Aoi Nakano

Laminin 5 mutations in junctional epidermolysis bullosa: molecular basis of Herlitz vs non-Herlitz phenotypes

Abstract. Junctional epidermolysis bullosa (JEB) is a group of heritable blistering diseases in which tissue separation occurs within the lamina lucida of the cutaneous basement membrane zone. Clinically, two broad subcategories have been recognized: The Herlitz variant (H-JEB; OMIM 226700) is characterized by early demise of the affected individuals, usually within the first year of life, while non-Herlitz (nH-JEB; OMIM 226650) patients show a milder phenotype with life-long blistering, yet with normal lifespan. In this study, we have examined a cohort of 27 families, 15 with Herlitz and 12 with non-Herlitz JEB, for mutations in the candidate genes, LAMA3, LAMB3, and LAMC2, encoding the subunit polypeptides of laminin 5. The mutation detection strategy consisted of PCR amplification of all exons in these genes, followed by heteroduplex scanning and nucleotide sequencing. We were able to identify pathogenic mutations in both alleles of each proband, the majority of the mutations being in the LAMB3 gene. Examination of the mutation database revealed that most cases with Herlitz JEB harbored premature termination codon (PTC) mutations in both alleles. In non-Herlitz cases, the PTC mutation was frequently associated with a missense mutation or a putative splicing mutation in trans. In three cases with putative splicing mutations, RT-PCR analysis revealed a repertoire of splice variants in-frame, predicting the synthesis of either shortened or lengthened, yet partly functional, polypeptides. These observations would explain the relatively mild phenotype in cases with splicing mutations. Collectively, these findings, together with the global laminin 5 mutation database, contribute to our understanding of the genotype/phenotype correlations explaining the Herlitz vs non-Herlitz phenotypes.

Epidermolysis Bullosa with Congenital Pyloric Atresia: Novel Mutations in the β4 Integrin Gene (ITGB4) and Genotype/Phenotype Correlations

Epidermolysis bullosa with pyloric atresia (EB-PA: OMIM 226730), also known as Carmi syndrome, is a rare autosomal recessive genodermatosis that manifests with neonatal mucocutaneous fragility associated with congenital pyloric atresia. The disease is frequently lethal within the first year, but nonlethal cases have been reported. Mutations in the genes encoding subunit polypeptides of the α6β4 integrin (ITGA6 and ITGB4) have been demonstrated in EB-PA patients. To extend the repertoire of mutations and to identify genotype-phenotype correlations, we examined seven new EB-PA families, four with lethal and three with nonlethal disease variants. DNA from patients was screened for mutations using heteroduplex analysis followed by nucleotide sequencing of PCR products spanning all β4 integrin-coding sequences. Mutation analysis disclosed 12 distinct mutations, 11 of them novel. Four mutations predicted a premature termination codon as a result of nonsense mutations or small out-of-frame insertions or deletions, whereas seven were missense mutations. This brings the total number of distinct ITGB4 mutations to 33. The mutation database indicates that premature termination codons are associated predominantly with the lethal EB-PA variants, whereas missense mutations are more prevalent in nonlethal forms. However, the consequences of the missense mutations are position dependent, and substitutions of highly conserved amino acids may have lethal consequences. In general, indirect immunofluorescence studies of affected skin revealed negative staining for β4 integrin in lethal cases and positive, but attenuated, staining in nonlethal cases and correlated with clinical phenotype. The data on specific mutations in EB-PA patients allows prenatal testing and preimplantation genetic diagnosis in families at risk.

Nephrotic Syndrome and Aberrant Expression of Laminin Isoforms in Glomerular Basement Membranes for an Infant With Herlitz Junctional Epidermolysis Bullosa

Herlitz junctional epidermolysis bullosa (H-JEB) is a hereditary bullous disease caused by absent expression of laminin-5, a component of anchoring filaments within the dermal-epidermal basement membrane zone. Affected individuals usually die during the first 1 year of life. We studied an infant with H-JEB who presented with nephrotic syndrome, a previously unreported complication that may contribute to early death in this disease. DNA analysis revealed a compound heterozygote for mutations 2379delG and Q995X in the LAMB3 gene. The patient had massive albuminuria, attributable to failure of the glomerular filtration barrier, and high urinary N-acetylglucosaminidase levels, indicating renal tubular involvement. Electron-microscopic examination of the renal tissue revealed diffuse fusion of the foot processes, irregular swelling of the lamina rara interna, and disappearance of endothelial cell fenestrations. Immunohistopathologic analysis of the patient’s renal tissue revealed compositional changes in laminin isoforms of the glomerular basement membrane and no detectable laminin-5 in the renal tubular basement membrane, which suggests that laminin-5 may play an important role in renal function. Our findings strongly suggest that H-JEB should be considered in the spectrum of congenital nephrotic syndromes. Combination therapy with meticulous skin care and treatment strategies established for congenital nephrotic syndromes may rescue patients with this disease.

Novel COL7A1 mutations in a Japanese family with transient bullous dermolysis of the newborn associated with pseudosyndactyly

Transient bullous dermolysis of the newborn (TBDN) is a neonatal blistering disease evident at birth or shortly thereafter. A key feature of TBDN is that the blistering decreases with increased age, and most lesions heal within several months to leave minimal scarring and pigmentation. Although the blistering can be widespread and occasionally affects the mucous membranes, TBDN rarely involves skeletal abnormalities such as pseudosyndactyly, so this subtype of epidermolysis bullosa (EB) is thought to be relatively benign. Retention of collagen VII within the epidermis is observed histologically, and dilated rough endoplasmic reticulum and inclusion bodies, known as stellate bodies, are visible by electron microscopy in basal ⁄suprabasal keratinocytes of the patient’s skin. We herein report novel COL7A1 gene mutations in a Japanese family with TBDN associated with pseudosyndactyly.

ZNT4 gene is not responsible for acrodermatitis enteropathica in Japanese families

We read with great interest a recent paper by Küry et al. (2001), describing the absence of mutations in the human ZNT4 gene (also known as SLC30A4) in 10 patients with acrodermatitis enteropathica (AE). AE is a rare autosomal recessive disease manifesting with acral dermatitis and low serum zinc level. ZNT4 encodes one of the zinc-transport proteins, Znt4, and is expressed in mammary epithelia and brain (Huang and Gitschier 1997). A previous study has revealed that the lethal milk mouse, a putative animal model for AE, is caused by a nonsense mutation (R297X) in mouse Znt4 (Huang and Gitschier 1997), suggesting a possible relationship between AE and human ZNT4, which has been mapped to chromosomal region 15q15 (Küry et al. 2001). In search of the candidate gene for AE, we have characterized the exon/intron organization of the human ZNT4 gene and screened ZNT4 for mutations in two Japanese AE families.

A novel H1 domain mutation in the keratin 2 gene in a Japanese family with ichthyosis bullosa of Siemens.

Ichthyosis bullosa of Siemens (IBS) is a rare autosomal dominant skin disorder characterized by blister formation in the upper suprabasal layers of the epidermis. Clinical features of the disease, which include blistering after mechanical trauma, lichenified hyperkeratosis over the flexural areas of the limbs, and superficial peeling of the skin, are similar to those of epidermolytic hyperkeratosis/bullous congenital ichthyosiform erythroderma (EHK/BCIE). This clinical resemblance makes diagnosis difficult. Recent molecular studies have identified mutations in the keratin 2 (K2) gene (KRT2; this new designation is used throughout this report according to the current nomenclature for mammalian keratins) in patients with IBS, in contrast to the keratin 1 or 10 mutations that have been detected in patients with EHK/BCIE, enabling us to differentiate between IBS and EHK/BCIE. Thus far, 29 missense mutations that result in amino acid substitutions in K2 have been identified in patients with IBS; all such mutations reside in the helix initiation or termination motifs of K2. Here, we describe a novel mutation in the H1 region of K2 in a Japanese family with IBS.

Dominant dystrophic epidermolysis bullosa caused by a novel G2037R mutation and by a known G2028R mutation in the type VII collagen gene (COL7A1)

An autosomal dystrophic epidermolysis bullosa (DDEB) is a hereditary mechanobullous disease characterized by blistering of the skin and the mucous membrane. DDEB is caused by a heterozygous mutation in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils, and phenotypically classified into several types. We experienced two boys with DDEB and examined the mutation analyses of the COL7A1 genes of the two patients and their fathers to clarify the relationship between the genotypes and phenotypes, that is, the mutation sites of COL7A1 gene and the clinical types of DDEB. The case 1 and 2 patients and their fathers revealed a heterozygous nucleotide G to A transition at position 6109 and 6082 in 73 exon of COL7A1, which resulted in a glycine to arginine substitution (G2037R and G2028R), respectively. G2037R found in the case 1 patient was a novel mutation. There was no clear relationship recognized between the two mutation sites in the COL7A1 gene and the clinical variations.