Whole-exome sequencing diagnosis of two autosomal recessive disorders in one family.

Autosomal recessive congenital ichthyosis (ARCI) is a genetically heterogeneous disorder for which subtyping through molecular analysis can help determine the eventual phenotype and prognosis. We used whole-exome sequencing to identify a new homozygous splice-site mutation in ST14 (IVS5+1G>A), encoding matriptase, in a 4-year-old girl with ARCI from a consanguineous Kuwaiti family. Clinically, she also had hypotrichosis, which supported a diagnosis of ARCI type 11. Only four previous examples of pathogenic mutations in ST14 have been reported, and our findings expand the genotype-phenotype correlation for this subtype of ARCI. Our patient was the second child born to these parents; the first (deceased) and third children had congenital brain and eye abnormalities, of uncertain aetiology and with no precise diagnosis. Further analysis of our patient's exome dataset revealed heterozygosity for a splice-site mutation in POMT1 (IVS4+1G>T), encoding the protein O-mannosyltransferase, a gene implicated in Walker-Warburg syndrome. DNA sequencing in the third child showed homozygosity for this mutation in POMT1. The first-cousin parents were both heterozygous for the splice-site mutations in ST14 and POMT1. In this family, whole-exome sequencing provided accurate subtyping of a form of ARCI in one child and provide an explanation for an undiagnosed developmental disorder in two other children, findings that improve the prospects for diagnostic accuracy and genetic counselling, and demonstrate the impact of next-generation sequencing technologies on clinical genetics.

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

BACKGROUND: 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. OBJECTIVES: Next-generation DNA sequencing (NGS) technologies offer a fast and efficient complementary diagnostic strategy, but the value of NGS in EB diagnostics has yet to be explored. The aim of this study was to undertake whole-exome sequencing (WES) in nine cases of EB in which established diagnostic methods failed to make a genetic diagnosis. METHODS: Whole-exome capture was performed using genomic DNA from each case of EB, followed by massively parallel sequencing. Resulting reads were mapped to the human genome reference hg19. Potentially pathogenic mutations were subsequently confirmed by Sanger sequencing. RESULTS: Analysis of WES data disclosed biallelic pathogenic mutations in each case, with all mutations occurring in known EB genes (LAMB3, PLEC, FERMT1 and COL7A1). This study demonstrates that NGS can improve diagnostic sensitivity in EB compared with current laboratory practice. CONCLUSIONS: With appropriate diagnostic platforms and bioinformatics support, WES is likely to increase mutation detection in cases of EB and improve EB diagnostic services, although skin biopsy remains an important diagnostic investigation in current clinical practice.

Founder mutation in dystonin-e underlying autosomal recessive epidermolysis bullosa simplex in Kuwait.

Only two homozygous nonsense mutations in the epidermal isoform of the dystonin gene, DST-e, have been reported previously in autosomal recessive epidermolysis bullosa simplex (EBS); the affected pedigrees were Kuwaiti and Iranian. This subtype of EBS is therefore considered to be a rare clinicopathological entity. In this study, we identified four seemingly unrelated Kuwaiti families in which a total of seven individuals had predominantly acral trauma-induced blistering since infancy. All affected individuals were homozygous for the mutation p.Gln1124* in DST-e, the same mutation that was identified in the originally reported family from Kuwait. Haplotype analysis in the five pedigrees (including the previous case) revealed a shared block of ~60 kb of genomic DNA across the site of the mutation, consistent with a founder effect. Most heterozygotes had no clinical abnormalities although one subject had mild transient skin fragility during childhood, an observation noted in the previously reported Iranian pedigree, suggesting that the condition may also be semidominant in some pedigrees rather than purely autosomal recessive. Our study reveals propagation of a mutant ancestral allele in DST-e throughout Kuwait, indicating that this subtype of EBS may be more common in Kuwait, and perhaps other Middle Eastern countries, than is currently appreciated.

Mutations in EXPH5 result in autosomal recessive inherited skin fragility.

Several different genes have been implicated in the pathophysiology of inherited blistering skin diseases. Recently, autosomal recessive loss-of-function mutations in EXPH5 (encoding exophilin-5, also known as Slac2-b, a protein involved in intracellular vesicle transport) were identified in a new mechanobullous disease resembling a form of epidermolysis bullosa simplex (EBS). Here, we searched for mutations in EXPH5 in a 4-year-old white boy with EBS in whom initial Sanger sequencing of known genes implicated in intraepidermal skin fragility failed to identify pathogenic mutations. Transmission electron microscopy of rubbed nonlesional patient skin revealed disruption of keratinocytes in the lower epidermis with cytolysis and acantholysis, keratin filament clumping and prominent perinuclear cytoplasmic vesicles, and provided the clue to the candidate gene pathology. Sanger sequencing of genomic DNA showed compound heterozygosity for two new mutations in EXPH5, c.1947dupC (p.Pro649fsPro*11) and c.2249C>A (p.Ser750*). Immunofluorescence microscopy of patient skin showed a complete absence of exophilin-5 labelling. This case represents the third pedigree with EXPH5 mutations resulting in inherited skin fragility. The clinical and molecular data expand genotype-phenotype correlation in this new form of EBS and demonstrate the important role of exophilin-5 in keratinocyte cell biology.

Plectin defects in epidermolysis bullosa simplex with muscular dystrophy.

Epidermolysis bullosa simplex with muscular dystrophy (EBS-MD, MIM 226670) is caused by plectin defects. We performed mutational analysis and immunohistochemistry using EBS-MD (n = 3 cases) and control skeletal muscle to determine pathogenesis. Mutational analysis revealed a novel homozygous plectin-exon32 rod domain mutation (R2465X). All plectin/HD1-121 antibodies stained the control skeletal muscle membrane. However, plectin antibodies stained the cytoplasm of type II control muscle fibers (as confirmed by ATPase staining), whereas HD1-121 stained the cytoplasm of type I fibers. EBS-MD samples lacked membrane (n = 3) but retained cytoplasmic HD1-121 (n = 1) and plectin staining in type II fibers (n = 3). Ultrastructurally, EBS-MD demonstrated widening and vacuolization adjacent to the membrane and disorganization of Z-lines (n = 2 of 3) compared to controls (n = 5). Control muscle immunogold labeling colocalized plectin and desmin to filamentous bridges between Z-lines and the membrane that were disrupted in EBS-MD muscle. We conclude that fiber-specific plectin expression is associated with the desmin-cytoskeleton, Z-lines, and crucially myocyte membrane linkage, analogous to hemidesmosomes in skin.

Beta defensin-3 engineered epidermis shows highly protective effect for bacterial infection.

Defensins are small cationic proteins that harbor broad-spectrum microbicidal activity against bacteria, fungi and viruses. This study examines the effects on pathogens of the epidermis engineered to express human beta-defensin 3 (HBD3) to combat bacterial infections. First, we examined the localization of HBD3 in the epidermis and observed HBD3 in the intercellular spaces and lamellar bodies of the upper epidermal layers. This result showed HBD3 expressed and assembled in the outer layers of the epidermis was suspected to counter the invading microorganisms. Next, we established a keratinocyte cell line that stably expressed HBD3 and found that the culture medium showed antibacterial activity. Furthermore, we prepared an epidermal sheet of these cells with the HBD3 gene and grafted this onto a dermal wound on a nude rat. The HBD3 engineered epidermis demonstrated significant antimicrobial activity. Skin ulcers without epidermis are constantly exposed to invading microorganisms. Biopsy samples of re-epithelizing epidermis from patients with skin ulcers were collected, and HBD3 mRNA level measured in the epidermis. The epidermal samples from the ulcer skin expressed 2.5 times higher levels of HBD3 transcript than those in the control skin. These results, taken together, indicate that the therapeutic introduction of the HBD3 gene into somatic cells may provide a new gene therapy strategy for intractable infectious diseases.

The ethics of research related to health care in developing countries.

The Ethics of Research Related to Health Care in Developing Countries by the Nuffield Council on Bioethics makes a number of innovative recommendations that depart from codes such as the Declaration of Helsinki. It recommends that standards of care might be relativised to the standard of that nation. It recommends that very good reasons need to be given for not giving post-trial access to medications but recognises that there may be justifiable instances of this. It is the view of the authors that these and other recommendations of the report are sensible pieces of advice given the complexities of the developing world.

Gap junction development in the human fetal hair follicle and bulge region.

BACKGROUND: Gap junctions, composed of connexin (Cx) subunits, are channels that allow intercellular communication between adjacent cells and are thought to play a key role in the regulation of cell proliferation and differentiation. The Cx expression pattern and formation of gap junctions in human fetal hair follicles has yet to be clarified, including the prominent follicular bulge region that is believed to be a site rich in stem cells. OBJECTIVES: To study the expression of two major Cxs, Cx26 and Cx43, in developing hair follicles in skin samples from a series of human fetuses of estimated gestational age (EGA) 88-163 days, and to determine quantitatively the presence of gap junctions. METHODS: We used immunofluorescence labelling to investigate the sequential expression pattern of Cx26 and Cx43 in developing human hair follicles. Gap junction formation was observed by electron microscopy and the numbers of gap junctions were analysed quantitatively. Results Both Cx26 and Cx43 expression were observed at 88 days' EGA in the inner part of the hair peg. At 135 days' EGA, Cx26 was expressed in the outer root sheath (ORS) and the inner root sheath (IRS), while Cx43 was expressed chiefly in the IRS, hair matrix and sebaceous glands. At 163 days' EGA, Cx26 expression was most intense in the outermost layer of the ORS, in contrast to Cx43 expression which was in the inner part of the ORS. In the bulge region, only Cx43 was expressed in a subset of cells in the bulge. Ultrastructurally, gap junctions were observed at 102 days' EGA in the hair peg, and the number of gap junctions increased as the hair follicle matured. Gap junctions were also observed between the bulge cells in considerable numbers. CONCLUSIONS: The changing expression patterns of Cx26 and Cx43 and the increasing gap junction numbers suggest a close association of Cx expression and gap junction formation with hair follicle morphogenesis. In addition, the present ultrastructural observations demonstrate that considerable numbers of the bulge cells, a putative site rich in hair follicle stem cells, form gap junctions during human hair follicle development.

Desmosomes: structure and function in normal and diseased epidermis.

Desmosomes are important epidermal adhesion complexes that are characterized by a cell-specific expression of transmembrane cadherins and plaque-associated molecules. Desmosomes have so far, been implicated in three main disease types: autoimmune diseases that involve desmosome components (such as pemphigus vulgaris and pemphigus foliaceus), congenital diseases that affect intracellular calcium channels (such as Hailey-Hailey disease and Darier disease) and congenital diseases that directly affect desmosomal structural components. The identification of the first congenital defect affecting a desmosome component was in the gene for plakophilin I which caused an autosomal recessive skin fragility-ectodermal dysplasia syndrome with skin, hair and nail defects. Subsequently, either a haploinsufficiency of desmoplakin or a defect in desmoglein 1 was found to underlie the autosomal dominant condition Striate Palmoplantar Keratoderma. In addition, plakoglobin has been shown to be defective in Naxos disease, which results in a cardiomyopathy and growth of abnormal hair. These findings pave the way for the discovery of further cell cohesion-related diseases and will help to greatly increase our understanding of the specific function of desmosome and other epithelial junction components.

Moderation of phenotypic severity in dystrophic and junctional forms of epidermolysis bullosa through in-frame skipping of exons containing non-sense or frameshift mutations.

Non-sense mutations on both alleles of either the type VII collagen gene (COL7A1) or the genes encoding laminin 5 (LAMA3, LAMB3, or LAMC2) usually result in clinically severe forms of recessive dystrophic or junctional epidermolysis bullosa, respectively. In this study we assessed two unrelated families whose mutations in genomic DNA predicted severe recessive dystrophic epidermolysis bullosa or junctional epidermolysis bullosa phenotypes but in whom the manifestations were milder than expected. The recessive dystrophic epidermolysis bullosa patients had a homozygous single base-pair frameshift mutation in exon 19 of COL7A1 (2470insG). Clinically, there was generalized blistering but only mild scarring. Skin biopsy revealed positive type VII collagen immunoreactivity and recognizable anchoring fibrils. The junctional epidermolysis bullosa patients were compound heterozygotes for a frameshift/non-sense combination of mutations in exons 3 and 17 of LAMB3 (29insC/Q834X). These patients did not have the lethal form of junctional epidermolysis bullosa but, as adults, displayed the milder generalized atrophic benign epidermolysis bullosa variant. There was undetectable laminin 5 staining at the dermal-epidermal junction using an antibody to the beta3 chain, but faintly positive alpha3 and gamma2 chain labeling, and there was variable hypoplasia of hemidesmosomes. To explain the milder recessive dystrophic epidermolysis bullosa and junctional epidermolysis bullosa phenotypes in these families, reverse transcription-polymerase chain reaction, using RNA extracted from frozen skin, was able to provide evidence for some rescue of mutant mRNA transcripts with restoration of the open- reading frame. In the recessive dystrophic epidermolysis bullosa patients, transcripts containing in-frame skipping of exon 19 of COL7A1 in the cDNA were detected, and in the junctional epidermolysis bullosa patients transcripts with in-frame skipping of exon 17 of LAMB3 were identified. The truncated proteins encoded by these transcripts are expected to lack certain critical domains involved in cell-matrix attachment, but may still be able to contribute to adhesion thereby moderating the severity of the skin blistering. This study shows the limitations in predicting phenotype in epidermolysis bullosa solely based on mutation analysis of genomic DNA and emphasizes the importance of immunohistochemistry, electron microscopy, and mRNA assessment as parallel investigations.

Skin fragility and hypohidrotic ectodermal dysplasia resulting from ablation of plakophilin 1.

We report a 2-year-old boy with an unusual autosomal recessively inherited skin disease comprising trauma-induced skin fragility and congenital ectodermal dysplasia affecting hair, nails and sweat glands. Skin biopsy showed widening of intercellular spaces between keratinocytes and ultrastructural findings of small, poorly formed desmosomes with reduced connections to the keratin filament cytoskeleton. Immunohistochemical analysis revealed a complete absence of staining for the accessory desmosomal plaque protein plakophilin 1 (PKP1; band 6 protein). The affected individual was a compound heterozygote for null mutations on both alleles of the PKP1 gene. Both mutations occurred within the amino terminus of PKP1, the domain which normally binds the cytoskeletal keratin filament network to the cell membrane. Apart from its localization within desmosomal plaques, PKP1 may also be present within the cytoplasm and nucleus and has putative roles in signal transduction and regulation of gene activity. The clinicopathological observations in this patient demonstrate the relevance of PKP1 to desmosome formation, cutaneous cell-cell adhesion and epidermal development and demonstrate the specific manifestations of human functional knockout mutations in this gene.

Acquired junctional epidermolysis bullosa associated with IgG autoantibodies to the beta subunit of laminin-5.

We report the case of a 72-year-old man with clinical features resembling those of non-lethal junctional epidermolysis bullosa associated with IgG autoantibodies to the beta chain of laminin-5. The patient presented with a sudden onset of blistering and severe fragility of the skin and mucous membranes resulting in atrophic scars. Electron microscopy showed that the blistering arose in the lamina lucida. Indirect immunofluorescence indicated that the autoantibodies bound to the dermal side of 1 mol/L NaCl-split skin, and both direct and indirect immunoelectron microscopy demonstrated antibody binding to the lamina densa. Postembedding immunogold electron microscopy also revealed labelling in the lamina lucida beneath the hemidesmosomes. On immunoblotting, we found the autoantibodies to comigrate with the beta chain of laminin-5. Following the nomenclature of inherited junctional epidermolysis bullosa with mutations of the laminin-5 gene, we propose the name acquired junctional epidermolysis bullosa for this newly recognized disease.

Hemidesmosomes show abnormal association with the keratin filament network in junctional forms of epidermolysis bullosa.

Junctional epidermolysis bullosa is a group of hereditary bullous disorders resulting from defects in several hemidesmosome-anchoring filament components. Because hemidesmosomes are involved not only in keratinocyte-extracellular matrix adherence, but also in normal anchorage of keratin intermediate filaments to the basal keratinocyte membrane, we questioned whether this intracellular function of hemidesmosomes was also perturbed in junctional epidermolysis bullosa. We used quantitative electron microscopic methods to assess certain morphologic features of hemidesmosome-keratin intermediate filaments interactions in skin from normal subjects (n = 11) and from patients with different forms of junctional epidermolysis bullosa (n = 13). In addition, skin from patients with autosomal recessive epidermolysis bullosa simplex with plectin defects (n = 3) or with autosomal recessive dystrophic epidermolysis bullosa (n = 4) were included as controls. Values were expressed as a percentage of the total number of hemidesmosomes counted. In normal skin 83.3% +/- 3.3 (SEM) hemidesmosomes were associated with keratin intermediate filaments and 90.1% +/- 1.9 had inner plaques. In Herlitz junctional epidermolysis bullosa (laminin 5 abnormalities, n = 4) these values were reduced to 45.3% +/- 11.5 (p < 0.001; analysis of variance) and 50.3% +/- 12.8 (p < 0.001), respectively. In junctional epidermolysis bullosa with pyloric atresia (alpha6beta4 abnormalities, n = 3) the values were also reduced [41.8% +/- 7.0 (p < 0.001) and 44.5% +/- 5.7 (p < 0.001), respectively]. In the non-Herlitz group (laminin 5 mutations, n = 3) the counts were 66.7% +/- 7.1 (p > 0.05) and 70.5% +/- 8.5 (p < 0.05), and in skin from patients with bullous pemphigoid antigen 2 mutations (n = 3) the counts were 54.3% +/- 13.8 (p < 0.01) and 57.1% +/- 13.9 (p < 0.01). In epidermolysis bullosa simplex associated with plectin mutations the values were 31.9% +/- 8.9 (p < 0.001) for keratin intermediate filaments association and 39.9% +/- 7.1 (p < 0.001) for inner plaques. Findings in recessive dystrophic epidermolysis bullosa patients' skin were indistinguishable from normal control skin with inner plaques (90.5% +/- 2.5) and keratin intermediate filaments attachment (86.3% +/- 2.1). These findings suggest that the molecular abnormalities underlying different forms of junctional epidermolysis bullosa appear to affect certain critical intracellular functions of hemidesmosomes, such as the normal connections with keratin intermediate filaments. This may have important implications for the maintenance of basal keratinocyte integrity and resilience in junctional epidermolysis bullosa.