Linda Ozoemena

Whole-exome sequencing improves mutation detection in a diagnostic epidermolysis bullosa laboratory

Subtypes of inherited epidermolysis bullosa (EB) vary significantly in their clinical presentation and prognosis. Establishing an accurate diagnosis is important for genetic counselling and patient management. Current approaches in EB diagnostics involve skin biopsy for immunohistochemistry and transmission electron microscopy, and Sanger sequencing of candidate genes. Although informative in most cases, this approach can be expensive and laborious and may fail to identify pathogenic mutations in ~15% of cases.

Impact of next generation sequencing on diagnostics in a genetic skin disease clinic.

Individuals with inherited skin diseases often pose one of the most difficult diagnostic challenges in dermatology. The hunt for the underlying molecular pathology may involve candidate gene screening or linkage analysis, which is usually determined by the initial history, the physical findings and laboratory tests. Recent technical advances in DNA sequencing, however, are shifting the diagnostic paradigm. Notably, next‐generation sequencing allows a more comprehensive approach to diagnosing inherited diseases, with potential savings of both time and money. In the setting of a paediatric dermatology genetics clinic in Kuwait, we therefore performed whole‐exome sequencing on seven individuals without a priori detailed knowledge of the patients’ disorders: from these sequencing data, we diagnosed X‐linked hypohidrotic ectodermal dysplasia (two cases), acrodermatitis enteropathica, recessive erythropoietic protoporphyria (two siblings) and localized recessive dystrophic epidermolysis bullosa (two siblings). All these groups of disorders are clinically and genetically heterogeneous, but the sequencing data proved inherently useful in improving patient care and avoiding unnecessary investigations. Our observations highlight the value of whole‐exome sequencing, in combination with robust bioinformatics analysis, in determining the precise molecular pathology and clinical diagnosis in patients with genetic skin disorders, notably at an early stage in the clinical evaluation of these often complex disorders and thereby support a new paradigm for future diagnostics.

Mutations in GRHL2 Result in an Autosomal-Recessive Ectodermal Dysplasia Syndrome

Grainyhead-like 2, encoded by GRHL2, is a member of a highly conserved family of transcription factors that play essential roles during epithelial development. Haploinsufficiency for GRHL2 has been implicated in autosomal-dominant deafness, but mutations have not yet been associated with any skin pathology. We investigated two unrelated Kuwaiti families in which a total of six individuals have had lifelong ectodermal defects. The clinical features comprised nail dystrophy or nail loss, marginal palmoplantar keratoderma, hypodontia, enamel hypoplasia, oral hyperpigmentation, and dysphagia. In addition, three individuals had sensorineural deafness, and three had bronchial asthma. Taken together, the features were consistent with an unusual autosomal-recessive ectodermal dysplasia syndrome. Because of consanguinity in both families, we used whole-exome sequencing to search for novel homozygous DNA variants and found GRHL2 mutations common to both families: affected subjects in one family were homozygous for c.1192T>C (p.Tyr398His) in exon 9, and subjects in the other family were homozygous for c.1445T>A (p.Ile482Lys) in exon 11. Immortalized keratinocytes (p.Ile482Lys) showed altered cell morphology, impaired tight junctions, adhesion defects, and cytoplasmic translocation of GRHL2. Whole-skin transcriptomic analysis (p.Ile482Lys) disclosed changes in genes implicated in networks of cell-cell and cell-matrix adhesion. Our clinical findings of an autosomal-recessive ectodermal dysplasia syndrome provide insight into the role of GRHL2 in skin development, homeostasis, and human disease.

Rapp-Hodgkin ectodermal dysplasia syndrome: the clinical and molecular overlap with Hay-Wells syndrome.

We report on the clinical and molecular abnormalities in a 7‐month‐old girl and her mother with an ectodermal dysplasia disorder that most closely resembles Rapp–Hodgkin syndrome (RHS). At birth, the child had bilateral cleft palate, a narrow pinched nose, small chin, and hypoplastic nipples, and suffered from respiratory distress, feeding difficulties, and poor weight gain, although developmental progress was normal. Her mother had a cleft palate, sparse hair, high forehead, dental anomalies, a narrow nose, dysplastic nails, and reduced sweating. Sequencing of the p63 gene in genomic DNA from both individuals revealed a heterozygous frameshift mutation, 1721delC, in exon 14. This mutation has not been described previously and is the seventh report of a pathogenic p63 gene mutation in RHS. The frameshift results in changes to the tail of p63 with the addition of 90 missense amino acids downstream and a delayed termination codon that extends the protein by 21 amino acids. This mutation is predicted to disrupt the normal repressive function of the transactivation inhibitory domain leading to gain‐of‐function for at least two isoforms of the p63 transcription factor. The expanding p63 mutation database demonstrates that there is considerable overlap between the molecular pathology of RHS and Hay–Wells syndrome, with identical mutations in some cases, and that these two disorders may in fact be synonymous.

Under-recognition of acral peeling skin syndrome: 59 new cases with 15 novel mutations

Acral peeling skin syndrome (APSS) is a rare skin fragility disorder usually caused by mutations in the transglutaminase 5 gene (TGM5).

Desquamative enteropathy and pyloric atresia without skin disease caused by a novel intracellular beta4 integrin mutation

Background: Mutations in α6 or β4 integrins (ITGA6, ITGB4) are known to cause junctional epidermolysis bullosa with pyloric atresia (JEB-PA), often lethal in infancy through skin desquamation. There is 1 report of pyloric atresia associated with a desquamatory enteropathy but without skin disease, of unknown molecular basis. Patients and Methods: We report 2 Kuwaiti siblings with pyloric atresia and life-threatening intestinal desquamation without significant skin abnormality. The older sibling died of intractable diarrhoea, and the younger sibling suffered episodes of massive protein-losing enteropathy, triggered by viral infections, in addition to obstructive uropathy. Mutation analysis was performed for ITGA6 and ITGB4 and expression of ITGA6 and ITGB4 protein was examined in skin and intestinal biopsies. Her serum also was incubated with normal intestine. Results: We identified a novel mutation in ITGB4, with homozygous deletion of a single residue (isoleucine 1314) within the intracellular plectin-binding domain. Expression of ITGA6 and ITGB4 within skin, duodenal, and colonic epithelium was normal or minimally reduced, in contrast to previous reports. Biopsies taken during relapse showed accumulation of immunoglobulin G and C1q within intestinal basement membrane, whereas immunoglobulin G from her serum bound to basement membrane of normal small intestine. Immunomodulatory therapy induced significant improvement following relapses. Conclusions: ITGB4 mutation may induce a desquamative enteropathy in infancy without significant skin disease. A history of pyloric atresia is important in infants with severe chronic diarrhoeal disease and should prompt investigation for JEB-PA associated mutations. Acquired immune responses may exacerbate primary genetic disorders of epithelial adhesion and immunomodulatory therapy may be beneficial.

A new homozygous nonsense mutation in LAMA3A underlying laryngo-onycho-cutaneous syndrome

Laryngo‐onycho‐cutaneous (LOC) syndrome is a subtype of autosomal recessive junctional epidermolysis bullosa in which there is prominent skin and mucosal granulation tissue that can lead to delayed wound healing, laryngeal obstruction and blindness. Thus far, all cases are of Punjabi ancestry and have been shown to result from a founder mutation in the LAMA3 gene, notably involving a single nucleotide insertion mutation in exon 39, which is specific to the LAMA3A (designated exon 1 of LAMA3A) and not the LAMA3B1 or LAMA3B2 isoforms. Here, we describe a new pedigree with LOC syndrome. Affected individuals (from Iran) have the characteristic clinicopathological and molecular features of LOC syndrome: prominent granulation tissue (especially affecting the eyes), normal intensity laminin‐332 immunostaining at the dermal–epidermal junction, and autosomal recessive mutations in the LAMA3A‐specific exon. The pathogenic mutation is a homozygous nonsense mutation, designated p.Gln57X, which just affects the laminin‐α3a transcript. These findings therefore expand the molecular basis of LOC syndrome.

Mutations in EXPH5 underlie a rare subtype of autosomal recessive epidermolysis bullosa simplex

DEAR EDITOR, Epidermolysis bullosa simplex (EBS) is a heterogeneous disorder with mutations in at least nine different genes underlying various autosomal dominant and recessive subtypes thereof. Making an accurate clinical and laboratory diagnosis in some of the less common forms of EBS can be challenging. A 9-year-old boy with first-cousin Pakistani parents presented with skin fragility from birth. He had scattered vesicles and bullae up to 1–2 cm in size on the limbs and trunk (Fig. 1a, b). Blistering was made worse by hot weather. Individual blisters healed within 1–2 weeks, leaving postinflammatory hypopigmentation. His blistering tendency persisted but ameliorated with age. Hair, teeth, nails and mucosae were normal. His 2-year-old brother had similar symptoms and signs from birth. Of note, the father of both children, whose parents were also first cousins, had similar skin fragility as a child that resolved completely by 10 years of age. The pattern of inheritance was consistent with autosomal recessive transmission, with possible pseudodominant inheritance, given the father’s history of blistering. Unfortunately, the father declined to participate in the study and was not available for further evaluation. To date, eight of the nine EBS genes have been implicated in the pathophysiology of autosomal recessive subtypes: KRT14, PLEC, DST-e, PKP1, DSP, JUP, TGM5 and EXPH5. With

Clinical subtypes and molecular basis of epidermolysis bullosa in Kuwait

Epidermolysis bullosa (EB) is a clinically and genetically heterogeneous blistering skin disease, but in countries such as Kuwait, there are very limited data on the clinical and molecular pathology of EB. To improve understanding of EB in Kuwait, we report the experience of a local tertiary referral center over a 17.5 year period (January 2000–June 2017) in establishing clinical and molecular diagnoses.

Limited ectrodactyly, ectodermal dysplasia and cleft lip–palate syndrome with a p63 mutation, associated with linear and whorled naevoid hypermelanosis

An 18-year old Malaysian student presented with pigmented marks on his trunk and limbs, which had been present since birth. He also had uncorrected congenital deformities of his hands and feet, and a history of surgically treated congenital cataracts and glaucoma. He was the third of four children born to nonconsanguineous parents, with no family history of similar problems or of orofacial clefting. On physical examination, the patient was found to have widespread macular hyperpigmentation in streaks and whorls along the lines of Blaschko (Fig. 1). He also had hypodontia, microdontia and ectrodactyly with median clefts of both hands and feet (Fig. 2), consistent with split-hand split-foot malformation (SHFM). The hair and nails appeared normal, and systemic examination was unremarkable. Blood samples were collected, and DNA extracted from lymphocytes. Mutation analysis identified a heterozygous cytosine to thymine transition at nucleotide 982 (c.982C>T) in the p63 gene, resulting in the missense mutation, p.Arg280Cys, within the DNA-binding domain. The proband’s parents were not available for genetic testing. p63-associated disorders encompass a spectrum of phenotypes with varying combinations of ectodermal dysplasia, orofacial clefting, and limb, ocular and other malformations. Heterozygous missense mutations affecting several amino acid residues of p63 have been reported. Interestingly, our patient’s missense mutation has been previously found in patients with both ectrodactyly, ectodermal dysplasia and cleft lip–palate (EEC) syndrome and non-syndromic SHFM. EEC syndrome, which is often incomplete, best describes our patient’s clinical phenotype of ectrodactyly and ectodermal dysplasia without orofacial clefting. No other p63-associated features were clinically identified in our patient (Table 1). The balance between the ΔNp63/ TAp63 isoforms may contribute to phenotypic variability in EEC syndrome, with TAp63 serving as genetic modifier. In addition, our patient had linear and whorled naevoid hypermelanosis (LWNH), a pigmentary pattern often associated with chromosomal mosaicism, and probably reflecting mosaicism for a mutation affecting a pigmentary gene. Of note, the only other reported patient with LWNH associated with SHFM had a son with EEC syndrome resulting from the p63 mutation R204W. Mosaicism for this mutation was found in the father, and was considered to be the likely cause of his pigmentary pattern and milder clinical phenotype. Although the identified mosaicism was for a mutation in p63 rather than in a pigmentary gene, the mutation might have disrupted expression of a pigmentary gene, as p63, a transcription factor, is known to regulate the expression of genes involved in epidermal differentiation. Thus, we did consider the possibility that somatic mosaicism for p63 may have caused the pigmentary mosaicism (PM) in our patient; however, we did not detect any mosaicism for the p63 mutation. DNA extracted from punch biopsies of both affected and unaffected skin contained the mutation p.Arg280Cys. Neither was there any evidence of chromosomal mosaicism to account for LWNH; cytogenetic analysis of adequate numbers of blood lymphocytes and skin fibroblasts was normal. Chromosomal mosaicism could not be excluded Correspondence: Dr Penelope Pratsou, Department of Dermatology, University Hospitals Coventry and Warwickshire NHS Trust, Clifford Bridge Road, Coventry, CV2 2DX, UK E-mail: p.pratsou@nhs.net