Ellen Pfendner

Epidermolysis bullosa. II. Type VII collagen mutations and phenotype–genotype correlations in the dystrophic subtypes

Background: The dystrophic forms of epidermolysis bullosa (DEB), a group of heritable blistering disorders, show considerable phenotypic variability, and both autosomal dominant and autosomal recessive inheritance can be recognised. DEB is derived from mutations in the type VII collagen gene (COL7A1), encoding a large collagenous protein that is the predominant, if not exclusive, component of the anchoring fibrils at the dermal–epidermal junction. Methods: The Dystrophic Epidermolysis Bullosa Research Association Molecular Diagnostics Laboratory (Philadelphia, Pennsylvania, USA), established in 1996, has analysed more than 1000 families with different forms of epidermolysis bullosa, among them 332 families with DEB. DNA specimens were subjected to mutation analysis by polymerase chain reaction (PCR) amplification of all 118 exons and flanking intronic sequences of COL7A1, followed either by heteroduplex scanning and sequencing of the PCR products demonstrating heteroduplexes or by direct nucleotide sequencing. Results: 355 mutant alleles out of the anticipated 438 (81.1%) were disclosed. Among these mutations, a total of 242 mutations were distinct and 138 were novel, previously unreported mutations. No evidence of mutations in any other gene was obtained. Discussion: Examination of the mutation database suggested phenotype–genotype correlations, contributing to the improved subclassification of DEB with prognostic implications. The mutation information also forms the basis for accurate genetic counselling and prenatal diagnosis in families at risk for recurrence.

Mutation Detection in the ABCC6 Gene and Genotype-Phenotype Analysis in a Large International Case Series Affected by Pseudoxanthoma Elasticum

Background: Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder with considerable phenotypic variability, mainly affects the eyes, skin and cardiovascular system, characterised by dystrophic mineralization of connective tissues. It is caused by mutations in the ABCC6 (ATP binding cassette family C member 6) gene, which encodes MRP6 (multidrug resistance-associated protein 6). Objective: To investigate the mutation spectrum of ABCC6 and possible genotype–phenotype correlations. Methods: Mutation data were collected on an international case series of 270 patients with PXE (239 probands, 31 affected family members). A denaturing high-performance liquid chromatography-based assay was developed to screen for mutations in all 31 exons, eliminating pseudogene coamplification. In 134 patients with a known phenotype and both mutations identified, genotype–phenotype correlations were assessed. Results: In total, 316 mutant alleles in ABCC6, including 39 novel mutations, were identified in 239 probands. Mutations were found to cluster in exons 24 and 28, corresponding to the second nucleotide-binding fold and the last intracellular domain of the protein. Together with the recurrent R1141X and del23–29 mutations, these mutations accounted for 71.5% of the total individual mutations identified. Genotype–phenotype analysis failed to reveal a significant correlation between the types of mutations identified or their predicted effect on the expression of the protein and the age of onset and severity of the disease. Conclusions: This study emphasises the principal role of ABCC6 mutations in the pathogenesis of PXE, but the reasons for phenotypic variability remain to be explored.

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. I. Molecular genetics of the junctional and hemidesmosomal variants

Introduction: Epidermolysis bullosa (EB), a group of autosomal heritable blistering diseases, is characterised by extensive phenotypic variability with considerable morbidity and mortality. EB is classified into distinct subtypes depending on the location of blistering within the cutaneous dermoepidermal basement membrane zone. Ten genes are known to harbour mutations in the major types of EB, and the level of expression of these genes within the cutaneous basement membrane zone and in extracutaneous tissues, as well as the types and combinations of the mutations, explain in general terms the phenotypic variability. Methods: The DebRA Molecular Diagnostics Laboratory, established in 1996 and supported in part by the patient advocacy organisation DebRA of America, has analysed over 1000 families with different forms of EB. Results: In total, 265 cases were submitted with the preliminary diagnosis of junctional or hemidesmosomal forms of EB. We found 393 mutant alleles in seven different genes, with 173 of the mutations being distinct and 71 previously unpublished. Discussion: These findings attest to the clinical and molecular heterogeneity of the junctional and hemidesmosomal subtypes of EB. The results also reveal exceptions to the general rules on genotype-phenotype correlations, unusual phenotypes, and surprising genetics. Collectively, mutation analysis in different forms of EB provides the basis for improved classification with prognostic implications and for prenatal and preimplantation diagnosis in families at risk for recurrence of EB.

Progress in epidermolysis bullosa: the phenotypic spectrum of plectin mutations

Abstract:  Plectin, a large multidomain adhesive protein with versatile binding functions, is expressed in a number of tissues and cell types. In the skin, plectin is a critical component of hemidesmosomes, interacting with keratin intermediate filaments and β4 integrin. Mutations in the plectin gene (PLEC1) result in fragility of skin, demonstrating blister formation at the level of hemidesmosomes. These blistering disorders belong to the spectrum of epidermolysis bullosa (EB) phenotypes, and three distinct variants because of plectin mutations have been identified. First, EB with muscular dystrophy, an autosomal recessive syndrome, is frequently caused by premature termination codon‐causing mutations leading to the absence of plectin both in the skin and in the muscle. Second, a heterozygous missense mutation (R2110W) in PLEC1 has been documented in patients with EB simplex of the Ogna type, a rare autosomal dominant disorder. Finally, recent studies have disclosed plectin mutations in patients with EB with pyloric atresia, an autosomal recessive syndrome, frequently with lethal consequences. Collectively, these observations attest to the phenotypic spectrum of plectin mutations, and provide the basis for accurate genetic counselling with prognostic implications, as well as for prenatal diagnosis in families at the risk of recurrence of the disease.

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.

Probing the fetal genome: progress in non-invasive prenatal diagnosis

Progress in our understanding of the molecular basis of heritable diseases, through identification of specific mutations, has provided a foundation for the development of DNA-based prenatal diagnosis. Genetic analysis of fetal DNA is now routinely performed from chorionic villus samples obtained as early as the tenth week of gestation or by amniocentesis from week 15 onwards. However, both of these approaches involve invasive procedures with increased risk of fetal loss. To avoid such complications, attempts have been made to develop non-invasive tests through the identification, characterization and isolation of fetal cells or free fetal DNA from the maternal circulation. Recently, progress has been made towards the development of novel strategies that are expected to provide non-invasive means for early prenatal diagnosis in pregnancy.

Cloning of multiple keratin 16 genes facilitates prenatal diagnosis of pachyonychia congenita type 1

Pachyonychia congenita type 1 (PC‐1) is an autosomal dominant ectodermal dysplasia characterized by severe nail dystrophy, focal non‐epidermolytic palmoplantar keratoderma (FNEPPK) and oral lesions. We have previously shown that mutations in keratin K16 cause fragility of specific epithelia resulting in phenotypes of PC‐1 or FNEPPK alone. These earlier analyses employed an RT‐PCR approach to avoid amplification of K16‐like pseudogenes. Here, we have cloned the K16 gene (KRT16A) and two homologous pseudogenes (ψKRT16B and ψKRT16C), allowing development of a genomic mutation detection strategy based on a long‐range PCR, which is specific for the functional K16 gene. We report a novel heterozygous 3 bp deletion mutation (388del3) in K16 in a sporadic case of PC‐1. The mutation was detected in genomic DNA and confirmed at the mRNA level by RT‐PCR, showing that our genomic PCR system is reliable for K16 mutation detection. Using this system, we carried out the first prenatal diagnosis for PC‐1 using CVS material, correctly predicting a normal fetus. This work will greatly improve K16 mutation analysis and allow predictive testing for PC‐1 and the related phenotype of FNEPPK. Copyright

Severe mucous membrane involvement in epidermolysis bullosa simplex with muscular dystrophy due to a novel plectin gene mutation

Epidermolysis bullosa simplex with muscular dystrophy (OMIM 226670) is an autosomal recessive disorder caused by mutations of the human plectin gene on chromosome 8q24. Here, we report a 3-year-old girl, offspring of a consanguineous Lebanese family, who presented with skin blistering and recurrent episodes of severe respiratory distress necessitating tracheotomy at the age of 2 years. Repeated examination did not provide any evidence of muscle involvement. Indirect immunofluorescence analysis of a diagnostic skin biopsy with four different domain specific plectin antibodies showed a complete absence of plectin staining. Mutation analysis revealed a novel homozygous single guanine insertion mutation (5588insG/5588insG) residing in the N-terminal part of exon 31 of the plectin gene. Conclusion: the complete lack of protein expression, which may be attributed to a nonsense-mediated plectin mRNA decay, is likely to cause muscular dystrophy and other multisystem involvement later in life.