Kenneth Fong

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.

Epithelial Inflammation Resulting from an Inherited Loss-of-Function Mutation in EGFR

Epidermal growth factor receptor (EGFR) signaling is fundamentally important for tissue homeostasis through EGFR/ligand interactions that stimulate numerous signal transduction pathways. Aberrant EGFR signaling has been reported in inflammatory and malignant diseases but thus far no primary inherited defects in EGFR have been recorded. Using whole-exome sequencing, we identified a homozygous loss-of-function missense mutation in EGFR (c.1283G>A; p.Gly428Asp) in a male infant with life-long inflammation affecting the skin, bowel and lungs. During the first year of life, his skin showed erosions, dry scale, and alopecia. Subsequently, there were numerous papules and pustules – similar to the rash seen in patients receiving EGFR inhibitor drugs. Skin biopsy demonstrated an altered cellular distribution of EGFR in the epidermis with reduced cell membrane labeling, and in vitro analysis of the mutant receptor revealed abrogated EGFR phosphorylation and EGF-stimulated downstream signaling. Microarray analysis on the patient’s skin highlighted disturbed differentiation/premature terminal differentiation of keratinocytes and upregulation of several inflammatory/innate immune response networks. The boy died aged 2.5 years from extensive skin and chest infections as well as electrolyte imbalance. This case highlights the major mechanism of epithelial dysfunction following EGFR signaling ablation and illustrates the broader impact of EGFR inhibition on other tissues.

Autosomal recessive epidermolysis bullosa simplex due to loss of BPAG1-e expression.

TO THE EDITOR In 2010, we identified a homozygous nonsense mutation in the dystonin (DST) gene in a Kuwaiti individual with a new form of autosomal recessive epidermolysis bullosa (EB) simplex (Groves et al., 2010). The mutation occurred within the coiled-coil rod domain of the BPAG1-e isoform (the hemidesmosomal 230-kDa bullous pemphigoid antigen) and the main symptom was mild trauma-induced predominantly acral blistering, although there was also some generalized skin fragility. However, additional neurological features were noted, including episodes of collapse, headaches, numbness, and weakness. Molecular studies also revealed a heterozygous mutation in the NOTCH3 gene consistent with concomitant autosomal dominant cerebral small-vessel arteriopathy (CADASIL; MIM125310; Joutel et al., 1996). Given that some DST isoforms may be expressed in the central nervous system (Young and Kothary, 2007; see Groves et al., 2010, for details of alternative splicing of DST), it was unclear whether the overall clinical features were solely due to the DST mutation or whether NOTCH3 might be involved. To address this further, we have now identified a second unrelated family with a different homozygous nonsense mutation in the same region of the DST gene. Notably, affected individuals have mild skin blistering but no neurological abnormalities. The proband was a 34-year-old Iranian woman who had lifelong skin blistering that was worse during summer. Blisters occurred on the soles, at sites of friction from clothing, and following removal of adhesive dressings. The mouth, eyes, and genital areas were never involved. No extracutaneous symptoms were reported. Her father and two of her three children also had mild blistering. Blisters occurred on the feet of her 15-year-old son following sporting activities, and her 6-month-old son developed blisters on the great toes after starting to crawl; her husband reported no skin fragility. Some consanguinity was reported in the family, but precise details were not forthcoming. On examination, she had a solitary blister on the instep of one foot (Figure 1a). The rest of her skin appeared normal apart from subtle atrophic scars affecting the shins, ankles, elbows, dorsal aspects of the hands, and lower back. Teeth, nails, and hair were normal, and there was no palmoplantar hyperkeratosis. Following written and informed consent, blood and skin samples were obtained from the patient in adherence to the Declaration of Helsinki Principles. The skin sampled was noninflamed and nonlesional. Initially, we performed immunofluorescence microscopy, as described elsewhere (Groves et al., 2010). Labeling for plectin, a6 and b4 integrin subunits, keratin 14, laminin-332, and collagens IV, VII, and XVII, however, showed no abnormalities. Next, we undertook transmission electron microscopy, as described previously (Groves et al., 2010). The morphology of individual hemidesmosomes was abnormal with poorly formed inner plaques, leading to a lucent zone between keratin filaments and outer hemidesmosomal plaques (Figure 1b). The keratin filaments extended to where the inner plaques should be, but did not associate with any attachment structures; no other abnormalities were noted. Further skin immunostaining with a mAb against BPAG1-e (clone BPC319, directed against the carboxyl terminus, Okumura et al., 2002) showed a complete absence of immunoreactivity compared with bright, linear labeling at the dermal– epidermal junction in control skin (Figure 1c). Sequencing of the patient’s genomic DNA identified a homozygous A4T transversion (c.3853A4T; GenBank NM_001723.4) that converts arginine to a stop codon, designated p.Arg1249X (Figure 1d). This mutation occurs within the coiled-coil domain in a region similar to the previously reported DST mutation, p.Gln1124X (Groves et al., 2010). The new mutation was not identified in screening 200 ethnically matched control chromosomes. Sequencing of the genes encoding keratins 5 and 14 revealed no pathogenic mutations. Skin biopsies and DNA samples were not available from other family members, but the data are consistent with autosomal recessive EB simplex due to a homozygous lossof-function mutation in the DST gene with a pseudodominant pattern of inheritance in this consanguineous family. The similar nature and sites of the DST mutations in this and the previously published case allow for clinicopathological refinement of the subtype of EB. BPAG1-e, or BP230, is a structural component of hemidesmosomal inner plaques in basal keratinocytes (Guo et al., 1995; Yang et al., 1996). The rod domain comprises a coiled-coil region that is involved in homodimerization, whereas the amino terminus is important for the recruitment of BPAG1-e into hemidesmosomes (Koster et al., 2003), and the carboxy-terminal region binds to keratin intermediate filaments (Yang et al., 1996; Fontao et al., 2003). Lossof-function mutations in the coiled-coil region in both cases led to similar ultrastructural pathology with an absence of hemidesmosomal inner plaques and a ‘‘lucent zone’’ where keratin filaments normally attach. Both mutations also led to a complete absence of BPAG1-e on skin immunostaining. However, skin immunohistochemistry in the Kuwaiti case also reported abnormalities in labeling for plectin, the b4 integrin subunit, and collagen XVII—changes that were not noted in the current case (data not shown). These inconsistencies require Abbreviations: DST, dystonin; EB, epidermolysis bullosa

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.

Spectrum of mutations in the ANTXR2 (CMG2) gene in infantile systemic hyalinosis and juvenile hyaline fibromatosis.

oped a greater skin colour change at the final stage of irradiation. Although the vitiligo-involved site also shows an increase of the area under the curve, the small magnitude of change was not significant to detect pigment formation clinically. Figure 2(b) displays the correlation of the constitutive melanin content of normal skin to the degree of formation of IPD. The amount of constitutive melanin is quantified by an area of differential apparent absorbance between normal and vitiligoinvolved skin at baseline in the spectral range of 390–450 nm in which the soluble fraction of epidermal melanin predominantly contributes to the apparent absorbance. The result shows that the degree of IPD response appears to be related to the constitutive pigment expressed at short wavelengths. In this study, we found that VIS-NIR radiation produces IPD only in normally pigmented skin and that the presence of constitutive pigment is required to induce IPD response. We conclude that the degree of formation of IPD from VIS-NIR radiation is related to the content of constitutive pigment expressed at short wavelengths (390–450 nm). This relation has been confirmed in an ongoing study on healthy subjects with various skin types.

BPAG1-e Restricts Keratinocyte Migration through Control of Adhesion Stability

Bullous pemphigoid antigen 1 (BPAG1-e, also known as BP230) is a member of the plakin family of hemidesmosome cytoskeletal linker proteins that is encoded by an isoform of the dystonin (DST) gene. Recently, we reported two unrelated families with homozygous nonsense mutations in this DST isoform that led to ultrastructural loss of hemidesmosomal inner plaques and clinical features of trauma-induced skin fragility. We now demonstrate that keratinocytes isolated from these individuals have significant defects in adhesion, as well as increased cell spreading and migration. These mutant keratinocytes also display reduced levels of β4 integrins at the cell surface but increased total protein levels of keratin-14 and β1 integrins. These alterations in cell behavior and protein expression were not seen in control keratinocytes in which BPAG1-e expression had been silenced by stable expression of short hairpin RNA to target DST. The failure of knockdown approaches to recapitulate the changes in morphology, adhesion, and migration seen in patient cells therefore suggests such approaches are not appropriate to study loss of this protein in vivo. The contrasting findings in keratinocytes harboring naturally occurring mutations, however, demonstrate a previously unappreciated key role for BPAG1-e in regulating keratinocyte adhesion and migration and suggest a requirement for this protein in controlling functional switching between integrin types in epithelial cells.

Novel and Recurrent FERMT1 Gene Mutations in Kindler Syndrome

Kindler syndrome (OMIM 173650) is an autosomal recessive condition characterized by skin blistering, skin atrophy, photosensitivity, colonic inflammation and mucosal stenosis. Fewer than 100 cases have been described in the literature. First reported in 1954, the molecular basis of Kindler syndrome was elucidated in 2003 with the discovery of FERMT1 (KIND1) loss-of-function mutations in affected individuals. The FERMT1 gene encodes kindlin-1 (also known as fermitin family homologue 1), a 77 kDa protein that localizes at focal adhesions, where it plays an important role in integrin signalling. In the current study, we describe five novel and three recurrent loss-of-function FERMT1 mutations in eight individuals with Kindler syndrome, and provide an overview of genotype-phenotype correlation in this disorder.

Schopf-Schulz-Passarge syndrome resulting from a homozygous nonsense mutation, p.Cys107X, in WNT10A

Schöpf‐Schulz‐Passarge syndrome (SSPS; MIM224750) is a rare autosomal recessive form of ectodermal dysplasia that was recently shown to result from mutations in the WNT10A gene. We now report a 59‐year‐old woman with SSPS in whom a homozygous nonsense mutation (p.Cys107X) in WNT10A was detected. Mutations in this gene may also underlie odonto‐onycho‐dermal dysplasia and other ectodermal dysplasia syndromes. To date, 16 different WNT10A mutations have been reported, although considerable clinical and molecular overlap exists. This report demonstrates the molecular basis of a further case of SSPS and highlights the clinical features of this unusual ectodermal dysplasia syndrome.

Acral Peeling Skin Syndrome: A Clinically and Genetically Heterogeneous Disorder

Abstract:  Acral peeling skin syndrome (APSS) is a rare, autosomal, recessive genodermatosis characterized by painless spontaneous exfoliation of the skin of the hands and feet at a subcorneal or intracorneal level. It usually presents at birth or appears later in childhood or early adulthood. Some cases result from mutations in the TGM5 gene that encodes transglutaminase 5, which has an important role in cross‐linking cornified cell envelope proteins. We report a new APSS pedigree from Jordan that contains at least 10 affected family members, although sequencing of the TGM5 gene failed to disclose any pathogenic mutation(s). On the basis of probable consanguinity, we performed homozygosity mapping and identified areas of homozygosity on chromosomes 1, 6, 10, 13, and 16, although none of the intervals contained genes of clear relevance to cornification. APSS is a clinically and genetically heterogeneous disorder, and this Jordanian pedigree underscores the likelihood of still further heterogeneity.

MBTPS2 mutation in a British pedigree with keratosis follicularis spinulosa decalvans

Keratosis follicularis spinulosa decalvans (KFSD; OMIM 308800) is an X‐linked disorder characterized by widespread hyperkeratotic follicular papules (including keratosis pilaris‐like lesions), facial erythema, hypotrichosis and scarring alopecia. KFSD results from mutations in the MBTPS2 gene. Mutations in this gene also underlie ichthyosis follicularis, alopecia and photophobia syndrome. We report a British pedigree with KFSD resulting from the mutation p.Asn508Ser. This particular mutation has been reported in three other pedigrees with KFSD (Dutch, American, British) and is the only pathogenic mutation reported in this disorder to date. However, the same mutation has also been reported in a Chinese pedigree with IFAP syndrome, highlighting the clinical heterogeneity and overlapping molecular pathology of these two disorders.