Yasuko Takizawa

Epidermolysis bullosa simplex associated with muscular dystrophy: Phenotype-genotype correlations and review of the literature ☆ ☆☆ ★

BACKGROUND Epidermolysis bullosa simplex associated with muscular dystrophy (EBS-MD; OMIM# 226670) is an autosomal recessive disorder caused by genetic defects in the plectin gene. Because EBS-MD is relatively rare, and gene defects have been elucidated only in a limited number of patients, the precise phenotype-genotype correlations have not yet been fully elucidated. OBJECTIVE The purpose of this study was to define clinical features of EBS-MD and to clarify its phenotype-genotype correlations. METHODS Clinical, ultrastructural, immunohistochemical, and molecular features of 4 unrelated Japanese patients with EBS-MD were recorded. In addition, 6 cases with defined plectin gene mutations reported in the literature were reviewed. RESULTS In skin of the EBS-MD patients, the blister formation always occurs just above the hemidesmosomes, and expression of plectin is absent or markedly reduced in all cases examined. All 10 patients, including 6 cases in the literature, showed generalized blistering at birth or soon thereafter, and experienced nail deformities. In addition, decayed teeth (5 cases), urethral strictures (3), mild palmoplantar hyperkeratosis (2), infantile respiratory complications (2), alopecia (1), and laryngeal webs (1) were present. All 8 patients who were older than 9 years demonstrated considerable muscle weakness, and the majority of them ended up being wheelchair bound. Among the 10 patients, 7 were products of consanguineous marriage, 9 have premature termination codon (PTC) mutations in both alleles of the plectin gene, and 7 cases were homozygous for the mutation. One patient who is homozygous for a 2719del9 in-frame deletion mutation that resulted in elimination of 3 amino acids, QEA, could still walk at the age of 46 and showed milder clinical severity. CONCLUSION EBS-MD reveals clinical features not only characteristic of EBS and MD, but also other manifestations including urethral, dental, and respiratory complications. The majority of patients are products of consanguineous marriage and have homozygous plectin gene mutations. Whereas patients with PTC mutations in both alleles typically showed severe clinical features of EBS-MD and ended up being wheelchair bound, a homozygous patient for an in-frame deletion mutation showed positive, yet attenuated, plectin expression and milder clinical phenotype. Thus plectin immunofluorescence, combined with identification of the underlying plectin mutations, is of value in predicting the severity of the muscle involvement that occurs later in life of patients with EBS-MD.

Glycine substitution mutations by different amino acids in the same codon of COL7A1 lead to heterogeneous clinical phenotypes of dominant dystrophic epidermolysis bullosa

Abstract Dystrophic epidermolysis bullosa (DEB), caused by mutations in the gene encoding type VII collagen (COL7A1), is known to show heterogeneous clinical phenotypes. Certain correlations between the nature or position of COL7A1 mutations and the resultant DEB phenotypes have been suggested, although such relationships may be more complex than initially thought. The purpose of the present study was to clarify the molecular basis of two different subtypes of dominant DEB (DDEB), EB pruriginosa and classical type. Interestingly, we found that both cases were caused by a missense glycine substitution mutation by different amino acids in the same codon of COL7A1 (G2028R and G2028A). These results further support the notion that different glycine substitution mutations in the same codon can lead to heterogeneous clinical phenotypes of DDEB, EB pruriginosa and classical type.

A recurrent keratin 14 mutation in Dowling-Meara epidermolysis bullosa simplex.

SIR, Epidermolysis bullosa simplex (EBS) is an inherited skinfragility disorder characterized by a cytolysis of basal keratinocytes and epidermal blistering, usually inherited in an autosomal dominant fashion in which various genetic mutations have been found in the keratin genes encoding K14 and K5. We present a Japanese family with the Dowling– Meara type of EBS (EBS–DM) caused by recurrent K14 mutation, in which four family members demonstrated heterogeneous clinical severity. The 5-year-old elder sister of the family showed the most severe clinical phenotype, with extensive generalized blisters and erosions typical of EBS–DM over the whole body from birth (Fig. 1a–d). Her 6-month-old younger sister presented with blisters at the age of 3 days, but they have always been restricted to the hands and feet (Fig. 1e). Their father (Fig. 1f) and paternal grandmother developed blisters mainly on the palms and soles in childhood, which improved markedly in adult life, and had only diffuse postinflammatory pigmentation on the abdomen. Electron microscopy revealed cytolysis of the basal cells and suprabasal blister formation in the skin of all the patients, while electrondense clumped tonofilaments were observed only in the skin of the two sisters, not in that of the father. For mutation analysis, genomic DNA extracted from peripheral leucocytes was subjected to extra-long polymerase chain reaction (PCR) amplification from the first to the seventh exon of the K14 gene using forward primer 5-GGTC CGAT GGGA AAGT GTAG CCTG CAGG CC-3 and backward primer 5-CGCT GTAG GGCC CTTG TTCA TGGT GCTG AC-3. Several pairs of PCR primers specific to the K14 gene were designed based on the sequence of K14. DNA fragments containing each exon were amplified using the PCR product obtained by extra-long PCR as a template. The primers used in the PCR assay for exon 1 of K14 were: 5-CTCT GGGG GAGC CTAT GGGT-3, 5-GAGC TAGC TGGA ATGG TGCC-3. Heteroduplex analysis of the PCR products covering exon 1 of the K14 gene exhibited abnormal bands in electrophoretic mobility in the samples from the patients (data not shown). Direct sequencing performed using an automatic sequencer (Perkin Elmer-Applied ABI 310A, Perkin-Elmer Corporation, CA, U.S.A.) and PRISM fluorescent dye-terminator system (Perkin-Elmer Corporation) revealed that all the patients carried a C→T transition at the first nucleotide of codon 125 of K14 gene that resulted in the substitution of a cysteine for arginine (K14R125C) (Fig. 2a). To confirm the nucleotide substitution, we carried out an allele-specific oligonucleotide hybridization assay (ASO). DNA probes used in ASO were: 5CTCA ATGA CCGC CTGG C-3 for the wild-type allele and 5CTCA ATGA CTGC CTGG C-3 for the mutant-type allele of exon 1 of K14. Southern hybridization of the PCR products for exon 1 of K14 with oligonucleotide probes labelled with P confirmed the presence of K14R125C in all the patients, but not in the unaffected individuals (Fig. 2b,c). In the literature, pathogenic mutations have been identified in 25 unrelated kindreds with EBS–DM, including five Japanese families. Of the 26 families, including the present case, 19 have mutations in K14 and seven in K5. Interestingly, eight of 26 families have the K14R125C mutation. Moreover, this K14R125C mutation was identified in four of six Japanese families. Our results further support the notion that K14R125C is indeed a recurrent mutation that might be particularly common among Japanese EBS–DM patients. British Journal of Dermatology 1999; 141: 747–776.

Absence of R42X and R635X mutations in the LAMB3 gene in 12 Japanese patients with junctional epidermolysis bullosa

Epidermolysis bullosa (EB), a heterogeneous group of inherited blistering skin diseases, is divided into three major categories on the basis of the level of tissue separation [4, 15]. In Junctional EB (JEB), the separation of the skin occurs within the lamina lucida of the cutaneous basement membrane zone. Conventionally, JEB has been divided into lethal (Herlitz) and nonlethal (non-Herlitz) types on the basis of the clinical findings, although there is considerable clinical heterogeneity [4]. Generalized atrophic benign EB (GABEB) is a rare form of nonlethal JEB. Laminin 5, a candidate gene/protein system for most patients with JEB, consists of three subunit polypeptide chains, α3, β3 and γ2, encoded by distinct genes LAMA3, LAMB3 and LAMC2, respectively. Abnormalities of laminin 5 expression have been shown to be present in most patients with JEB, and mutations in all three genes encoding laminin 5 (LAMA3, LAMB3 and LAMC2) have been reported in different JEB families [1, 5, 8–10]. Recent studies have delineated two recurrent mutations in the LAMB3 gene, R42X and R635X, which account for approximately 50% of all mutant laminin 5 alleles in European/North American families with JEB [6]. The objective of the present study was to assess the prevalence of these recurrent mutations in Japanese patients with different subtypes of JEB. Included in this study were 12 Japanese families with JEB (Table 1). The diagnosis was based on a combination of clinical observations, electron microscopy and immunofluorescence staining with a range of basement membrane zone monoclonal antibodies. The subtypes of JEB among these 12 patients are shown in Table 1, and included lethal Herlitz variant (n = 5), GABEB (n = 2), and other nonlethal variants of JEB (n = 5). In all cases, tissue separation at the level of the lamina lucida was confirmed by electron microscopy. In nine cases, fresh skin samples were available for indirect immunofluorescence as described previously [14]. Briefly, skin samples obtained from the patients were embedded in OCT compound, snap-frozen in isopentane cooled with liquid nitrogen and stored at H. Shimizu · Y. Takizawa · J. A. McGrath · L. Pulkkinen · A. M. Christiano · J. Uitto · R. E. Burgeson · K. Iwatsuki · N. Niimi · M. Noguchi · S. Imayama · Y. Abe · Y. Shirakata · S. Hagiwara · T. Saida · H. Ogawa · I. Hashimoto · T. Nishikawa

A novel homozygous mutation 371delA in TGM1 leads to a classic lamellar ichthyosis phenotype

Malformation of the cornified cell envelope (CCE) arising from mutations of the transglutaminase (TGase) 1 gene (TGM1) is the cause of some cases of lamellar ichthyosis (LI). However, genotype/phenotype correlation in TGM1 mutations has not yet been fully clarified. We report a typical case of LI caused by a novel mutation in TGM1. The patient, a 33‐year‐old woman, showed thick, lamellar scales on the entire body surface. Immunofluorescence labelling with anti‐TGase 1 antibodies was negative in the patients epidermis. In situ TGase activity assay detected markedly reduced TGase activity in granular layers of the patients epidermis. Electron microscopy revealed incomplete thickening of the CCE during keratinization in the epidermis. Sequencing of the entire exons and exon‐intron borders of TGM1 revealed that the patient was a homozygote for a novel deletion mutation 371delA in exon 3. This mutation leads to a frameshift resulting in a premature termination codon 43 bp downstream from the mutation site. According to the protein modelling of TGase 1, the truncated protein from this mutated allele loses the entire catalytic core domain of TGase 1. Thus, the present homozygous mutation is expected to cause total loss of TGase 1 activity, resulting in large, dark, lamellar scales on the entire body, the classic phenotype of LI, in this patient.

Pyloric atresia‐junctional epidermolysis bullosa syndrome showing novel 594insC/Q425P mutations in integrin β4 gene (ITGB4)

Abstract:  Pyloric atresia‐junctional epidermolysis bullosa syndrome (PA‐JEB) is an autosomal recessive inherited rare blistering disorder caused by mutations in ITGA6 or ITGB4, genes encoding integrin α6 or β4, respectively. In this study, we have disclosed the mutations in ITGB4 in a Korean patient with PA‐JEB. The proband, who showed skin blisters, was diagnosed as having pyloric atresia and died 2 years after birth. Mutational analysis showed a novel 594insC maternal mutation in exon 7, which led to premature termination codon (PTC), and a novel Q425P paternal mutation in exon 11. Q425P mutation was not detected in 200 alleles obtained from a normal healthy Korean control, and was shown to reduce α‐helix forming ability in integrin β4 a by Garnier α‐helicity plot of the protein, indicating that this mutation is pathogenic but not polymorphism. The phenotype in the present case can be explained by (1) the combination of PTC and missense mutation, and (2) amino‐acid substitution occurring for the amino acid not preserved in the integrin β family. Our results contribute to further the accumulation of mutation data for better understanding of the genotype/phenotype correlation in PA‐JEB, and may give profound insight into the role of integrins α6 and β4.

Electron microscopic DOPA reaction test for oculocutaneous albinism

Abstract Oculocutaneous albinism (OCA) is an autosomal recessive disorder in which the biosynthesis of melanin is reduced or absent in skin, hair and eyes. Tyrosinase-related OCA (OCA1) is caused by mutations in the tyrosinase gene. Tyrosinase-negative OCA (OCA1A) is the most severe phenotype in which tyrosinase catalytic activity is completely lost, resulting in no mature melanin pigment. Yellow OCA (OCA1B) varies from very little pigment associated with whitish-blond hair to nearly normal pigment with dark-blond hair and skin. We determined the tyrosinase activity in melanocytes by the electron microscopic dihydroxyphenylalanine (EM-DOPA) reaction test using skin samples and analyzed tyrosinase gene mutations in nine Japanese patients with OCA. In 18 alleles of nine patients, the OCA1A-associated mutations, P310insC, R77Q and R278X, were found in seven, three and one alleles, respectively. Five patients who had these mutations in both alleles showed white hair, blue eyes and white skin and demonstrated no tyrosinase activity by the EM-DOPA reaction test. Three patients who had no tyrosinase gene mutation showed tyrosinase activity and heterogeneous clinical features. One patient in whom only an R77Q OCA1A mutation was found in one allele demonstrated a reduced tyrosinase activity, indicating OCA1B. This patient had white hair at birth, but it had turned blond by the age of 1 year. These results indicate that the EM-DOPA reaction test provides clear information on the status of tyrosinase activity which is essential for the identification of the disease subtype which in turn is important for the prognosis of patients with OCA.

Structural, enzymatic and molecular studies in a series of nonbullous congenital ichthyosiform erythroderma patients

Causative gene defects have not been demonstrated in the majority of nonbullous congenital ichthyosiform erythroderma (NBCIE) cases. The purpose of this study was to further elucidate the pathogenesis of NBCIE. Immunohistochemical and ultrastructural observations, transglutaminase activity assays and sequencing of TGM1 were performed in five patients from four NBCIE families. Transglutaminase 1 (TGase 1), involucrin and loricrin expression and in situ transglutaminase activity were present in all of the cases. Ultrastructurally, two cases out of five showed incomplete thickening of the cornified cell envelope (CCE) during keratinization and the other three exhibited abnormal lipid droplets in the cornified cells and malformed lamellar granules. No TGM1 mutation was found in any of the four families by direct sequence analysis. NBCIE cases with normal TGase 1 seemed to have two distinct patterns of abnormality, one with abnormal lipid droplets and malformed lamellar granules and the other with defective CCE formation.