Molecular Genetics of Keratinization Disorders - What\u27s New About Ichthyosis

The heritable forms of keratinization disorders, including various forms of ichthyosis and keratodermas, comprise a phenotypically heterogeneous group of diseases which can be divided into syndromic and non-syndromic forms. In the non-syndromic forms, the clinical manifestations are limited to the cutaneous structures while the syndromic ones are associated with a spectrum of extracutaneous manifestations. The inheritance in different families can be autosomal dominant, autosomal recessive or either X-linked dominant or recessive. Currently at least 67 distinct genes have been associated with different forms of ichthyosis. These genes can be grouped on the basis of their physiological involvement, including genes encoding structural components of epidermis, those involved in epidermal lipid metabolism, or those critical for cell-cell adhesion, and keratinocyte differentiation. This overview highlights some of the recent progress made in understanding the molecular genetics of keratinization disorders, and presents selected, recently characterized cases as representative of different forms of heritable ichthyosis

Dystrophic Epidermolysis Bullosa: COL7A1 Mutation Landscape in a Multi-Ethnic Cohort of 152 Extended Families with High Degree of Customary Consanguineous Marriages

Dystrophic epidermolysis bullosa is a heritable skin disease manifesting with sub-lamina densa blistering, erosions, and chronic ulcers. COL7A1, encoding type VII collagen, has been identified as the candidate gene for dystrophic epidermolysis bullosa. In this study, we have identified COL7A1 mutations in a large multi-ethnic cohort of 152 extended Iranian families with high degree of consanguinity. The patients were diagnosed by clinical manifestations, histopathology, and immunoepitope mapping. Mutation detection consisted of a combination of single nucleotide polymorphism-based whole-genome homozygosity mapping, Sanger sequencing, and gene-targeted next-generation sequencing. A total of 104 distinct mutations in COL7A1 were identified in 149 of 152 families (98%), 56 (53%) of them being previously unreported. Ninety percent of these mutations were homozygous recessive, reflecting consanguinity in these families. Three recurrent mutations were identified in five or more families, and haplotype analysis suggested a founder effect in two of them. In conclusion, COL7A1 harbored mutations in the overwhelming majority of patients with dystrophic epi-dermolysis bullosa, and most of them in this Iranian cohort were consistent with autosomal recessive inheri-tance. The mutation profile attests to the impact of consanguinity in these families

Gene-Targeted Next Generation Sequencing Identifies PNPLA1 Mutations in Patients with a Phenotypic Spectrum of Autosomal Recessive Congenital Ichthyosis: The Impact of Consanguinity

Heritable forms of ichthyoses, also referred to as generalized Mendelian disorders of cornification, are phenotypically a highly heterogeneous group of conditions caused by mutations in a number of genes playing a role in keratinocyte differentiation and epidermal barrier function (Baden and Digiovanna, 2013; Schmuth et al., 2013). These diseases are characterized by scaling and hyperkeratosis with associated cutaneous and extracutaneous features. This group of disorders is also genetically heterogeneous, with autosomal dominant, autosomal recessive, and X-linked inheritance being described. A specific subgroup of inherited ichthyoses is the autosomal recessive congenital ichthyosis (ARCI), with many newborns presenting as collodion babies, but the subsequent clinical presentation and the spectrum of severity can be highly variable (Richard and Bale, 2014). In the most severe forms, such as harlequin ichthyosis, the disease is often fatal during the early postnatal period, whereas at the other end of the continuum of the spectrum, the disease may present with a relatively mild scaling and variable degree of palmoplantar keratoderma. There is considerable genetic heterogeneity in ARCI, and as many as nine different genes are known to harbor biallelic mutations; these include TGM1, ALOXE3, ALOX12B, NIPAL4, ABCA12, CYP4F22, PNPLA1, LIPN, and CERS3. Previous reports have suggested that mutations in TGM1 account for 30e65% of patients with ARCI, whereas mutations in LIPN, PNPLA1, and CERS3 have been reported only in a few consanguineous families (Richard and Bale, 2014). With the advent of next generation sequencing (NGS), there has been tremendous progress in facilitating the mutation detection in various heritable skin disorders, including ichthyosis (South et al., 2015; Takeichi et al., 2013). In fact, at least 38 different genes have now been suggested to be associated with the ichthyotic phenotypes, either as the primary mutated genes or modifying the phenotypic presentation. To elucidate the genetic basis of ichthyosis in Iran, a country of approximately 80 million people with high prevalence of customary consanguineous marriages, we developed a gene-targeted NGS array consisting of 38 genes reported in association with ichthyosis phenotypes. Identification of specific mutations in a large number of families has allowed us to examine phenotype/genotype correlations with respect to both intra- and interfamilial heterogeneity, in part because of extensive consanguinity in these families. In this study, we identified six distinct and, to our knowledge, previously unreported mutations in the PNPLA1 gene in nine families

Hypotrichosis with juvenile macular dystrophy: Combination of whole-genome sequencing and genome-wide homozygosity mapping identifies a large deletion in CDH3 initially undetected by whole-exome sequencing-A lesson from next-generation sequencing.

BACKGROUND: Hypotrichosis with juvenile macular dystrophy (HJMD) is an autosomal recessive disorder characterized by abnormal growth of scalp hair and juvenile macular degeneration leading to blindness. We have explored the genetic basis of HJMD in a large consanguineous family with 12 affected patients, 1-76 years of age, with characteristic phenotypes. METHODS: We first applied genome-wide homozygosity mapping to 10 affected individuals for linkage analysis to identify the genomic region of the defective gene. All affected individuals shared a 7.2 Mb region of homozygosity on chromosome 16q21-22.3, which harbored 298 genes, including CDH3, previously associated with HJMD. However, whole-exome sequencing (WES) failed to identify the causative mutation in CDH3. RESULTS: Further investigation revealed a missense variant in a gene closely linked to CDH3 (1.4 Mb distance: FHOD1: c.1306A\u3eG, p.Arg436Gly). This variant was homozygous in all affected individuals and heterozygous in 18 out of 19 obligate carriers. While this variant was found by bioinformatics predictions to be likely pathogenic, a knock-in mouse for this variant, made by the CRISPR/Cas, showed no disease phenotype. However, using whole-genome sequencing (WGS), we were able to identify a novel Alu recombination-mediated deletion in CDH3:c.del161-811_246 + 1,044. CONCLUSION: WGS was able to identify a deep intronic deletion mutation, not detected by WES

Recessive mutation in tetraspanin CD151 causes Kindler syndrome-like epidermolysis bullosa with multi-systemic manifestations including nephropathy

Epidermolysis bullosa (EB) is caused by mutations in as many as 19 distinct genes. We have developed a next-generation sequencing (NGS) panel targeting genes known to be mutated in skin fragility disorders, including tetraspanin CD151 expressed in keratinocytes at the dermal-epidermal junction. The NGS panel was applied to a cohort of 92 consanguineous families of unknown subtype of EB. In one family, a homozygous donor splice site mutation in CD151 (NM_139029; c.351 + 2T > C) at the exon 5/intron 5 border was identified, and RT-PCR and whole transcriptome analysis by RNA-seq confirmed deletion of the entire exon 5 encoding 25 amino acids. Immunofluorescence of proband's skin and Western blot of skin proteins with a monoclonal antibody revealed complete absence of CD151. Transmission electron microscopy showed intracellular disruption and cell-cell dysadhesion of keratinocytes in the lower epidermis. Clinical examination of the 33-year old proband, initially diagnosed as Kindler syndrome, revealed widespread blistering, particularly on pretibial areas, poikiloderma, nail dystrophy, loss of teeth, early onset alopecia, and esophageal webbing and strictures. The patient also had history of nephropathy with proteinuria. Collectively, the results suggest that biallelic loss-of-function mutations in CD151 underlie an autosomal recessive mechano-bullous disease with systemic features. Thus, CD151 should be considered as the 20th causative, EB-associated gene

Whole Transcriptome-Based Skin Virome Profiling in Typical Epidermodysplasia Verruciformis Reveals α-, β-, and γ-HPV Infections

HPVs are DNA viruses include approximately 450 types that are classified into 5 genera (α-, β-, γ-, μ-, and ν-HPV). The γ- and β-HPVs are present in low copy numbers in healthy individuals; however, in patients with an inborn error of immunity, certain species of β-HPVs can cause epidermodysplasia verruciformis (EV), manifesting as recalcitrant cutaneous warts and skin cancer. EV presents as either typical or atypical. Manifestations of typical EV are limited to the skin and are caused by abnormal keratinocyte-intrinsic immunity to β-HPVs due to pathogenic sequence variants in TMC6, TMC8, or CIB1. We applied a transcriptome-based computational pipeline, VirPy, to RNA extracted from normal-appearing skin and wart samples of patients with typical EV to explore the viral and human genetic determinants. In 26 patients, 9 distinct biallelic mutations were detected in TMC6, TMC8, and CIB1, 7 of which are previously unreported to our knowledge. Additionally, 20 different HPV species, including 3 α-HPVs, 16 β-HPVs, and 1 γ-HPV, were detected, 8 of which are reported here for the first time to our knowledge in patients with EV (β-HPV-37, -47, -80, -151, and -159; α-HPV-2 and -57; and γ-HPV-128). This study expands the TMC6, TMC8, and CIB1 sequence variant spectrum and implicates new HPV subtypes in the pathogenesis of typical EV

Investigating the Molecular Genetics and Viral Repertoire of Patients with Epidermodysplasia verruciformis

Epidermodysplasia verruciformis (EV) is a rare autosomal recessive skin disorder with a phenotype conditional on human papillomavirus (HPV). HPV infections are primarily universal and asymptomatic in the general population; however, in individuals with EV, they lead to the development of flat-like warts and red/brownish papules or pityriasis versicolor-like skin lesions, most often from childhood onwards. Most patients eventually develop non-melanoma skin cancer, mainly in areas of sun-exposed skin, later in life. Less than 500 cases with confirmed diagnoses have been characterized. At least half of the cases of typical EV (skin-limited manifestations) are caused by bi-allelic loss-of-function mutations in TMC6/EVER1 or TMC8/EVER2 or CIB1. EVER1, EVER2, and CIB1 form a complex, and the cellular and molecular basis of disease in CIB1-TMC/EVER-deficient patients is poorly understood. A defect of keratinocyte-intrinsic immunity to HPV (mostly β form) is suspected. Indeed, these patients are not susceptible to other infectious diseases and have apparently normal leukocyte development. In contrast, patients with an atypical form of EV due to inborn errors of T-cell immunity invariably develop clinical symptoms of EV in the context of other infectious diseases. The features of the typical and atypical forms of EV thus suggest that the control of β-HPV infections requires both EVER1/EVER2-dependent keratinocyte-intrinsic immunity and T cell-dependent adaptive immunity. This study aimed to analyze, in greater depth, the molecular and cellular basis of EV in an exceptionally large cohort of 50 patients with an initial diagnosis of EV. We performed a systematic, stepwise process of sequencing by total RNA sequencing (RNA-Seq). We modified existing bioinformatic pipelines for mutation detection from RNA-Seq and significantly increased the diagnostic rate. Homozygosity mapping was embedded into our pipelines to add another level of confidence in identifying pathogenic variants. The utilization of RNA-Seq as a first-tier diagnostic method in this study allowed us to profile the transcriptome for the consequences of variants of unknown significance (VUS) on the mRNA splicing process, providing information on altered gene expression and, in particular, profile the existing viruses with tropism to the skin such as HPVs. We developed a sequencing-based method called VirPy, an automated pipeline for concurrent detection of 926 viruses, including more than 400 HPVs, and corresponding human mutations. Identification of mutations will assist in genetic counseling, particularly in the case of multiplex consanguineous families with high recurrence risk, as illustrated by the extensive family pedigrees in our study. Knowledge of the mutations also provides basis for prenatal testing through invasive chorionic villus sampling, non-invasive fetal DNA analysis in the maternal circulation, or through preimplantation genetic testing. Additionally, these genetic results allow for the application of allele-specific therapies