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Allele-Specific Small Interfering RNA Corrects Aberrant Cellular Phenotype in Keratitis-Ichthyosis-Deafness Syndrome Keratinocytes.

ABSTRACT: Keratitis-ichthyosis-deafness (KID) syndrome is a severe, untreatable condition characterized by ocular, auditory, and cutaneous abnormalities, with major complications of infection and skin cancer. Most cases of KID syndrome (86%) are caused by a heterozygous missense mutation (c.148G>A, p.D50N) in the GJB2 gene, encoding gap junction protein Cx26, which alters gating properties of Cx26 channels in a dominant manner. We hypothesized that a mutant allele-specific small interfering RNA could rescue the cellular phenotype in patient keratinocytes (KCs). A KID syndrome cell line (KID-KC) was established from primary patient KCs with a heterozygous p.D50N mutation. This cell line displayed impaired gap junction communication and hyperactive hemichannels, confirmed by dye transfer, patch clamp, and neurobiotin uptake assays. A human-murine chimeric skin graft model constructed with KID-KCs mimicked patient skin in vivo, further confirming the validity of these cells as a model. In vitro treatment with allele-specific small interfering RNA led to robust inhibition of the mutant GJB2 allele without altering expression of the wild-type allele. This corrected both gap junction and hemichannel activity. Notably, allele-specific small interfering RNA treatment caused only low-level off-target effects in KID-KCs, as detected by genome-wide RNA sequencing. Our data provide an important proof-of-concept and model system for the potential use of allele-specific small interfering RNA in treating KID syndrome and other dominant genetic conditions.

SUBMITTER: Lee MY

PROVIDER: S-EPMC7395648 | biostudies-literature |

REPOSITORIES: biostudies-literature

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RNA-sequencing-based off-target analysis of allele-specific siRNA in keratitis-ichthyosis-deafness syndrome patient-derived keratinocytes

Project description:Purpose: RNA-sequencing (RNA-Seq)-based transcriptomic analysis has facilitated identification of off-target effects of drug of interest. The goals of this study are to investigate off-target effects of the therapeutic, allele-specific siRNA, S7. Methods: mRNA profiles of KID syndrome patient-derived keratinocytes (with c.148G>A mutation in GJB2) with or without S7 treatment were generated by RNA-Seq, in triplicate, using an Illumina NextSeq 500 instrument. The sequence reads that passed quality filters were processed using RNA-STAR (v2.5b) and BioConductor SARTools package (v1.3.2). Real-time qRT–PCR validation was performed using SYBR Green. Results: We mapped about 15 million sequence reads per sample to the human genome (build hg38) and identified expression of 19,667 genes in the samples. Only 6 genes (MMP10, MMP1, NSA2, AFAP1L1, GLB1L2, GPR137) showed differential expression between the treated and untreated cells, with |log2 fold change| ≥ 1 and adjusted p-value (padj) <0.05. Altered expression of the top 10 genes (TMEM109, MMP9, ANGPTL4, CXCL5, in addition to the 6 genes above) was confirmed using real-time qRT–PCR. Conclusions: Our RNA-Seq findings suggest low-level off-target effects of S7, the allele-specific siRNA targeted to the most common GJB2 mutant associated with KID syndrome.

2022-05-23 | GSE131709 | GEO

Keratitis-ichthyosis-deafness syndrome and hidradenitis suppurativa.

Project description: Not available

| S-EPMC10404597 | biostudies-literature

Pathological hemichannels associated with human Cx26 mutations causing Keratitis-Ichthyosis-Deafness syndrome.

Project description:Connexin (Cx) proteins form intercellular gap junction channels by first assembling into single membrane hemichannels that then dock to connect the cytoplasm of two adjacent cells. Gap junctions are highly specialized structures that allow the direct passage of small molecules between cells to maintain tissue homeostasis. Functional activity of nonjunctional hemichannels has now been shown in several experimental systems. Hemichannels may constitute an important diffusional exchange pathway with the extracellular space, but the extent of their normal physiological role is currently unknown. Aberrant hemichannel activity has been linked to mutations of connexin proteins involved in genetic diseases. Here, we review a proposed role for hemichannels in the pathogenesis of Keratitis-Ichthyosis-Deafness (KID) syndrome associated with connexin26 (Cx26) mutations. Continued functional evaluation of mutated hemichannels linked to human hereditary disorders may provide additional insights into the mechanisms governing their regulation in normal physiology and dysregulation in disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

| S-EPMC3245810 | biostudies-literature

More than keratitis, ichthyosis, and deafness: Multisystem effects of lethal GJB2 mutations.

Project description:BackgroundInfant death in keratitis-ichthyosis-deafness (KID) syndrome is recognized; its association with specific genotypes and pathophysiology is inadequately understood.ObjectiveWe sought to discover characteristics that account for poor outcomes in lethal KID syndrome.MethodsWe collected 4 new cases and 9 previously reported, genotyped cases of lethal KID syndrome. We performed new molecular modeling of the lethal mutants GJB2 p.A88V and GJB2 p.G45E.ResultsInfant death occurred in all patients with GJB2 p.G45E and p.A88V; it is unusual with other GJB2 mutations. Early death with those 2 "lethal" mutations is likely multifactorial: during life all had ≥1 serious infection; most had poor weight gain and severe respiratory difficulties; many had additional anatomic abnormalities. Structural modeling of GJB2 p.G45E identified no impact on the salt bridge previously predicted to account for abnormal central carbon dioxide sensing of GJB2 p.A88V.LimitationsThis clinical review was retrospective.ConclusionGJB2 p.G45E and p.A88V are the only KID syndrome mutations associated with uniform early lethality. Those electrophysiologically severe mutations in GJB2 reveal abnormalities in many organs in lethal KID syndrome. All patients with KID syndrome may have subtle abnormalities beyond the eyes, ears, and skin. Early genotyping of KID syndrome births will inform prognostic discussion.

| S-EPMC6372339 | biostudies-literature

Missense mutations in GJB2 encoding connexin-26 cause the ectodermal dysplasia keratitis-ichthyosis-deafness syndrome.

Project description:Keratitis-ichthyosis-deafness syndrome (KID) is a rare ectodermal dysplasia characterized by vascularizing keratitis, profound sensorineural hearing loss (SNHL), and progressive erythrokeratoderma, a clinical triad that indicates a failure in development and differentiation of multiple stratifying epithelia. Here, we provide compelling evidence that KID is caused by heterozygous missense mutations in the connexin-26 gene, GJB2. In each of 10 patients with KID, we identified a point mutation leading to substitution of conserved residues in the cytoplasmic amino terminus or first extracellular domain of Cx26. One of these mutations was detected in six unrelated sporadic case subjects and also segregated in one family with vertical transmission of KID. These results indicate the presence of a common, recurrent mutation and establish its autosomal dominant nature. Cx26 and the closely related Cx30 showed differential expression in epidermal, adnexal, and corneal epithelia but were not significantly altered in lesional skin. However, mutant Cx26 was incapable of inducing intercellular coupling in vitro, which indicates its functional impairment. Our data reveal striking genotype-phenotype correlations and demonstrate that dominant GJB2 mutations can disturb the gap junction system of one or several ectodermal epithelia, thereby producing multiple phenotypes: nonsyndromic SNHL, syndromic SNHL with palmoplantar keratoderma, and KID. Decreased host defense and increased carcinogenic potential in KID illustrate that gap junction communication plays not only a crucial role in epithelial homeostasis and differentiation but also in immune response and epidermal carcinogenesis.

| S-EPMC447609 | biostudies-literature

Alterations in connexin 26 protein structure from lethal keratitis-ichthyosis-deafness syndrome mutations A88V and G45E.

Project description: Not available

| S-EPMC7263394 | biostudies-literature

Heterozygous p.Asp50Asn mutation in the GJB2 gene in two Cameroonian patients with keratitis-ichthyosis-deafness (KID) syndrome.

Project description:Keratitis-Ichthyosis-Deafness (KID) syndrome (OMIM 148210) is a congenital ectodermal defect that consists of an atypical ichthyosiform erythroderma associated with congenital sensorineural deafness. KID appears to be genetically heterogeneous and most cases are caused by GJB2 mutations. Mutations in African patients have been rarely described.We report on two unrelated Cameroonian individuals affected with sporadic KID, presenting with the classic phenotypic triad. The two patients were heterozygous for the most frequent p.Asp50Asn mutation. This first report in patients from sub-Saharan African origin supports the hypothesis that the occurrence of KID due to p.Asp50Asn mutation in GJB2 seems not to be population specific.Our finding has implication in medical genetic practice, specifically in the molecular diagnosis of KID in Africans. These cases also reveal and emphasize the urgent need to develop appropriate policies to care for patients with rare/orphan diseases in Sub-Saharan Africa, as many of these cases become more and more recognizable.

| S-EPMC3750395 | biostudies-literature

Connexin 26 (GJB2) mutation in an Argentinean patient with keratitis-ichthyosis-deafness (KID) syndrome: a case report.

Project description:BackgroundKeratitis-Ichthyosis-Deafness (KID) syndrome is a rare condition characterized by pre-lingual sensorineural deafness with skin hyperkeratinization. The primary cause of the disease is a loss-of-function mutation in the GJB2 gene. Mutations in Argentinean patients have not been described.Case presentationWe studied a 2 year-old boy with bilateral congenital sensorineural deafness with dry skin over the entire body, hypotrichosis of the scalp, thin and light-blond hair. Analysis of the GJB2 gene nucleotide sequence revealed the substitution of guanine-148 by adenine predicted to result in an Asp50Asn amino acid substitution.ConclusionThis is the first KID report in a patient from Argentina. This de novo mutation proved to be the cause of keratitis-ichthyosis-deafness syndrome (KID-syndrome) in the patient, and has implications in medical genetic practice.

| S-EPMC4855445 | biostudies-literature

RNA-sequencing-based off-target analysis of allele-specific siRNA in keratitis-ichthyosis-deafness syndrome patient-derived keratinocytes

Project description:RNA-sequencing-based off-target analysis of allele-specific siRNA in keratitis-ichthyosis-deafness syndrome patient-derived keratinocytes

| PRJNA544483 | ENA

Antibody gene transfer treatment drastically improves epidermal pathology in a keratitis ichthyosis deafness syndrome model using male mice.

Project description:BackgroundKeratitis ichthyosis deafness (KID) syndrome is a rare disorder caused by hemichannel (HC) activating gain-of-function mutations in the GJB2 gene encoding connexin (Cx) 26, for which there is no cure, or current treatments based upon the mechanism of disease causation.MethodsWe applied Adeno Associated Virus (AAV) mediated mAb gene transfer (AAVmAb) to treat the epidermal features of KID syndrome with a well-characterized HC blocking antibody using male mice of a murine model that replicates the skin pathology of the human disease.FindingsWe demonstrate that in vivo AAVmAb treatment significantly reduced the size and thickness of KID lesions, in addition to blocking activity of mutant HCs in the epidermis in vivo. We also show that AAVmAb treatment eliminated abnormal keratinocyte proliferation and enlarged cell size, decreased apoptosis, and restored the normal distribution of keratin expression.InterpretationOur findings reinforce the critical role played by increased HC activity in the skin pathology associated with KID syndrome. They also underscore the clinical potential of anti-HC mAbs coupled with genetic based delivery systems for treating the underlying mechanistic basis of this disorder. Inhibition of HC activity is an ideal therapeutic target in KID syndrome, and the genetic delivery of mAbs targeted against mutant HCs could form the basis of new therapeutic interventions to treat this incurable disease.FundingFondazione Telethon grant GGP19148 and University of Padova grant Prot. BIRD187130 to FM; Foundation for Ichthyosis and Related Skin Types (FIRST) and National Institutes of Health grant EY 026911 to TWW.

| S-EPMC9926223 | biostudies-literature

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