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.

Malignant ovarian germ cell tumors in pediatric patients: The AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) study.

OBJECTIVE: Malignant ovarian germ cell tumors (MOGCT) carry an excellent prognosis, and the treatment aims to achieve results with the least possible treatment-related morbidity. The aim of this study was to assess the outcomes of pediatric patients with MOGCT. METHODS: Patients were treated according to their stage: surgery and surveillance for stage I; a modified bleomycin-etoposide-cisplatin (BEP) regimen for stages II (three cycles), III, and IV (three cycles) with surgery on residual disease. RESULTS: Seventy-seven patients were enrolled (median age 11.8 years), 26 with dysgerminoma (Dysg), 13 with immature teratoma and elevated serum alpha-fetoprotein levels (IT + AFP), and 38 with nondysgeminoma (Non-Dysg) staged as follows: 27 stage I, 13 stage II, 32 stage III, 5 stage IV. Among evaluable patients in stage I (5-year event-free survival [EFS] 72.1% [95% CI: 56.4-92.1%]; 5-year overall survival [OS] 100%), seven relapsed (three patients with Dysg and four patients with Non-Dysg) and were rescued with chemotherapy (plus surgery in three patients). Among the evaluable patients with stages II-IV, 48 (98%) achieved complete remission after chemotherapy ± surgery, one (IT + AFP, stage IV) had progressive disease. In the whole series (median follow-up 80 months), the 5-year OS and EFS were 98.5% (95% CI: 95.6-100%) and 84.5% (95% CI: 76.5-93.5%). CONCLUSIONS: We confirm the excellent outcome for MOGCT. Robust data are lacking on surgical staging, surveillance for Non-Dysg with stage I, the management of IT + AFP, and the most appropriate BEP regimen. As pediatric oncologists, we support the role of surveillance after proper surgical staging providing cases are managed by experts at specialized pediatric centers.

A truncating mutation in the laminin-332α chain highlights the role of the LG45 proteolytic domain in regulating keratinocyte adhesion and migration.

BACKGROUND: Altered function of laminin-332 (α3ß3γ2) consequent to mutations in the LAMA3, LAMB3 and LAMC2 genes causes junctional epidermolysis bullosa non-Herlitz (JEB-nH). JEB-nH patients suffer from skin blistering and have an increased risk of developing aggressive skin carcinomas in adulthood. Laminin-332 is proteolytically processed and its extracellular mature form lacks the α3 chain C-terminal globules 4 and 5 (LG45). The LG45 tandem has cell adhesion and protumorigenic properties. However, mutations that affect this domain are very rare and their functional effects in patients have not been explored to date. OBJECTIVE: To characterize molecularly an adult patient with JEB-nH and altered laminin-332 expression presenting multiple skin carcinomas, and to analyse LG45-mediated biological functions using keratinocytes from the patient. METHODS: A mutational search in laminin-332 genes was performed by hetero-duplex analysis. LAMA3 mRNA and laminin-332 protein levels in patient keratinocytes were investigated by real-time reverse transcriptase polymerase chain reaction and radioimmunoprecipitation assay, respectively. Keratinocyte migration was examined by scratch and Boyden chamber assays. RESULTS: We identified a homozygous LAMA3 mutation, p.Leu1648TrpfsX32, which truncates the last 45 amino acids of the carboxyl terminal LG5 subdomain. Gene expression studies revealed that the mutant transcripts were stable and even increased, precursor laminin-332 molecules were retained intracellularly and the amount of mature extracellular heterotrimers was reduced to about 50%. Finally, the patient's keratinocytes migrated faster than normal keratinocytes. CONCLUSIONS: Structural disruption of LG5 highlights the critical functions of the LG45 proteolytic region in precursor laminin-332 secretion and keratinocyte adhesion and migration. Perturbation of LG45 function might explain the non-aggressive behaviour of carcinomas in this patient.

Five new species of Camelobaetidius Demoulin, 1966 (Ephemeroptera: Baetidae), and redescription of Camelobaetidius mexicanus (Traver & Edmunds, 1968).

The genus Camelobaetidius Demoulin, 1966 has 38 species distributed throughout the Neotropical Region, being one of the most studied genera of the family Baetidae. Based on material from Brazil, the aim of the present paper is to describe five new species of Camelobaetidius with the terminal filament as long as cerci, and to redescribe the nymph of C. mexicanus (Traver & Edmunds, 1968) based on a paratype from Mexico.

A new species of Corinnella Thomas & Dominique, 2006 (Ephemeroptera: Baetidae).

The monotypic genus Corinnella Thomas & Dominique, 2006, was described based on nymphs from French Guyana, and since the original description nothing has been added to knowledge of its systematics. The aim of the present paper is to describe a new species of the genus from Northern Brazil, to improve its diagnoses, and to report the genus for the first time from Brazil. The new species can be recognized by: labrum with discernible anteromedial emargination and process; posterior margin of terga IV with rounded spines; and tarsal claw with seven to nine denticles, with the outer and inner denticles distinctly longer than the others.

The adults of Camelobaetidiusjanae Dominique & Thomas, 2001 and C. yacutinga Nieto, 2003, with a new synonym for C. leentvaari Demoulin, 1966 (Ephemeroptera: Baetidae).

The genus Camelobaetidius Demoulin, 1966 has 39 species distributed throughout the Nearctic and Neotropical Regions, being one of the most studied genera of the family Baetidae. Based on material from Brazil, the aim of the present paper is to describe adults of C. janae Dominique & Thomas, 2001 and C. yacutinga Nieto, 2003, and to recognize a new synonym for Camelobaetidius leentvaari Demoulin, 1966 (= C. mantis Traver & Edmunds, 1968 nov. syn.) based on the examination of type material.

Diversity of EPT (Ephemeroptera, Plecoptera, Trichoptera) Along Streams Fragmented by Waterfalls in the Brazilian Savanna.

In stream ecosystems, waterfalls intercept the fluvial continuum and limit the dispersion of aquatic organisms. However, the barrier effect may be minimized or absent for insects whose life cycle is not restricted to the aquatic environment (i.e., winged adults), such as the orders Ephemeroptera, Plecoptera and Trichoptera (EPT). In this study, we investigated EPT diversity (abundance, richness, and composition) in savanna headwater streams (Cerrado Domain, Brazil) to investigate the hypothesis that large waterfalls have little effect on instream assemblage structure. During 2017 and 2018, we sampled EPTs and measured environmental conditions at reaches adjacent (downstream and upstream) to waterfalls (5 to 70 m high). We collected 2532 immature specimens of 33 genera, including the first record of 14 genera of Ephemeroptera, 11 Trichoptera, and three Plecoptera for the Tocantins State. Hydro-geomorphic and streambed conditions varied among sites, but they were not related to the presence of waterfalls. Assemblage structure was similar between downstream and upstream reaches, with no significant difference in taxa richness, abundance, and composition. This pattern was consistently observed considering all data combined, and each order separately. These results support the hypothesis that physical barriers have weak effects on EPT assemblages at small spatial scales, where strong dispersal and habitat similarity probably homogenizes the structure of local assemblages in stream corridors adjacent to large waterfalls. The study provides important information about factors driving the distribution of biodiversity in savanna streams, which are relevant to support conservation and management plans in the Brazilian Cerrado.

A new species of Camelobaetidius Demoulin, 1966 (Ephemeroptera: Baetidae), from the Colombian Orinoco River basin.

Three species of Camelobaetidius Demoulin, 1966 have been reported from Colombia until now, based on nymphs. We describe a fourth species based on nymphs from the Colombian Orinoco river basin. The new species can be recognized by: 1) labrum narrowly rounded anteriorly; 2) segment II of labial palp with a short rounded distomedial projection; 3) incisors of right mandible with eleven denticles; 4) gills absent from the bases of coxae; 5) tarsal claws with ten denticles; 6) outer margin of forefemur with a row of about 23 long, spine-like setae; 7) posterior margin of tergum IV with truncate spines, and 8) terminal filament almost as long as cerci. [Zoobank URL: urn:lsid:zoobank.org:act:500CF998-8EF3-43E3-BA8B-F062B92768F3].

Corinnella lourii, a rare new species of Baetidae (Ephemeroptera) from central Brazil.

A new species of the rare genus Corinnella is described based on nymphs from the Rio Pindaíba, a river located close to the Cerrado - Amazonian border. The new species is more related to C. eximia than to C. thomasi, but can be distinguished from them by the following combination of characters: 1) distomedial emargination of labrum broad and with very small process; 2) maxillary palp 2.0× length of galea-lacinia; 3) posterior margin of terga I-IX with pointed spaced spines; 4) claws with four denticles.

Major myofibrillar changes in early onset myopathy due to de novo heterozygous missense mutation in lamin A/C gene.

Mutations in the lamin A/C gene (LMNA) have been associated with neuromuscular diseases and more complex syndromes, involving bone and adipose tissue. We report on a case of early onset myopathy due to a heterozygous LMNA mutation in exon 9, characterized by the presence of a marked number of cytoplasmic bodies with extensive myofibrillar abnormalities and Z-disk disruption in skeletal muscle. This case suggests there is a need to increase the list of genes to be screened in patients with myofibrillar myopathy.

The male imago of Paramaka incognita Domínguez, Grillet, Nieto, Molineri and Guerrero, 2014 (Ephemeroptera: Leptophlebiidae).

The genus Paramaka Savage & Domínguez, 1992 (Ephemeroptera: Leptophlebiidae) is restricted to South America, and it is comprised of four species. The male imago of P. incognita is described for the first time based on material from the state of Roraima in northern Brazil. The male adult differs from other described species of the genus by having the following combination of characteristics: Forewing with costal and subcostal areas basally washed with light yellowish brown; hind wing with base of wing washed with yellowish brown; abdominal terga II-V and VI-IX with lateral margins washed with reddish brown; abdominal sterna VI-VIII with lateral blackish stripes; styliger plate median portion with a pointed, posterior projection that is oriented in a posterior direction; and penes with ventral spines length subequal to length of penes, located on the distal third of the penes.

First record of the genus Lumahyphes Molineri, 2004 (Ephemeroptera: Leptohyphidae) from Brazil with description of a new species.

The genus Lumahyphes Molineri, 2004 (Ephemeroptera: Leptohyphidae) currently is comprised of three species. Lumahyphes cocal sp. nov. is described based on material from the state of Roraima in northern Brazil. The new species differs from the other described species by having the following combination of characteristics: greyish wing membrane; terga base color whitish and terga I-III & VIII-X washed completely with gray; dorsal structure of penes rounded; spines of penes S-shaped in lateral view; and apex of spines directed anteriorly.

Mechanisms of osteoclast dysfunction in human osteopetrosis: abnormal osteoclastogenesis and lack of osteoclast-specific adhesion structures.

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast-like cells.

Clinical and molecular findings in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome.

The authors report the clinical and molecular findings in eight patients with hyperornithinemia, hyperammonemia, and homocitrullinuria (HHH) syndrome. The most consistent neurologic finding was spastic paraparesis, seen in five of the eight patients. However, all showed signs of pyramidal tract involvement. A broad spectrum of pathogenetic mutations (including missense, nonsense, splice site, insertion, and deletions) were identified in the ORNT1 gene.

Fatal infantile leukodystrophy: a severe variant of CACH/VWM syndrome, allelic to chromosome 3q27.

OBJECTIVE: To describe clinical and neuropathologic studies and linkage analysis on two sisters with a severe form of leukodystrophy. METHODS: A detailed study was performed on the second sister. Genotyping markers for chromosome 3, including eight additional markers surrounding the vanishing white matter (VWM) locus, were used. RESULTS: During the first year of life, two sisters developed a severe neurologic condition after an intercurrent infection. It was accompanied by irritability and stupor with rapid loss of their motor abilities. Results of extensive metabolic studies were negative. Brain MRI showed severe and diffuse abnormalities of the encephalic white matter. Neuropathologic examination showed a severe lack of myelin with diffuse vacuolating white matter lesions in the brain, associated with an increased density of oligodendrocytes and a reduced number of astrocytes on morphometric analysis. In sharp contrast, the spinal cord white matter was preserved. The affected sibpairs shared a common haplotype for a broad region in chromosome 3. They were homozygous between markers D3S1565 and D3S3669, including the VWM locus. CONCLUSIONS: This condition is an unusual variant of childhood ataxia with diffuse central hypomyelination (CACH)/VWM, with characteristic shrinking and perivascular clustering of astrocytes. Haplotype analysis suggests that this variant is allelic to the VWM locus located on chromosome 3q27.

Complete transposition of the great arteries: visualization of left and right outflow tract obstruction by oblique subcostal two-dimensional echocardiography.

Subcostal oblique 2-dimensional echocardiography was performed in 64 infants younger than 2 years with complete transposition of the great arteries (TGA) (situs solitus, concordant atrioventricular and discordant ventriculoarterial connections). All patients examined before cardiac catheterization had a correct diagnosis by 2-dimensional echocardiography using the subcostal oblique views. Twelve patients had associated left ventricular (LV) outflow tract obstruction and 7 had right ventricular (RV) outflow obstruction. The standard parasternal views failed to diagnose obstruction in 1 patients with LV outflow obstruction and 5 with RV outflow obstruction; the subcostal left oblique cut and long axis of the left ventricle visualized all left-sided obstructions, and right-sided obstructions were correctly displayed in 5 of 7 cases using a combination of left oblique and right oblique cuts. Two-dimensional echocardiographic subcostal oblique views allow an excellent definition of the morphologic characteristics of RV and LV outflow tracts in patients with TGA and improve the diagnosis of the outflow obstruction in these malformations.

Agenesis of the corpus callosum, combined immunodeficiency, bilateral cataract, and hypopigmentation in two brothers.

We describe 2 brothers with a malformation syndrome consisting of agenesis of the corpus callosum, cutaneous hypopigmentation, bilateral cataract, cleft lip and palate, and combined immunodeficiency. The clinical history of both patients was characterized by severe psychomotor retardation, seizures, recurrent severe respiratory infections, and chronic mucocutaneous candidiasis. The children died of bronchopneumonia at age 2 and 3 years, respectively. Immunological investigations showed, in one sib studied, skin anergy to recall antigens, profound depletion of T4+ lymphocytes, and serum IgG2 deficiency. Necropsy showed agenesis of the corpus callosum, hypoplasia of the cerebellar vermis, and profound hypoplasia of the thymus and of the peripheral lymphoid tissue. The distinctive features of these sibs appear to define a previously undescribed hereditary MCA/MR syndrome. The clinical and pathological findings seem to indicate, as a pathogenetic mechanism, a defect involving the embryonic organization of the central nervous system and of the immune system.