Acquired dermal melanocytosis of the face and extremities.

Acquired dermal melanocytosis (ADM) is a relatively rare, but well-described disease among adolescent to middle-aged East Asian women, particularly those of Japanese and Chinese descent. Clinically, ADM manifests as multiple punctate and greyish-brown pigmented areas 1-3 mm in diameter occurring on both sides of the forehead and zygomatic region. The subtype of ADM affecting the face and extremities is extremely rare even in East Asian women. We describe three patients with ADM of the face and extremities (ADMFE) and their characteristic clinical features. All patients were Japanese women, and showed multiple greyish-brown pigmentations on both nasal wings and on the extensor surface of the extremities. We found that the clinical features were strikingly uniform, and that a pigmented lesion on the nasal wing can be an important clue to distinguish ADMFE from other hyperpigmented diseases of the hands and feet. One patient was treated with Q-switched ruby laser with excellent outcome. Increased awareness of ADMFE can lead to earlier diagnosis and potential treatment.

Immunoglobulin G deposition to nonhemidesmosomal lamina lucida and early neutrophil involvement are characteristic features in a case of anti-p200 pemphigoid.

The ultrastructural characteristics and immunolocalization of in vivo bound immunoglobulin G (IgG) in skin affected by anti-p200 pemphigoid have not been elucidated. To give insight into the mechanism of blister formation we report a new case of anti-p200 pemphigoid, studied with stage-oriented morphological analysis and immunoelectron microscopy. Skin biopsy specimens were evaluated ultrastructurally and histologically with immunohistochemistry. By observing the nonblister site, the blister edge and centre of the blister, we determined that neutrophil infiltration increases gradually at the dermoepidermal junction in association with the destruction of type IV collagen. Ultrastructurally, many neutrophils were observed under the lamina densa, with vacuole formation in the dermis. At the periphery of the blister, the lamina densa became fragmented and was observed either at the roof or the floor of the blister. At the centre of the blister, the lamina densa was mainly observed at the blister floor. Postembedding immunoelectron microscopy demonstrated that the IgG, bound in vivo, localized at the lamina lucida, while the area beneath the hemidesmosomes was spared. Together with the early involvement of neutrophils and the destruction of the basal lamina, we suggest that the binding of autoantibodies to the nonhemidesmosomal lamina lucida may induce inflammation with neutrophils, resulting in blister formation.

Malignant eccrine spiradenoma: case report and review of the literature, including 15 Japanese cases.

Malignant eccrine spiradenoma (MES) is an extremely rare cutaneous malignant tumour. An 86-year-old man presented at our hospital with an enlarging tumour on the dorsum of the left hand. An excisional biopsy was taken and histological examination showed a solid island of cells of two distinct types: cells with abundant cytoplasm and oval vesicular nuclei, and small round cells with less cytoplasm and hyperchromatic nuclei with a high frequency of mitosis. We diagnosed this tumour as MES. Although we did not perform further treatment because of the patient's age, there was no sign of recurrence or metastasis in the 2 years of follow-up after excisional biopsy. We reviewed cases of malignant eccrine spiradenoma in the English and Japanese literature and found that 'sarcomatous' or 'squamous' change in histopathology was significantly correlated with a poorer prognosis. It is therefore important for treatment planning to evaluate the entire specimen histologically.

Novel mutation p.Gly59Arg in GJB6 encoding connexin 30 underlies palmoplantar keratoderma with pseudoainhum, knuckle pads and hearing loss.

BACKGROUND: Connexins, components of the gap junction, are expressed in several organs including the skin and the cochlea. Mutations in connexin genes including GJB2 (Cx26), GJB3 (Cx31), GJB4 (Cx30.3), GJB6 (Cx30) and GJA1 (Cx43) are responsible for various dermatological syndromes and/or inherited hearing loss, frequently showing overlapping phenotypes. OBJECTIVES: To clarify the spectrum of clinical phenotypes caused by connexin mutations. METHODS: We report a 32-year-old Japanese woman with mild palmoplantar keratoderma (PPK) with severe sensorineural hearing loss, knuckle pads and pseudoainhum of her toes. RESULTS: Direct sequencing revealed no mutation in GJB2, but a novel heterozygous missense mutation p.Gly59Arg in GJB6. Electron microscopy revealed no apparent morphological abnormality of gap junctions in the patient's lesional epidermis. CONCLUSIONS: The patient harboured the novel GJB6 missense mutation p.Gly59Arg in the first extracellular loop of Cx30. Mutations in glycine 59 of Cx26 are associated with PPK-deafness syndrome, and the similar phenotype here supports the observed heteromeric channel formation; the dominant nature of the mutation suggests an effect on gap junctions similar to that of the comparable mutation in Cx26.

Indeterminate cell histiocytosis successfully treated with ultraviolet B phototherapy.

Indeterminate cell histiocytosis (ICH) is a rare disorder, characterized by infiltration of the skin by neoplastic cells that are characteristically positive for S-100 and CD1a, but lack Birbeck's granules. A 75-year-old man presented with a 4-year history of multiple papules on the trunk, limbs, face and neck. Skin biopsy revealed dense infiltration of histiocytic cells that were CD1a+/S100+, but lacked Birbeck's granules. No other abnormality was seen during a general examination including a computed tomography scan of the body, gallium scintigraphy, and an abdominal sonography. Broadband ultraviolet B (UVB) treatment was used for the skin lesions, and partial but almost complete remission was obtained. The case suggests that UVB phototherapy is an option for treatment of ICH.

Novel mutation of the Notch3 gene in a Japanese patient with CADASIL.

We report a novel missense mutation of the Notch3 gene in a Japanese family with CADASIL. The Cys49Gly mutation in this family is located in exon 2 of the Notch3 gene. Most of the documented Notch3 gene mutations occur in exons 3 or 4. On the other hand, there are few reports around the world of mutations in exon 2 of the Notch3 gene, and this is the first report of a mutation in exon 2 of the gene in a Japanese family. In general, CADASIL mutations involve a cysteine residue. Such mutations may influence the tertiary structure of the Notch3 protein, resulting in protein dysfunction. Thus, the CADASIL in the present case may be a consequence of the mutation in exon 2 causing a structural change in the Notch3 protein.

Prenatal exclusion of harlequin ichthyosis; potential pitfalls in the timing of the fetal skin biopsy.

BACKGROUND: Harlequin ichthyosis (HI) is a severe and usually fatal congenital skin disorder with autosomal recessive inheritance. Several cases of HI prenatal diagnosis have been performed using fetal skin biopsy, mainly at around 23 weeks estimated gestational age (EGA), and reported in the literature. However, prenatal testing must be done earlier than 21 weeks EGA in several countries including Japan where the present HI families live, because termination is legally allowed only until 22 weeks EGA. OBJECTIVES: We report the successful prenatal exclusion of HI in two fetuses from two independent families and discuss the technical difficulties and potential pitfalls in the prenatal exclusion of HI at early gestation stages. METHODS: Fetal skin biopsy specimens and amniotic fluid samples at 19 and 20 weeks EGA from two fetuses at risk of HI were examined by light and electron microscopy. RESULTS: For the prenatal diagnosis in case 1, the fetal skin biopsy samples were obtained at 20 weeks EGA and showed normal keratinization in the hair canals; no abnormalities were observed in the keratinized cells. In case 2, the interfollicular epidermis and the hair follicles in the samples obtained at 19 weeks EGA had not differentiated enough to show proper keratinization. However, lamellar granules were normally formed in the inner root sheath cells of the late bulbous hair pegs. From these ultrastructural findings, the case 1 fetus was diagnosed as unaffected with HI, and the case 2 fetus was diagnosed as unlikely to be affected. Subsequently, both were born as healthy, unaffected babies. CONCLUSIONS: The timing of biopsies at 19 weeks EGA is not ideal for fetal skin biopsy because the samples are not always sufficiently differentiated for the prenatal diagnosis of HI. However, morphological observations of lamellar granules gives us important additional information useful for HI prenatal diagnosis.

Personal identification using Y-chromosomal short tandem repeats from bodily fluids mixed with semen.

The male-specific, human Y-chromosomal short tandem repeats (Y-STRs) are very useful in forensic analysis. The authors report a sexual crime case in which the direct Y-STR haplotype analysis of several mixtures of various bodily fluids including semen was very effective for identifying the perpetrator of the crime. The typing of three Y-STRs (DYS19, DYS389II, and DYS390) could be detected from the mixed DNA of sperm and female cells in the victim's vagina, vaginal orifice, and anus. These haplotypes originated from one man and matched those of the suspect. Accordingly, the combination of direct extraction of DNA and Y-STR haplotype analysis is considered to be very useful for mixtures of bodily fluids, including semen or other male cells.

Compound heterozygosity for a point mutation and a deletion located at splice acceptor sites in the LAMB3 gene leads to generalized atrophic benign epidermolysis bullosa.

An autosomal recessive disorder, generalized atrophic benign epidermolysis bullosa, is a rare form of nonlethal type junctional epidermolysis bullosa. It is associated not only with skin fragility but also with other unique clinical features including widespread atrophic skin changes, alopecia, reduced axillary and pubic hair, dysplastic teeth, and dystrophic nails. The majority of generalized atrophic benign epidermolysis bullosa cases are caused by mutations in the COL17A1 gene coding for type XVII collagen (or the 180 kDa bullous pemphigoid antigen). Another candidate gene for mutations in some forms of generalized atrophic benign epidermolysis bullosa is LAMB3 encoding the beta3 chain of laminin 5. This report documents compound heterozygosity for novel mutations in LAMB3 of a Japanese patient showing typical clinical features of generalized atrophic benign epidermolysis bullosa. One is an A-to-G transversion at the splice acceptor site of intron 14, which is designated as a 1977-2A-->G mutation; the other is a deletion of 94 bp located at the junction of intron 18 and exon 19, which is a 2702-29del94 mutation. Reverse transcriptase polymerase chain reaction analysis suggested skipping of exon 19 in LAMB3 mRNA produced from the allele with 2702-29del94 and impaired stability of the aberrant mRNA transcribed from the second allele with the 1977-2A-->G mutation.

Detection of antigens by immunofluorescence on ultrathin cryosections of skin.

Cryoultramicrotomy was originally established to provide ultrathin cryosections as substrates for on-section immunolabeling in immunoelectron microscopy. Recently, we recognized that ultrathin cryosections of skin (0.2 micrometer thick) could serve as substrates for immunofluorescence (IF) with excellent resolution. To assess the advantages and the limitations of IF on ultrathin cryosections, we compared the labeling of IF on 0.2-micrometer ultrathin cryosections of skin with those of routine IF on 6-micrometer cryostat sections, confocal laser scanning microscopy (LSM), and immunogold electron microscopy using several markers of keratinocyte cell surface and basement membrane zone molecules. IF on ultrathin cryosections clearly demonstrated a lack of bullous pemphigoid antigens beneath the melanocytes, desmosomal antigens as discontinuous dot-like labeling, and nondesmosomal plasma membrane antigen as a ladder-like pattern. IF on ultrathin cryosections provided convincing images with higher resolution than confocal LSM, which corresponded well to those of immunogold electron microscopy. IF on ultrathin cryosections had superior resolution compared to routine IF or confocal LSM and should serve as a powerful tool in future studies for the analysis of skin antigens. (J Histochem Cytochem 46:1455-1460, 1998)

Melanogenesis, biosynthetic phenotype of fibronectin and collagen, and migrating activity in cloned B16 mouse melanoma cells.

Three clones, melanotic (M3), amelanotic (A4 and A7) cells were isolated from B16 F10 mouse melanoma cell line. These clones exhibited different biosynthetic activities of fibronectin and collagen: A4 clone showed relatively active synthesis of both collagen and fibronectin, and A7 clone exhibited most active fibronectin synthesis, whereas no significant synthesis of these molecules was observed in M3 clone. No significant difference in growth rate was observed in these three clones. Migrating activities measured by basement membrane matrix-coated dishes were greater in A7 clone than in A4 and M3 clones. Messenger RNA levels of collagen and fibronectin paralleled collagen and fibronectin synthesis in these clones whereas tyrosinase mRNA level was unaltered between melanotic (M3) and amelanotic (A4 and A7) cells. These results indicate that B16 melanoma cells have heterogeneous cell populations consisting of different biosynthetic and metastatic properties. These clones may provide good tools for studying the relationship between the phenotypes of melanogenesis biosynthesis of extracellular macromolecules and migrating activity of melanoma cells.

Desmoyokin/AHNAK protein localizes to the non-desmosomal keratinocyte cell surface of human epidermis.

Desmoyokin, a high-molecular-weight protein of 680 kD with a 170-nm-long dumbbell shape, was originally thought to be localized to the desmosomal attachment plaque and to work as a kind of stabilizer of desmosomes. Recently, desmoyokin was shown to be widely detected in many types of cells that do not possess desmosomes. The purpose of the present study was to elucidate the precise localization and possible function of desmoyokin in human epidermis. In 0.2-micron ultrathin cryosections of human skin for immunofluorescence, anti-desmoyokin antibody showed a ladder-like staining pattern along the cell surface, whereas anti-desmocollin and anti-desmoplakin antibodies as controls showed a discontinuous dotted staining pattern, indicating their distinct localization. Post-embedding immunoelectron microscopy with cryofixation and cryosubstitution revealed that desmoyokin was localized mainly along the non-desmosomal and non-hemidesmosomal plasma membrane, but not to the desmosomes and hemidesmosomes themselves. This localization was further confirmed by double-labeling immunoelectron microscopy with antibodies against desmocollin, desmoplakin, or bullous pemphigoid antigen. Results indicate that desmoyokin was not localized to the desmosomes themselves as previously considered. Desmoyokin was localized to the non-desmosomal and non-hemidesmosomal epidermal keratinocyte cell surface as a plasma membrane-associated protein, and might play a role in cell adhesion that is not directly associated with desmosomes or hemidesmosomes.

Autoantibodies from patients with cicatricial pemphigoid target different sites in epidermal basement membrane.

Indirect immunogold electron microscopy studies of cryofixed, freeze-substituted, and post-embedded normal human skin were performed to localize precisely the ultrastructural binding site of circulating autoantibodies from two groups of patients with cicatricial pemphigoid. One group of patients had circulating IgG autoantibodies that bound the dermal side of 1 M NaCl-split skin and immunoprecipitated epiligrin. The other group of patients had circulating IgG autoantibodies directed against the epidermal side of 1 M NaCl-split skin and showed no specific reactivity to any keratinocyte polypeptide by immunoprecipitation. IgG autoantibodies from all patients with anti-epiligrin cicatricial pemphigoid bound the lowermost aspect of the lamina lucida at its interface with the lamina densa; the greatest staining was seen beneath and beside hemidesmosomes. In contrast, IgG from cicatricial pemphigoid patients whose autoantibodies bound the epidermal side of 1 M NaCl-split skin localized to hemidesmosomes and the junction between hemidesmosomes and the plasma membranes of basal keratinocytes. Although the latter staining pattern is similar to that observed with anti-BPAG2 autoantibodies, sera from our patients with cicatricial pemphigoid did not bind BPAG2 in immunoprecipitation studies of radiolabeled human keratinocyte extracts or show immunoblot reactivity to a fusion protein corresponding to the immunodominant epitope of this polypeptide. These studies demonstrate the following: 1) Autoantibodies from patients with anti-epiligrin cicatricial pemphigoid consistently bind the lower lamina lucida at its interface with the lamina densa; and 2) other patients with the same phenotype may have IgG autoantibodies against yet-unknown epitopes in basal keratinocytes.