Immunofluorescence mapping, electron microscopy and genetics in the diagnosis and sub-classification of inherited epidermolysis bullosa: a single-centre retrospective comparative study of 87 cases with long-term follow-up.

BACKGROUND: Epidermolysis bullosa (EB) comprises a heterogeneous group of skin fragility disorders, classified in four major types based on skin cleavage level, i.e. EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), Kindler EB, and in more than 30 subtypes defined by the combination of laboratory and clinical data, including disease course. OBJECTIVES: Our aims were to address whether, in the age of genomics, electron microscopy (TEM) has still a role in diagnosing EB, and whether the genotype per se may be sufficient to sub-classify EB. METHODS: A thoroughly characterized single-centre EB case series was retrospectively evaluated to compare the power of TEM with immunofluorescence mapping (IFM) in establishing the EB type, and the ability of TEM, IFM and genetics to predict selected EB subtypes, i.e. severe dominant EBS (DEBS), severe JEB, severe recessive DEB (RDEB) and DEB self-improving, using genetic and final diagnosis, respectively, as gold standard. RESULTS: The series consisted of 87 patients, including 44 newborns, with a median follow-up of 54 months. Ninety-five mutations were identified in EB-associated genes, including 25 novel variants. Both IFM and TEM were diagnostic in about all cases of JEB (21/21 for both) and DEB (43/44 for IFM, 44/44 for TEM). TEM sensitivity was superior to IFM for EBS (19/20 vs. 16/19). As to EB subtyping, IFM performed better than genetics in identifying severe JEB cases due to laminin-332 defect (14/14 vs. 10/14) and severe RDEB (eight/nine vs. seven/nine). Genetics had no role in self-improving DEB diagnosis; it almost equalled TEM in predicting severe DEBS (eight/nine vs. nine/nine) and enabled to discriminate dominant from recessive non-severe DEB phenotypes and to identify special subtypes, e.g. DEBS with KLHL24 mutations. CONCLUSIONS: Transmission electron microscopy remains relevant to the diagnosis of EBS. IFM and genetics are essential and complementary tools in the vast majority of EB cases.

Retrospective comparison of qualitative and quantitative reverse transcriptase polymerase chain reaction in diagnosing and monitoring the ALL1-AF4 fusion transcript in patients with acute lymphoblastic leukaemia.

We compared quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) to qualitative RT-PCR in determining response to therapy and predicting clinical outcome in 18 retrospectively selected patients with ALL positive for the ALL1-AF4 fusion and with frozen RNA samples collected at diagnosis and during follow-up (96 samples analysed). The ALL1-AF4 junction was detected by qualitative RT-PCR in 18 patients and by Q-RT-PCR in 17 patients (one patient harboured the rare e10-e6 ALL1-AF4 junction, which falls outside of the primer and probe location designed for the Q-RT-PCR). In three of the 12 patients negative to qualitative RT-PCR after induction therapy, a small number of ALL1-AF4 copies was detected by Q-RT-PCR. Thus nine patients were negative and eight positive. Seven of the eight positive patients suffered a relapse, including two of the three patients positive to Q-RT-PCR yet negative to qualitative RT-PCR. Moreover, we found two (5%) discordant results among the 39 follow-up tests of the nine patients who converted to a negative qualitative-quantitative PCR status. The results suggest that qualitative RT-PCR is more appropriate for the routine diagnosis of this genetic alteration. However, Q-RT-PCR is more accurate in assessing the molecular response after induction treatment and could be more useful in clinical decision-making in ALL1-AF4-positive ALL patients.

Herlitz junctional epidermolysis bullosa: laminin-5 mutational profile and carrier frequency in the Italian population.

BACKGROUND: Herlitz junctional epidermolysis bullosa (HJEB; MIM 226700) is a rare epithelial adhesion disorder caused by null mutations in any of the three genes encoding the alpha3, beta3 and gamma2 chains of laminin-5, and is mainly characterized by extensive mucocutaneous blistering, recurrent infections and early lethality. OBJECTIVES: To perform immunoepitope mapping, electron microscopy and molecular analysis of five Italian patients with HJEB in order to complete the clinical and molecular characterization of patients with HJEB collected in the Italian Registry of hereditary epidermolysis bullosa (IRHEB) and to calculate the HJEB carrier frequency in this population. METHODS: Skin biopsies from perilesional skin of all patients were employed for immunoepitope mapping and electron microscopy examination. Blood genomic DNA was used for mutation analysis in the LAMA3, LAMB3 and LAMC2 genes by heteroduplex scanning, preceded by a search for Italian recurrent mutations. Carrier frequency calculation was performed assuming Hardy-Weinberg equilibrium. RESULTS: Two novel mutations in the LAMA3 (p.R782X) and LAMC2 (c.3235delA) genes, as well as three known and recurrent mutations in the LAMB3 (c.31insC and p.R81X) and LAMC2 (p.Y355X) genes were identified. Based on disease incidence reported in the IRHEB and the prevalence of mutations in each laminin-5 gene, the population carrier risk for HJEB was calculated to be one in 375. CONCLUSIONS: Our delineation of a laminin-5 mutational spectrum in the general Italian population provides a solid basis for expedited diagnosis, accurate genetic counselling and DNA-based prenatal testing for Italian families at risk for recurrence of HJEB.

Dystrophic epidermolysis bullosa pruriginosa in Italy: clinical and molecular characterization.

Dystrophic epidermolysis bullosa (DEB) pruriginosa (DEB-Pr) is a rare variant of DEB due to COL7A1 dominant and recessive mutations, which is characterized by severe itching and lichenoid or nodular prurigo-like lesions, mainly involving the extremities. Less than 30 patients have been described showing variable disease expression, and frequently, delayed age of onset. We report the clinical and molecular characterization of seven Italian DEB patients, three affected with recessive DEB-Pr and four with dominant DEB-Pr. In all the patients, the signs were typical of a mild DEB phenotype, until the onset of pruritus, which was followed by worsening of the clinical picture, with appearance of the distinctive lichenified lesions of DEB-Pr. Nine mutations were found in the COL7A1 gene, three of which were novel and one was de novo. DEB-Pr patients with either dominant or recessive mutations were shown to synthesize a normal or variably reduced amount of type VII collagen, which was correctly deposited at the dermal-epidermal junction. Since six of these mutations have been reported in DEB patients in the absence of intense pruritus, these data implicate a role of yet unidentified phenotype-modifying factors in the pathogenesis of DEB-Pr.