figure 2), while NCBI HomoloGene (https://www.ncbi.nlm.nih.gov/Homologene/) was utilized to investigate the conservation of amino acids across various orthologs (Figure 3(d)).

Clinical Features.
Te family had four afected individuals including two males and two females.Tey had sparse and thin hair on the scalp and had sparse eyebrows and eyelashes.In addition, partial cutaneous syndactyly involving toes 2-3 and fngers 3-4, short fngers, and large palm size were also observed in the afected individuals.Conical teeth with pig-like shapes, enamel ridges, and pits characterized the dental features of all afected subjects, along with noticeable spacing.Aberrant sweating rates were evident across the hands, face, and scalp.A previously unreported clinical phenotype, an upper lip cleft, was observed in an afected individual (III-1) (Figure 1(a)).Detailed clinical features are given in Table 1.

Analysis of Exome
Sequencing.An initial analysis of VCF of WES data revealed 71,630 variants.After fltering all synonymous and common variants with MAF threshold ≥0.001 in 1000 Genomes Project, ExAC, and dbSNP, 5,114 variants were selected for further analysis.Since the pedigree revealed an autosomal recessive inheritance pattern as shown in Figure 1(a), we searched the VCF fle specifcally     Genetics Research

Discussion
Te nectin family is comprised of Ca2+-independent immunoglobulin-like cellular adhesion molecules, including nectins 1 and 4. Tese proteins play a crucial role in cell adhesion via homophilic and heterophilic interactions [23].
Any defects in cell-cell adhesion molecules may cause various types of ectodermal dysplasias [24,25].More specifcally, mutations in NECTIN 1 and NECTIN4 cause cleft lip/palate ED (CLPED1; OMIM#225060) and EDSS1, respectively [5,9,24,25].Te expression of NECTIN4 has been seen in the adherens junctions of keratinocytes of suprabasal epidermal layers in the interfollicular skin, the inner root sheath, and the shaft cortex of the hair follicle.In addition, the presence of NECTIN4 was also detected in the interdigital skin during embryogenesis [5].In this study, we report a clinical and genetic investigation of a consanguineous family of Kashmiri origin, segregating EDSS1 in an autosomal recessive manner.Afected individuals displayed clinical features including sparse and very thin scalp hair, sparse mustache, very thin eyelashes and eyebrows, conical and widely spaced peg-shaped teeth, along with bilateral cutaneous syndactyly, palmoplantar keratoderma, fat discolored thickened hypoplastic fnger and toe nails, and upper lip cleft Figure 1(b)-1(i).
Te protein has many domains counting a transmembrane domain, a cytoplasmic domain, three subdomains of immunoglobulin in the extracellular segment, and an Nterminal signal peptide.Te candidate variant is situated in the V-type1 immunoglobulin-like segment of NECTIN4 (amino acids 32-144), as described earlier by Raza et al. [11], which results in the truncated protein [11] (Figure 4(a)).
Numerous tight junctional adhesion proteins complexes, desmosomes, and some other adhesive junctions play an important role in cell-cell adhesion, and the dynamic nature of this communication is vital for wound healing, tissue renewal, and establishing new cell contacts with other adjacent cells.Nectins (N-1 to N-4) are involved in cell-cell interaction through a calcium-independent adhesion mechanism and are considered as a supporting element for cell-to-cell adhesion by developing adhesive junctions (AJs).Tis phenomenon is based on heterophilic or homophilic interactions through Ig-like domains.Trans-heterophilic interactions are usually stronger than trans-homophilic interactions, and nectins engage in both homophilic and heterophilic interactions with other nectins or proteins on neighboring cells.Tese interactions trigger the creation of adherens junctions, which subsequently lead to the   6 Genetics Research formation of tight junctions [26].Te regulation of the Rac1 gene activity is also linked to these nectins.In Rac1-defcient mice, interdigital webbing, defective skin, and severe hair loss have been observed, which are similar to the clinical manifestations seen in both EDSS1 and CLEPD1 patients.However, no disease-causing mutations in Rac1 have been identifed in humans as of yet [22], and primary data for nectin in endothelium are scant.In summary, the discovery of the "upper lip cleft," a novel trait in EDSS1, is signifcant for future diagnosis and aids in developing a reference database specifc to the Kashmiri population, as there is a lack of publicly available reference databases for identifying genetic mutations in this population.Moreover, fndings could also lead to further clinical studies investigating the correlations between specifc NECTIN4 mutations and clinical features, potentially improving the diagnosis and treatment of EDSS1 patients.Also, this study raises awareness of EDSS1 and related syndromes among researchers, healthcare providers, and the general public, facilitating prenatal diagnosis, genetic counseling, and timely interventions to improve the quality of life for afected individuals.

2. 1 .
Human Subject and Ethics Statement.Te current study describes three generations of a consanguineous family from Neelum, Azad Jammu and Kashmir, Pakistan, who have clinical symptoms of EDSS1.Ethical approval to conduct this research was obtained from the Director of Advanced Study and Research (DSAR) Board of the University of Azad Jammu and Kashmir, Muzafarabad, Pakistan.All ethical principles of the Declaration of Helsinki (October 2013)

Figure 1 :
Figure 1: Pedigree and clinical manifestations of individuals afected with EDSS.(a) Te pedigree displays the index patient used for whole exome sequencing, marked with ( * ).All parents of the afected individuals have consanguineous relationships.(b-e) Te clinical manifestations of individual (III-3) include hypotrichosis, with sparse or absent scalp hairs, eyebrows, and eyelashes, an upper lip cleft with spaced and pointed teeth, cutaneous syndactyly afecting fngers 3-4 on both hands and toes 2-3 on both feet, and a discolored nail palate.(f-i) Te afected individual (III-5) displays clinical features consistent with EDSS.

Figure 3 :
Figure 3: Electropherogram of the patient showing the site of nucleotide change.Black arrows indicate the position of nucleotide change (stop gain).(a) Wild type, (b) heterozygous carrier, (c) homozygous afected with a stop gain at position c.181C > T, and (d) comparison of the amino acid sequences of human NECTIN4 protein with orthologs from other species, indicating conservation of the p. Q61 residue across all species.

4
Filtration stepsNumber of variants detected Total variants detected in afected individuals (III-3) 71,630 Te number of variants remaining after fltering out synonymous variants 35,466 Te number of variants remaining after fltering out common variants with a minor allele frequency (MAF) of less than 0

Figure 4 :
Figure 4: 3D model of the NECTIN4 wild type and mutant protein.A: 3D model of NECTIN4 protein indicating the position of the start codon and a stop gain at position p.(Gln61 * ).B: mutant protein after stop gain.

Table 1 :
Te clinical features observed in the current family and those reported in previous families.

Table 2 :
Te process of identifying the disease-causing variant involved several fltering steps.