Keratitis-ichthyosis-deafness Syndrome Research Articles

O18 Precision genome editing for targeted correction of pathogenic D50N mutation in keratitis–ichthyosis–deafness syndrome using CRISPR/Cas9 and homology-directed repair

Abstract Introduction and aims Keratitis–ichthyosis–deafness (KID) syndrome is an ectodermal disorder that causes blindness, skin inflammation and deafness. It is caused by autosomal dominant mutations in the gap junction beta 2 (GJB2) gene with 86% being a hotspot mutation on c.148G>A, resulting in the amino acid replacement (D50N) in its coding protein connexin 26 (Cx26). There is currently no curative treatment for KID syndrome. We aim to correct the hotspot mutation D50N using a genome-editing approach. Methods Keratinocytes generated from the patient with KID who had a D50N mutation (KID-KCs) were genetically corrected by homologous-directed repair/CRISPR-Cas9 using an electroporation approach. The correction efficiency was determined and analysed using Sanger sequencing and Synthego Inference of CRISPR Edits analysis. The off-target effects were confirmed by whole-exome sequencing [exome next-generation sequencing (NGS)]. Furthermore, the functional recovery following genome editing was assessed by GJB2 mRNA production, Cx26 protein expression, and trafficking and hemichannel activity. Results The correction efficiencies ranged from 95% to 100% with different doses of CRISPR-Cas9 and 8µg CRISPR-Cas9 was the most optimal concentration with the efficiency of 100% and approximately 0% insertion-deletion (INDEL). Fifty days after gene editing, the INDEL increased to only 2%, indicating a minimum residual CRISPR-Cas9 effect in edited cells. The exome-NGS analysis also showed no off-target effects in the outside target area. Functional studies showed that there was a significant increase in wildtype GJB2 mRNA expression and a reduction of mutant GJB2 expression (n = 5, P < 0.01). Increased Cx26 membranous localization in KID-KCs was also observed (n = 3, P < 0.01). Additionally, Cx26 hemichannel activity assessment using the neurobiotin assay showed a significant decrease in ‘leaky’ hemichannel in gene-edited KID-KCs compared with unedited cells (n = 3, P < 0.05). Conclusions We can genome-correct KID keratinocytes harbouring the mutation D50N with 100% editing efficiency, 0–2% INDEL, and undetectable off-target effects. This result indicates a promising gene therapy for KID syndrome with the hotspot mutation D50N.

Suspect dyskeratotic neoformations in a 7-year-old child with keratitis-ichthyosis-deafness syndrome: diagnostic, surgical and wound care management

Keratitis-ichthyosis-deafness syndrome (KID) is a rare genetic disorder characterized by the triad of hyperkeratosis, ichthyosis, and congenital prelingual sensorineural deafness, with less than 100 cases described in the literature. In addition to many other extra-cutaneous manifestations, these patients are burdened by two principal increased risk factors involving the skin: the risk of developing infections and the risk of developing malignant skin tumors, especially Squamous Cell Carcinoma and Trichilemmal tumors. We present the case of a 7-year-old girl with a unique genetic variant described to date, who developed 4 dyskeratotic neoformation. Since the dermatoscopic examination was not sufficient to exclude malignancy, excisional and punch biopsies were used. Nonetheless, healing was challenging, with wound dehiscence and infection onset. Ad hoc wound care management resorting to negative pressure therapy and advanced medical honey-based dressings was necessary to achieve complete wound healing.

High rate of molecular diagnosis among individuals with alopecia with known or suspected inborn errors of immunity

Background and aimsAlopecia is a multifactorial disorder causing hair loss of the scalp, face, and/or body. It affects an estimated 0.1–0.2% of individuals worldwide and can be visually stigmatizing and have significant quality of life consequences. While most cases of alopecia lack an identifiable genetic explanation, this condition is enriched in individuals with Mendelian inborn errors of immunity (IEI). The purpose of this study was to determine Mendelian contributions to alopecia in a mixed cohort of patients with suspected IEI. MethodsThe cohort was comprised of 3,072 unrelated participants with suspected IEI or related inflammatory disorders who underwent exome and/or genome sequencing from 2017–2022. From this pool, we identified 110 (3.58%) probands with alopecia. ResultsProbands were predominantly adult (mean 37.2 years, range 4–76), female (67%), and of European ancestry (64.5%). Alopecia was characterized as universalis (3/110, 2.7%), totalis (4/110, 3.6%), areata (5/110, 4.5%), scarring alopecia of the scalp (2/110, 1.8%), and other or not otherwise specified (96/100, 87.3%). Overall, 33/110 probands (30.0%) were found to have a Mendelian molecular diagnosis. Almost half of the participants with molecular diagnoses (15/33, 45%) had biallelic defects in AIRE, consistent with a molecular diagnosis of autoimmune polyendo-crinopathy-candidiasis-ectodermal dystrophy (APECED). In addition, 4/33 (12.1%) had a molecular diagnosis of STAT3-related hyper-IgE syndrome, 2/33 (6.1%) had STAT1 deficiency, and 2/33 (6.1%) had RAG1 deficiency. The remaining 10/33 (30.3%) included defects in the following genes: CXCR4, FAM111B, FOXN1, GATA2, IL2RG, GJB2, NFKB2, PIK3CD, PLCG2, and PSMB8. Multiple linear regression found that when controlling for sex and ancestry, younger age and greater phenotypic complexity was significantly associated with the likelihood of receiving a molecular diagnosis related to alopecia (OR = 0.93, p-value = 0.004 and OR = 1.27, p-value = 0.034, respectively). ConclusionsIn summary, a high proportion of individuals with alopecia in an IEI cohort received a molecular diagnosis via WES/WGS. Further, there was a small but significant association between younger age and higher phenotypic complexity and likelihood of receiving a molecular diagnosis. Most molecular diagnoses for alopecia in this cohort were disorders of the immune system, while two (hereditary fibrosing poikiloderma and keratitis-ichthyosis deafness syndrome) involved primarily non-immune etiologies.

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

Keratitis 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. We 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. We demonstrate that invivo AAVmAb treatment significantly reduced the size and thickness of KID lesions, in addition to blocking activity of mutant HCs in the epidermis invivo. We also show that AAVmAb treatment eliminated abnormal keratinocyte proliferation and enlarged cell size, decreased apoptosis, and restored the normal distribution of keratin expression. Our 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. Fondazione 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.

Keratitis-ichthyosis-deafness Syndrome with Heterozygous p.D50N in the GJB2 Gene in Two Serbian Adult Patients.

Keratitis-ichthyosis-deafness (KID) syndrome is a rare congenital ectodermal dysplastic syndrome presenting with keratitis, ichthyosis and sensorineural hearing loss. The most common causes of KID syndrome are heterozygous missense mutations in the GJB2 gene that codes for connexin 26. During the ophthalmological examination, two adult females complained of recent worsening of visual acuity in both eyes. Anamnesis revealed that their eyes were red and irritated from early childhood onwards. Both of them had thickening and keratinisation of eyelid margins, lash loss, diffuse opacification of cornea and conjunctiva caused by keratinisation of eye surface, superficial and deep corneal vascularisation and corneal oedema. Partial sensorineural hearing loss and difficulties in speech were also noted along with typical ichthyosiform erythroderma. Genetic testing of the GJB2 gene revealed a heterozygous p.D50N mutation in both patients.Patients were treated with a combined topical corticosteroid and artificial tears therapy, with steroid therapy being intensified during the last month. The therapy increased the visual acuity by decreasing corneal oedema and by forming a more regular air-tear interface during the six months follow up. Subsequently, the disease progressed despite the continuation of the therapy. This is the first report of Serbian patients with KID syndrome. Despite the administration of the combined topical corticosteroid and artificial tears therapy the disease is relentlessly progressive and therapeutic success of ophthalmological signs with local therapeutic modalities used so far had been disappointing.

Expression of KID syndromic mutation Cx26S17F produces hyperactive hemichannels in supporting cells of the organ of Corti.

Some mutations in gap junction protein Connexin 26 (Cx26) lead to syndromic deafness, where hearing impairment is associated with skin disease, like in Keratitis Ichthyosis Deafness (KID) syndrome. This condition has been linked to hyperactivity of connexin hemichannels but this has never been demonstrated in cochlear tissue. Moreover, some KID mutants, like Cx26S17F, form hyperactive HCs only when co-expressed with other wild-type connexins. In this work, we evaluated the functional consequences of expressing a KID syndromic mutation, Cx26S17F, in the transgenic mouse cochlea and whether co-expression of Cx26S17F and Cx30 leads to the formation of hyperactive HCs. Indeed, we found that cochlear explants from a constitutive knock-in Cx26S17F mouse or conditional in vitro cochlear expression of Cx26S17F produces hyperactive HCs in supporting cells of the organ of Corti. These conditions also produce loss of hair cells stereocilia. In supporting cells, we found high co-localization between Cx26S17F and Cx30. The functional properties of HCs formed in cells co-expressing Cx26S17F and Cx30 were also studied in oocytes and HeLa cells. Under the recording conditions used in this study Cx26S17F did not form functional HCs and GJCs, but cells co-expressing Cx26S17F and Cx30 present hyperactive HCs insensitive to HCs blockers, Ca2+ and La3+, resulting in more Ca2+ influx and cellular damage. Molecular dynamic analysis of putative heteromeric HC formed by Cx26S17F and Cx30 presents alterations in extracellular Ca2+ binding sites. These results support that in KID syndrome, hyperactive HCs are formed by the interaction between Cx26S17F and Cx30 in supporting cells probably causing damage to hair cells associated to deafness.

Keratitis Ichthyosis Deafness Syndrome and Communication Disorders: A Case Report

Keratitis-Ichthyosis Deafness (KID) syndrome is an atypical genetic, multi-system disorder characterised by defects of corneas’ surface, thickened plaques of skin and sensorineural hearing impairment. As it is a multifaceted syndrome, children with KID require a comprehensive examination of all the affected systems, including continuous monitoring of speech and language abilities, as it is associated with sensorineural hearing impairment. However, the KID syndrome being a rare condition, the speech, language and hearing abilities are less explored. Thus, it is attempted to portray those speech, language and hearing abilities affected in them by describing a case report of a 5-year old child diagnosed with KID syndrome. This case report also highlighted on the importance of early identification and rehabilitation in such cases.

The Enigma of a Keratitis-Ichthyosis-Deafness with Silence, Scaling, and Related Syndrome

Abstract Ichthyosis is a group of genetic disorders characterized by generalized dry skin and scaling, and it can be frequently associated with erythroderma. Here, we report a case of ichthyosis associated with a rare syndrome. A 6-year-old congenitally deaf female presented with severe scaling and generalized erythema for 6 months which began with a scaly plaque over the face at the age of 18 months and gradually covered the whole body. Three similar episodes happened after that with a gap of 6 months. There were multiple scaly erythematous plaques present over the trunk and limbs and thick hyperkeratotic plaques were present in axillary and inguinal folds. The scalp hairs were sparse. Histopathology showed psoriasiform changes. Salivary sample sent for genetic analysis revealed an heterozygous mutation c.42C>G (p.Asn14Lys) in GJB2 gene that is known to harbor pathogenic mutations associated with keratitis-ichthyosis-deafness (KID) syndrome. KID syndrome presents with the triad of keratitis, progressive erythrokeratoderma, and hearing loss.

The Hurried Child Syndrome: An Overview

A syndrome described as "hurried child syndrome"—a combination of stress-related behaviors—may develop when a child's parents expect performance levels much above his or her mental, social, or emotional capacity. In essence, parents overbook their children's calendars, put pressure on them to succeed academically, and expect them to act and react like young adults. The current situation of trade is another factor. Many people that market to children today are aware that their parents no longer have complete control over what their children eat, wear, or engage in for entertainment. The consumerist economy is taking advantage of this reality, and it affects children. The following statistics serve as an example of the "hurried child syndrome": The suicide and homicide rates have increased by three times over the previous 20 years, childhood obesity has increased by half, and 15 to 25 percent of young children are "failing" kindergarten, according to recent research on the effects of rushed kid syndrome. In an effort to "manage" them better at home and at school, millions of children take drugs. According to recent data, 70% of virgin girls will not remain virgins by the time they reach maturity, and 40% of adolescent girls who engage in sexual activity will become pregnant by then. Addiction to drugs and alcohol is the main cause of death for young people today, but suicide rates are also rising. Every year, 5,000 teenagers take their own lives.

A Brief Review on Hurried Child Syndrome

When a kid's parents demand performance levels much beyond his or her mental, social, or emotional capacity, the result might be a syndrome known as "hurried child syndrome"—a collection of stress-related behaviors. In essence, parents overbook their kids' calendars, pressure them to do well in school, and demand that they behave and respond like small adults. Another factor is the state of trade at the moment. Nowadays, a lot of people that market to children are aware that their parents no longer control what kids consume for food, clothing, or entertainment. This reality is being exploited by the consumerist system, and it has an impact on kids. The hurried child syndrome is represented by the following statistics: Recent research on the effects of rushed kid syndrome shows that the suicide and homicide rates have tripled over the past 20 years, childhood obesity has grown by 50%, and 15–25% of young children are "failing" kindergarten. Millions of children receive medications in an effort to "manage" them better at home and in school. According to recent statistics, 40% of adolescent girls who engage in sexual activity will become pregnant by the time they reach maturity, and 70% of virgin girls will not be virgins. The leading cause of mortality for youths today is drug and alcohol abuse, but suicide rates are also rising. Five thousand youngsters commit suicide each year.

Ichthyosis follicularis syndromes in patients with mutations in GJB2.

Ichthyosis follicularis (IF) manifests as generalized spiny follicular projections found in syndromic diseases secondary to SREBF1 and MBTPS2 mutations. We sought the genetic cause of IF in two distinct families from a cohort of 180 patients with ichthyosis. In Family 1, the proband (Patient 1) presented with IF, bilateral sensorineural hearing loss and punctate palmoplantar keratoderma. Using DNA from peripheral blood lymphocytes, two compound heterozygous mutations, c.526A>G and c.35delG, were discovered in GJB2. In Family 2, the proband (Patient 2) presented with a previously unreported IF phenotype in the context of keratitis-ichthyosis-deafness syndrome, and whole-exome sequencing found a de novo heterozygous mutation, c.148G>A in GJB2. Histopathology was consistent with porokeratotic eccrine ostial and dermal duct naevus (PEODDN) and IF in Patients 1 and 2, respectively. Our findings add to the clinical and histopathological spectrum of IF and emphasize the association of PEODDN-like entities with GJB2 variants.

Phenotypic spectrum of autosomal recessive Keratitis-Ichthyosis-Deafness Syndrome (KIDAR) due to mutations in AP1B1

Inborn errors in copper metabolism result in a diverse set of abnormalities such as Wilson disease and MEDNIK syndrome. Homozygous pathogenic variants in AP1B1 lead to KIDAR (Keratitis-Ichthyosis-Deafness Syndrome). The main phenotypic features of KIDAR are ichthyosis, keratitis, erythroderma, and progressive hearing loss accompanied by developmental delay and failure to thrive. Herein, we describe a six-and-a-half-year-old boy with KIDAR caused by a novel pathogenic variant in AP1B1 (NM_001127.4:c.1263C > A, p.Tyr421*). The proband presented with ichthyosis, erythroderma, palmoplantar keratoderma, hearing loss, and corneal scarring. He also had hypotonia, global developmental delay, and photophobia. Lastly, we review all of the previously reported cases and the clinical features associated with KIDAR.

Клиническое наблюдение ребенка с КИД (Кератит — Ихтиоз — Глухота) синдромом

Приведен клинический случай кератита — ихтиоза — глухоты (КИД-синдром) у ребенка грудного возраста. Статья знакомит врачей-педиатров и семейных врачей с трудностями диагностики этой редкой генетической патологии у детей грудного возраста.

Keratitis-Ichthyosis-Deafness Syndrome Caused by Missense Mutation in GJB2 Encoding Connexin 26 in a Chinese Patient

Introduction: Keratitis-ichthyosis-deafness (KID) syndrome is a rare congenital ectodermal disorder caused by a heterozygous missense mutation in GJB2 or GJB6, which encode connexin 26 and connexin 30, respectively. To date, three cases of KID syndrome caused by an N14Y mutation of GJB2 have been reported globally, but there is no relevant report in domestic literature. Case presentation: A 36-year-old man presented with scalp alopecia and absence of eyelashes, pubic hair, and axillary hair. Yellow-brown highly keratinized verrucous plaques were present on the perineum, palms, and soles. All 20 nails were severely abnormal. Keratitis, blepharitis, and profound sensorineural hearing loss were also observed. Foot biopsy revealed ichthyosis changes. Sequencing of GJB2 showed that the patient was heterozygous for a nucleotide change, c.40A > T, in exon 2, resulting in the substitution of asparagine for tyrosine at position 14 (N14Y) located in the N-terminal domain of connexin 26. Notably, the mutation site of KID syndrome has not been reported in domestic literature. The patient's father was heterozygous for the M195 V (c.583A > G) mutation, and his mother was heterozygous for the V37I (c.109G > A) mutation. The patient received oral retinoic acid, antibiotics and topical moisturizer packs, but was subsequently lost to follow-up. Discussion: KID syndrome manifests as the classic triad of chronic vascularizing keratitis, ichthyosiform erythroderma, and sensorineural hearing loss. Early and long-term multidisciplinary monitoring of affected individuals is essential. Genetic testing could help us detect the mutation site of connexin. Conclusion: These findings expand the mutation spectrum of KID syndrome in the Chinese population and provide a new understanding of the genotype–phenotype correlation. Functional studies on the N14Y mutation will help to reveal the underlying pathogenic mechanism.

KID Syndrome: A Rare Congenital Ichthyosiform Disorder.

KID Syndrome: A Rare Congenital Ichthyosiform Disorder.

Use of Systemic Mycophenolate Mofetil Therapy in Ocular Surface Inflammatory Pathologies at the Initiative and Responsibility of the Ophthalmologist.

The purpose of the study was to evaluate the efficacy and safety of systemic mycophenolate mofetil (MMF) treatment in ocular surface inflammatory diseases. For this retrospective study, patients who were treated with systemic MMF for ocular surface inflammatory diseases between March 2020 and March 2022 were evaluated. Apart from demographic data, examination notes including MMF treatment indication and systemic side effect interrogation and routine laboratory examinations during drug treatment were extracted from the patient records. Detailed staging scores were performed according to the diagnosis including Foster and Mondino for ocular mucous membrane pemphigoid (MMP) and limbal stem cell deficiency scoring for limbal transplantation. For thorough evaluation, anterior segment pictures were used. Fourteen patients were enrolled to the study, with a mean age of 58 ± 12. MMP (6, 42.8%) and limbal allograft transplantation (6, 42.8%) constituted the main indications for the MMF treatment, followed by keratitis-ichthyosis-deafness (KID) syndrome (1, 7.2%) and Mooren's ulcer (1, 7.2%). Five of six patients with MMP regressed according to both staging systems. Only one remained stable which was evaluated as Stage 3. Furthermore, while all limbal transplant groups (6) stabilized and showed regression according to the individualized limbal stem cell deficiency staging system with no rejection during follow-up. Furthermore, patients with Mooren's ulcer and KID syndrome showed control of the inflammation and stabilization after MMF treatment. No significant systemic side effects apart from constipation and nausea (3) were observed in patients whose routine laboratory tests were stable throughout the follow-up. MMF has the potential to be a valuable and safe systemic agent of first choice in the control of ocular surface inflammatory disorders, especially when topical treatment is not effective. With such studies, it is predicted that MMF may reach wider usage areas with the increase in its effectiveness and safety in its use for ocular surface inflammatory pathologies.

Connexin hemichannel inhibition ameliorates epidermal pathology in a mouse model of keratitis ichthyosis deafness syndrome

Mutations in five different genes encoding connexin channels cause eleven clinically defined human skin diseases. Keratitis ichthyosis deafness (KID) syndrome is caused by point mutations in the GJB2 gene encoding Connexin 26 (Cx26) which result in aberrant activation of connexin hemichannels. KID syndrome has no cure and is associated with bilateral hearing loss, blinding keratitis, palmoplantar keratoderma, ichthyosiform erythroderma and a high incidence of childhood mortality. Here, we have tested whether a topically applied hemichhanel inhibitor (flufenamic acid, FFA) could ameliorate the skin pathology associated with KID syndrome in a transgenic mouse model expressing the lethal Cx26-G45E mutation. We found that FFA blocked the hemichannel activity of Cx26-G45E in vitro, and substantially reduced epidermal pathology in vivo, compared to untreated, or vehicle treated control animals. FFA did not reduce the expression of mutant connexin hemichannel protein, and cessation of FFA treatment allowed disease progression to continue. These results suggested that aberrant hemichannel activity is a major driver of skin disease in KID syndrome, and that the inhibition of mutant hemichannel activity could provide an attractive target to develop novel therapeutic interventions to treat this incurable disease.

197 Revertant mosaicism as a clue for the role of keratinocytes in innate immunity against Candidaspecies

The role of epidermal keratinocytes (KC) in the innate immunity against pathogens has not been proven in human, aside from several lines of evidence in vitro. Cutaneous Candidiasis is limited to intertriginous area in immune-competent individuals. In absence of systemic immune-deficiency, however, patients with keratitis-ichthyosis-deafness (KID) syndrome caused by mutations in GJB2 coding a gap-junction protein Connexin (Cx) 26 develop chronic cutaneous Candidiasis (CCC) in extremities or trunk. A patient with KID syndrome harboring a germline heterozygous mutation in GJB2 (p.D50N) developed numerous patches of apparently normal skin only after the remission of CCC. We collected 5 revertant patches and separated the epidermis. Exome sequencing identified an additional mutation in GJB2 in each sample. These five mutations were independent to each other, suggesting emergence of independent clones harboring additional mutations in GJB2. All of the four mutations that were phase-able were in cis with D50N. Given that D50N causes hyperactivation of Cx26, these mutations are likely to dampen the effect of D50N in KC. The appearance of revertant patches indicates the impaired survival of KC with D50N in CCC, compared to revertant KC. Since revertant KC are predicted to be similar to wild type KC in the channel function of Cx26, KC with D50N can also be suspected to have impaired survival ability in CCC than wild type KC. Thus, we hypothesized that CCC in KID syndrome is based on the vulnerability of KC with germline mutations to Candida sp. We are currently addressing this hypothesis by analyzing the in vitro survival of KC with different genotypes of GJB2 under the presence of Candida sp. and establishing an in vivo model of CCC using inducible KID mouse model using K14-CreERT mice. In conclusion, we suspected the impaired survival of KC against Candida sp. in KID syndrome through the analysis of revertant patches. The presented case might be the first human phenotype showing the role of wild type KC in the resistance against Candida sp. in unaffected individuals.

Ichthyosiform Erythroderma, a Multifaceted Syndromic Entity.

Ichthyosiform Erythroderma, a Multifaceted Syndromic Entity.

Trichilemmal cyst of the neck: case report and review of the literature

Introduction: Trichilemmal cysts are lesions originating in the isthmus of the hair follicle. They are more common in women and occur in 90% on the scalp. Presentation of case: A 10-year-old female patient presented with a hard, slightly painful, nonexophytic mass in the right cervical region (Ia). After surgical resection in healthy margins, the diagnosis of trichilemmal cyst was made on histopathological examination. Discussion: The age of the patient as well as the cervical location of this type of lesion is an extremely rare presentation of trichilemmal cyst. Follow-up is rigorous in search of a triad: ichthyosis, keratosis, deafness, particularly in this patient. Conclusion: Close follow-up in this clinical case is indicated because the risk of malignant degeneration and development of similar lesions is present. Keywords: Follicular cyst; KID syndrome; Trichilemmal cyst; Neck surgery; Trichilemmal carcinoma