Amniotic membrane dressings for treatment of aplasia cutis in newborns.

BACKGROUND: Aplasia cutis congenita (ACC) is a rare congenital skin defect characterized by a focal or extensive absence of the epidermis, dermis, and occasionally, subcutaneous tissue. When the wound caused by this defect is wide or deep, various treatments are used, including skin grafting. The amniotic membrane (AM) is a biological dressing that facilitates re-epithelialization as it contains mesenchymal cells and numerous growth factors. OBJECTIVE: To report the efficacy of AM dressings in treating the skin defects of ACC. METHOD: This study was conducted on five neonates diagnosed with ACC born between 2018 and 2022, referred to the Children's Medical Center in Tehran, Iran. AM dressings were applied on wounds larger than 1 cm2. The wounds were assessed weekly and, if required, an additional AM dressing was applied. RESULTS: The skin defects gradually re-epithelialized after application of the AM. The complete healing process took around 3.5 weeks on average. No hypertrophic scarring was observed. CONCLUSION: The application of AM dressing resulted in satisfactory cosmetic outcomes, with no hypertrophic scar formation. Complete healing occurred in all cases except one. The length of the hospital stay ranged from 2 to 6 weeks, depending on the size of the wound.

Mutation update: The spectra of PLEC sequence variants and related plectinopathies.

Plectin, encoded by PLEC, is a cytoskeletal linker of intermediate filaments expressed in many cell types. Plectin consists of three main domains that determine its functionality: the N-terminal domain, the Rod domain, and the C-terminal domain. Molecular defects of PLEC correlating with the functional aspects lead to a group of rare heritable disorders, plectinopathies. These multisystem disorders include an autosomal dominant form of epidermolysis bullosa simplex (EBS-Ogna), limb-girdle muscular dystrophy (LGMD), aplasia cutis congenita (ACC), and an autosomal recessive form of EBS, which may associate with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and/or congenital myasthenic syndrome (EBS-MyS). In this study, genotyping of over 600 Iranian patients with epidermolysis bullosa by next-generation sequencing identified 15 patients with disease-causing PLEC variants. This mutation update analyzes the clinical spectrum of PLEC in our cohort and in the literature and demonstrates the relationship between PLEC genotype and phenotypic manifestations. This study has integrated our seven novel PLEC variants and phenotypic findings with previously published data totaling 116 variants to provide the most complete overview of pathogenic PLEC variants and related disorders.

Whole-Transcriptome Analysis by RNA Sequencing for Genetic Diagnosis of Mendelian Skin Disorders in the Context of Consanguinity.

BACKGROUND: Among the approximately 8000 Mendelian disorders, >1000 have cutaneous manifestations. In many of these conditions, the underlying mutated genes have been identified by DNA-based techniques which, however, can overlook certain types of mutations, such as exonic-synonymous and deep-intronic sequence variants. Whole-transcriptome sequencing by RNA sequencing (RNA-seq) can identify such mutations and provide information about their consequences. METHODS: We analyzed the whole transcriptome of 40 families with different types of Mendelian skin disorders with extensive genetic heterogeneity. The RNA-seq data were examined for variant detection and prioritization, pathogenicity confirmation, RNA expression profiling, and genome-wide homozygosity mapping in the case of consanguineous families. Among the families examined, RNA-seq was able to provide information complementary to DNA-based analyses for exonic and intronic sequence variants with aberrant splicing. In addition, we tested the possibility of using RNA-seq as the first-tier strategy for unbiased genome-wide mutation screening without information from DNA analysis. RESULTS: We found pathogenic mutations in 35 families (88%) with RNA-seq in combination with other next-generation sequencing methods, and we successfully prioritized variants and found the culprit genes. In addition, as a novel concept, we propose a pipeline that increases the yield of variant calling from RNA-seq by concurrent use of genome and transcriptome references in parallel. CONCLUSIONS: Our results suggest that "clinical RNA-seq" could serve as a primary approach for mutation detection in inherited diseases, particularly in consanguineous families, provided that tissues and cells expressing the relevant genes are available for analysis.

Genomics-based treatment in a patient with two overlapping heritable skin disorders: Epidermolysis bullosa and acrodermatitis enteropathica.

Next-generation sequencing (NGS) is helpful in diagnosing complex genetic disorders and phenotypes, particularly when more than one overlapping condition is present. From a large cohort of 362 families with clinical manifestations of skin and mucosal fragility, referred by several major medical centers, one patient was found by NGS to have two overlapping heritable skin diseases, recessive dystrophic epidermolysis bullosa (RDEB; COL7A1 mutations) and acrodermatitis enteropathica (AE; SLC39A4 mutations). The pathogenicity of the variants was studied at gene expression as well as ultrastructural and tissue levels. Although there is no specific treatment for RDEB except avoiding trauma, supplementation with oral zinc (3 mg·kg-1 ·day-1 ) for the AE resulted in rapid amelioration of the skin findings. This case demonstrates the power of NGS in identifying two genetically unlinked diseases that led to effective treatment with major clinical benefits as an example of genomics-guided treatment.

Cutaneous Granulomatosis and Class Switching Defect as a Presenting Sign in Ataxia-Telangiectasia: First Case from the National Iranian Registry and Review of the Literature.

Ataxia-telangiectasia (A-T) is a rare autosomal recessive syndrome characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency and cancer predisposition, caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The clinical and immunological manifestations of A-T are very heterogeneous, especially at an early age, leading to frequent misdiagnosis. Cutaneous granulomas with unknown pathogenesis occur uncommonly in a minority of A-T patients. We herein report an unusual case of a 13-year-old girl with A-T who presented severe clinical manifestations, including multiple granulomatous lesions of the skin and a class switch defect phenotype. This patient is the first Iranian A-T case with cutaneous granulomatosis and immunodeficiency. In addition, the literature on skin granulomas in all previously reported A-T patients is reviewed indicating an increased frequency of elevated IgM level and female dominancy in this selected group of patients.

Autosomal recessive congenital ichthyosis: Genomic landscape and phenotypic spectrum in a cohort of 125 consanguineous families.

Autosomal recessive congenital ichthyosis (ARCI), a phenotypically heterogeneous group of non-syndromic Mendelian disorders of keratinization, is caused by mutations in as many as 13 distinct genes. We examined a cohort of 125 consanguineous families with ARCI for underlying genetic mutations. The patients' DNA was analyzed with a gene-targeted next generation sequencing panel comprising 38 ichthyosis associated genes. The interpretations of results of genomic data were assisted by genome-wide homozygosity mapping and transcriptome sequencing. Sequence data analysis identified biallelic mutations in 106 families out of a total of 125 (85%), most of them (102, 96.2%) being homozygous, reflecting consanguinity in these families. Among the 85 distinct mutations in 10 different genes, 45 (53%) were previously unreported. Phenotype-genotype correlations allowed assignment of specific genes in the majority of the families to a specific subtype of ARCI, lamellar ichthyosis (LI) versus congenital ichthyosiform erythroderma (CIE). Interestingly, mutations in several genes could give rise to an overlapping phenotype consistent with either LI or CIE. Also, this is the third report for SDR9C7 and SULT2B1, and fourth report for CERS3 mutations. Direct comparison of our results with previously published regional cohorts highlights the global mutation landscape of ARCI, however, population specific differences were noted.

Inherited non-alcoholic fatty liver disease and dyslipidemia due to monoallelic ABHD5 mutations.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is a multifactorial condition and the most common liver disease worldwide, affecting more than one-third of the population. So far there have been no reports on mendelian inheritance in families with NAFLD. METHODS: We performed whole-exome or targeted next-generation sequencing on patients with autosomal dominant NAFLD. RESULTS: We report a heritable form of NAFLD and/or dyslipidemia due to monoallelic ABHD5 mutations, with complete clinical expression after the fourth decade of life, in 7 unrelated multiplex families encompassing 39 affected individuals. The prevalence of ABHD5-associated NAFLD was estimated to be 1 in 1,137 individuals in a normal population. CONCLUSION: We associate a Mendelian form of NAFLD and/or dyslipidemia with monoallelic ABHD5 mutations. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a common multifactorial disorder with a strong genetic component. Inherited forms of NAFLD have been suspected but, their molecular pathogenesis has not been disclosed. Here we report a heritable form of NAFLD with clinical expression after 40 years of age, associated with monoallelic ABHD5 mutations.

Genome-wide single nucleotide polymorphism-based autozygosity mapping facilitates identification of mutations in consanguineous families with epidermolysis bullosa.

Autozygosity mapping (AM) is a technique utilised for mapping homozygous autosomal recessive (AR) traits and facilitation of genetic diagnosis. We investigated the utility of AM for the molecular diagnosis of heterogeneous AR disorders, using epidermolysis bullosa (EB) as a paradigm. We applied this technique to a cohort of 46 distinct EB families using both short tandem repeat (STR) and genome-wide single nucleotide polymorphism (SNP) array-based AM to guide targeted Sanger sequencing of EB candidate genes. Initially, 39 of the 46 cases were diagnosed with homozygous mutations using this method. Independently, 26 cases, including the seven initially unresolved cases, were analysed with an EB-targeted next-generation sequencing (NGS) panel. NGS identified mutations in five additional cases, initially undiagnosed due to the presence of compound heterozygosity, deep intronic mutations or runs of homozygosity below the set threshold of 2 Mb, for a total yield of 44 of 46 cases (95.7%) diagnosed genetically.

Next generation sequencing identifies double homozygous mutations in two distinct genes (EXPH5 and COL17A1) in a patient with concomitant simplex and junctional epidermolysis bullosa.

Epidermolysis bullosa (EB) is a heterogeneous group of heritable blistering diseases. We developed a next generation sequencing (NGS) panel covering 21 genes associated with skin fragility disorders, and it was applied to DNA from 91 probands with the diagnosis of EB. In one patient, novel homozygous mutations were disclosed in two different, unlinked EB-associated genes: EXPH5, chr11 g.108510085G > A; p.Arg1808Ter and COL17A1, chr10 g.104077423delT; p.Thr68LeufsTer106. Consequences of the COL17A1 mutation were examined by RNAseq which revealed a complex splicing pattern predicting synthesis of a truncated polypeptide (85%) or in-frame deletion of exon 4 (15% of transcripts). Transmission electron microscopy (TEM) and immunostaining revealed findings consistent with EB simplex (EBS) and junctional EB (JEB), and clinical examination revealed a complex phenotype with features of both subtypes. This case illustrates the power of next generation sequencing in identifying mutations in patients with complex EB phenotype, with implications for genotype-phenotype correlations, prenatal testing, and genetic counseling of families at risk for recurrence.

DNA repair-related heritable photosensitivity syndromes: Mutation landscape in a multiethnic cohort of 17 multigenerational families with high degree of consanguinity.

Inherited photosensitivity syndromes are a heterogeneous group of genetic skin disorders with tremendous phenotypic variability, characterized by photosensitivity and defective DNA repair, especially nucleotide excision repair. A cohort of 17 Iranian families with heritable photosensitivity syndromes was evaluated to identify their genetic defect. The patients' DNA was analyzed with either whole-exome sequencing or RNA sequencing (RNA-Seq). The interpretations of the genomic results were guided by genome-wide homozygosity mapping. Haplotype analysis was performed for cases with recurrent mutations. RNA-Seq, in addition to mutation detection, was also utilized to confirm the pathogenicity. Thirteen sequence variants, including six previously unreported pathogenic variants, were disclosed in 17 Iranian families, with XPC as the most common mutated gene in 10 families (59%). In one patient, RNA-Seq, as a first-tier diagnostic approach, revealed a non-canonical homozygous germline variant: XPC:c.413-9 T > A. The Sashimi plot showed skipping of exon 4 with dramatic XPC down-expression. Haplotype analysis of XPC:c.2251-1 G>C and XPC:1243 C>T in four families showed common haplotypes of 1.7 Mb and 2.6 Mb, respectively, denoting a founder effect. Lastly, two extremely rare cases were presented in this report: a homozygous UVSSA:c .1990 C>T was disclosed, and ERCC2-related cerebro-oculo-facio-skeletal (COFS) syndrome with an early childhood death. A direct comparison of our data with the results of previously reported cohorts demonstrates the international mutation landscape of DNA repair-related photosensitivity disorders, although population-specific differences were observed.

An Unusual TEN-Like Presentation of Juvenile Bullous Pemphigoid: A Diagnostic Challenge.

Bullous pemphigoid (BP) is an acquired autoimmune bullous disorder rarely seen in the pediatric population. It usually presents as large and tense bullae, predominantly distributed in the acral areas. Herein, we describe a case of childhood BP with an atypical presentation mimicking toxic epidermal necrolysis (TEN). This case shows us that juvenile BP should be considered in the differential diagnosis of TEN in children, particularly if there are unusual features and an intractable course.

Whole-transcriptome sequencing-based concomitant detection of viral and human genetic determinants of cutaneous lesions.

Severe viral infections of the skin can occur in patients with inborn errors of immunity (IEI). We report an all-in-one whole-transcriptome sequencing-based method by RNA-Seq on a single skin biopsy for concomitantly identifying the cutaneous virome and the underlying IEI. Skin biopsies were obtained from healthy and lesional skin from patients with cutaneous infections suspected to be of viral origin. RNA-Seq was utilized as the first-tier strategy for unbiased human genome-wide rare variant detection. Reads unaligned to the human genome were utilized for the exploration of 926 viruses in a viral genome catalog. In 9 families studied, the patients carried pathogenic variants in 6 human IEI genes, including IL2RG, WAS, CIB1, STK4, GATA2, and DOCK8. Gene expression profiling also confirmed pathogenicity of the human variants and permitted genome-wide homozygosity mapping, which assisted in identification of candidate genes in consanguineous families. This automated, online, all-in-one computational pipeline, called VirPy, enables simultaneous detection of the viral triggers and the human genetic variants underlying skin lesions in patients with suspected IEI and viral dermatosis.

Kindler epidermolysis bullosa-like skin phenotype and downregulated basement membrane zone gene expression in poikiloderma with neutropenia and a homozygous USB1 mutation.

Epidermolysis bullosa (EB) is a genotypically heterogeneous group of disorders characterized by cutaneous blistering and erosions with a tremendous spectrum of severity. One of the distinct forms of EB, Kindler EB (KEB), manifests with blistering and poikiloderma; this subtype of EB is caused by mutations in the FERMT1 gene encoding kindlin-1. In this study, we investigated a patient clinically diagnosed as KEB with reduced FERMT1 gene expression and intensity of immunostaining for kindlin-1. Transmission electron microscopy showed lamina densa reduplication, frequently observed in KEB. However, no mutations were identified in FERMT1 in this patient with consanguineous parents, and this gene resided outside of genomic regions of homozygosity (ROH). Instead, whole-exome sequencing and homozygosity mapping identified a homozygous sequence variant at the +4 position of intron 2 in the USB1 gene, encoding an exoribonuclease required for processing of U6 snRNA, a critical component of spliceosomes. Examination of the patient's RNA by RNA-Seq confirmed the pathogenicity of this variant, causing aberrant splicing predicted to result in loss of function of USB1. Mutations in this gene have been reported in patients with poikiloderma and neutropenia, with a few reported cases in association with skin fragility, a condition distinct from the KEB phenotype. Transcriptome analysis revealed that several genes, expressed in the cutaneous basement membrane zone and previously associated with different subtypes of EB, were differentially downregulated at the mRNA level. EB-associated mRNA downregulation was confirmed at protein levels by skin immunofluorescence. These observations provide a novel mechanism for blistering and erosions in the skin as a result reduced presence of adhesion complexes critical for stable association of epidermis and dermis at the level of cutaneous basement membrane zone.

Arrhythmogenic right ventricular cardiomyopathy in patients with biallelic JUP-associated skin fragility.

Arrhythmogenic right ventricular cardiomyopathy (ARVC), with skin manifestations, has been associated with mutations in JUP encoding plakoglobin. Genotype-phenotype correlations regarding the penetrance of cardiac involvement, and age of onset have not been well established. We examined a cohort of 362 families with skin fragility to screen for genetic mutations with next-generation sequencing-based methods. In two unrelated families, a previously unreported biallelic mutation, JUP: c.201delC; p.Ser68Alafs*92, was disclosed. The consequences of this mutation were determined by expression profiling both at tissue and ultrastructural levels, and the patients were evaluated by cardiac and cutaneous work-up. Whole-transcriptome sequencing by RNA-Seq revealed JUP as the most down-regulated gene among 21 skin fragility-associated genes. Immunofluorescence showed the lack of plakoglobin in the epidermis. Two probands, 2.5 and 22-year-old, with the same homozygous mutation, allowed us to study the cross-sectional progression of cardiac involvements in relation to age. The older patient had anterior T wave inversions, prolonged terminal activation duration (TAD), and RV enlargement by echocardiogram, and together with JUP mutation met definite ARVC diagnosis. The younger patient had no evidence of cardiac disease, but met possible ARVC diagnosis with one major criterion (the JUP mutation). In conclusion, we identified the same biallelic homozygous JUP mutation in two unrelated families with skin fragility, but cardiac findings highlighted age-dependent penetrance of ARVC. Thus, young, phenotypically normal patients with biallelic JUP mutations should be monitored for development of ARVC.

Filaggrin gene polymorphisms in Iranian ichthyosis vulgaris and atopic dermatitis patients.

BACKGROUND: Filaggrin is a key structural epidermal protein in terminal differentiation and formation of skin barrier. The important role of filaggrin and its effects in various cutaneous and noncutaneous disorders initiated a cascade of considerable research in recent years. Loss-of-function mutations in FLG, the human gene encoding profilaggrin/filaggrin, is the cause of the common skin condition ichthyosis vulgaris (IV) and major genetic predisposing factor for atopic dermatitis (AD). Several null mutations in the FLG gene that lead to a decrease or absence of filaggrin in skin and predispose these conditions have been described. OBJECTIVE: The aim of this study was to investigative genetic polymorphism of FLG in Iranian patients with IV and AD. METHODS: In the current study, we carried out full sequencing of the entire FLG coding region in 30 IV patients and 30 AD patients, and also 60 healthy controls. RESULTS: In our research, we identified 43 variants reported previously and two novel variants. CONCLUSION: In our study, in the AD and IV patients, loss-of-function FLG mutation was not found. This means that another mechanism other than FLG nonsense mutation is involved in the pathogenesis of these patients.

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 epidermolysis bullosa, and most of them in this Iranian cohort were consistent with autosomal recessive inheritance. 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.

Autosomal recessive congenital ichthyosis is a heterogeneous group of disorders associated with mutations in at least nine distinct genes. To ascertain the molecular basis of ichthyosis patients in Iran, a country of approximately 80 million people with a high prevalence of customary consanguineous marriages, we have developed a gene-targeted next generation sequencing array consisting of 38 genes reported in association with ichthyosis phenotypes. In a subset of nine extended consanguineous families, we found homozygous missense mutations in the PNPLA1 gene, six of them being distinct and, to our knowledge, previously unpublished. This gene encodes an enzyme with lipid hydrolase activity, important for development and maintenance of the barrier function of the epidermis. These six mutations, as well as four previously published mutations, reside exclusively within the patatin-like subdomain of PNPLA1 containing the catalytic site. The mutations clustered around the active center of the enzyme or resided at the surface of the protein possibly involved in the protein-protein interactions. Clinical features of the patients showed considerable intra- and interfamilial heterogeneity. Knowledge of the specific mutations allows identification of heterozygous carriers, assisting in genetic counseling, prenatal testing, and preimplantation genetic diagnosis in extended families at risk of recurrence of this disorder, the incidence of which is significantly increased in consanguineous marriages.

Autosomal recessive congenital ichthyosis: CERS3 mutations identified by a next generation sequencing panel targeting ichthyosis genes.

There are at least 38 mutant genes known to be associated with the ichthyosis phenotypes, and autosomal recessive congenital ichthyosis (ARCI) is a specific subgroup caused by mutations in 13 different genes. Mutations in some of these genes, such as CERS3 with only two previous reports, are rare. In this study, we identified mutations in candidate genes in consanguineous families with ARCI with a next generation sequencing (NGS) array that incorporates 38 ichthyosis associated genes. We applied this sequencing array to DNA from 140 ichthyosis families with high prevalence of consanguinity. Among these patients we identified six distinct, previously unreported mutations in CERS3 in six Iranian families. These mutations in each family co-segregated with the ichthyosis phenotype. The patients demonstrated collodion membrane at birth, acrogeria, generalized scaling, and hyperlinearity of the palms and soles. The presence of a significant percentage of CERS3 mutations in our cohort depicts a marked difference between the etiology of ichthyoses in genetically poorly characterized regions and well-characterized western populations. Also, it shows that rare alleles are more prevalent in the gene pool of consanguineous populations and emphasizes the importance of these population studies for better understanding of ichthyosis pathogenesis.

Association between single nucleotide polymorphisms of the interleukin-4 gene and atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin disease in which both genetic and environmental factors seem to be involved. Several studies investigated the association of certain genetic factors with AD in different ethnic groups, but conflicting data were obtained. This study was performed to check the possible association between single nucleotide polymorphisms (SNPs) of interleukin 4 (IL-4) and the IL-4 receptor α chain (IL-4Rα) and AD in a group of Iranian patients. The allele and genotype frequencies of genes encoding for IL-4 and IL-4Rα were investigated in 89 patients with AD in comparison with 139 healthy controls, using methods based on polymerase chain reaction sequence-specific primers. The most frequent alleles of IL-4 in patients were T at -1098 (P<0.001, odds ratio (OR)=2.35), C at -590 (P<0.001, OR=4.84) and C at -33 (P=0.002, OR=2.08). The most frequent genotypes of IL-4 in patients were TT, CC, and CC at positions -1098 (P<0.001, OR=3.59), -590 (P<0.001, OR=31.25) and -33 (P<0.001, OR=3.46), respectively. We found a significant lower frequency of GT at -1098 GT, TC at -590, and TC at -33 in patients. There were no statistically significant differences in the frequency of alleles and genotypes of IL-4Rα gene at position +1902. A strong positive association was seen between TCC haplotype and AD (68% in patients vs. 23.4% in controls, P<0.001, OR=8.91). We detected a significantly lower frequency of TTC, GCC, and TTT haplotypes (P<0.001, OR=0.02, P<0.001, OR=0.40, P<0.001, OR=0.39, respectively) in patients compared to controls. A significant association between the polymorphisms of the IL-4 gene promoter at positions -1098, -590, and -33 and AD was detected in the Iranian population.