Skin fragility caused by biallelic KRT10 mutations: an intriguing form of self-improving epidermolytic ichthyosis.

Autosomal recessive epidermolytic ichthyosis is a rare skin condition associated with KRT10 loss-of-function mutations. It presents with severe life-threatening clinical manifestations. Here we describe a case of autosomal recessive epidermolytic ichthyosis with an unusually mild, spontaneously improving phenotype. Erythroderma and superficial blistering were present at birth, but the skin recovered and remained almost intact at the age of 1 year. Mild scaling on the neck and skin fragility manifesting as superficial erosions after scratching were the only clinical features as the child grew. As a cause, previously unreported compound heterozygous KRT10 pathogenic variants were found: a nonsense mutation leads to mRNA decay, while the other synonymous variant induces a leaky splice site, explaining the residual keratin 10 expression and mild clinical phenotype. What's already known about this topic? Autosomal recessive epidermolytic ichthyosis is a rare skin condition caused by loss-of-function KRT10 mutations. The clinical phenotype is severe with superficial skin blistering, scaling and hyperkeratosis. What does this study add? Here we extend the mutational and phenotypic spectrum of autosomal recessive epidermolytic ichthyosis. Our case presented with erythroderma and superficial blistering at birth, but the skin recovered and was almost intact at the age of 1 year. The only disease manifestations were mild scaling on the neck and skin fragility appearing as superficial erosions after scratching. The causative factors were found to be one nonsense mutation in KRT10 that leads to mRNA decay, and one synonymous variant that affects the donor splice site of exon 3. We hypothesize that this leaky splice site explains the residual keratin 10 expression and self-improving clinical phenotype.

Mosaicism due to postzygotic mutations in women with focal dermal hypoplasia.

Focal dermal hypoplasia (FDH, Goltz syndrome, MIM #305600) constitutes a rare multisystem genetic disorder of the skin, skeleton, teeth and eyes with considerable variation in the clinical features. FDH is transmitted as an X-linked dominant trait and is caused by mutations in PORCN. In male children, hemizygous PORCN mutations are lethal in utero. Around 300 cases have been reported in the literature to date. About 10% of them are male patients presenting with either Klinefelter syndrome (karyotype 47, XXY) or mosaicism of a postzygotic mutation. Here we describe four cases of women with typical features of FDH, in whom a PORCN mutation was found in DNA from affected cutaneous tissue but not in DNA from peripheral blood. This study suggests that mosaicism caused by a postzygotic mutation occurs more often than assumed to date in female patients with FDH. A negative analysis performed on peripheral blood DNA does not exclude the diagnosis of FDH and it is therefore of practical importance to analyse DNA from the affected skin in order to identify low-level mosaicism and thus to improve diagnostic precision. In total, we found two missense variants, one novel indel and one novel splice-site variant. Individuals harbouring postzygotic mosaicism run a risk of transmitting the disorder to their daughters, because the maternal mosaic could also affect the gonads.

Sixteen novel mutations in PNPLA1 in patients with autosomal recessive congenital ichthyosis reveal the importance of an extended patatin domain in PNPLA1 that is essential for proper human skin barrier function.

BACKGROUND: Autosomal recessive congenital ichthyosis (ARCI) is a genetically heterogeneous group of rare Mendelian skin disorders characterized by cornification and differentiation defects of keratinocytes. Mutations in nine genes including PNPLA1 are known to cause nonsyndromic forms of ARCI. To date, only 10 distinct pathogenic mutations in PNPLA1 have been reported. OBJECTIVES: To identify new causative PNPLA1 mutations. METHODS: We screened genetically unresolved cases, including our ARCI collection, comprising more than 700 families. Screening for mutations was performed either by direct Sanger sequencing or in combination with a multigene panel, followed by sequence and mutation analysis. RESULTS: Here we report on 16 novel mutations present in patients from 17 families. While all previously reported mutations and most of our novel mutations are located within the core patatin domain, we report five novel PNPLA1 mutations that are downstream of this domain. Thus, as recently described for PNPLA2, we hypothesize that a region larger than the core domain is required for full enzymatic activity of PNPLA1 in human skin barrier formation. CONCLUSIONS: We estimate the frequency of PNPLA1 mutations among patients with ARCI to be around 3%. Most of our patients were born as collodion babies and showed a relatively mild ichthyosis phenotype. In four unrelated patients we observed a cyclic scaling course, which seems to be a potential phenotypic variation in a small percentage of patients with PNPLA1 mutations. The variability of the clinical manifestations and the lack of typical clinical features are specific for patients with PNPLA1 mutations, and emphasize the importance of DNA sequencing for differential diagnosis of ARCIs.

Use of fluorescent probes as a useful tool to identify resistant Schistosoma mansoni isolates to praziquantel.

The use of chemotherapy on a mass scale in endemic areas may lead to the appearance of resistant isolates through the mechanism of selective drug pressure. Studies have demonstrated that praziquantel (PZQ) is able to inhibit the excretory activity and to cause tegumental damage in Schistosoma mansoni adult worms. The use of the probe resorufin to evaluate excretory activity, as well as the probe Hoechst 33258 to detect tegumental damage in adult worms, may represent a method to identify resistant (or less susceptible) isolates. The purpose of the present work was to compare the changes caused by PZQ in the function of the excretory system and in the integrity of the tegument of adult worms from the LE isolate (susceptible to PZQ) and the LE-PZQ isolate (less susceptible to PZQ). Worms from the isolate LE-PZQ showed less severe tegumental lesions, in both in vitro and in vivo experiments, detected by labelling with Hoechst 33258 and continued to have a functional excretory system as shown by labelling with resorufin in vitro.

Pathways for the influx of molecules into cercariae of Schistosoma mansoni during skin penetration.

It has been observed that fluorescent membrane-impermeant molecules can enter the cercariae as they penetrate mouse skin. The hypothesis to be tested was that such molecules, which included Lucifer Yellow and a variety of fluorescent dextrans, entered the parasite through the nephridiopore and excretory tubules as well as through the surface membrane. FITC-labelled poly-L-lysine (molecular weight 10 kDa), added at 4 degrees C during syringe transformation, was found to enter the nephridiopore and labelled the excretory bladder and sometimes the excretory tubules. This finding indicates that macromolecules (10 kDa) can enter the nephridiopore. It was found that linoleic acid (a normal constituent of skin) greatly stimulated uptake of Lucifer Yellow and dextrans into the excretory/subtegumental region of 2-h-old schistosomula. This correlated with an increased uptake of membrane-impermeant propidium iodide at 37 degrees C. Since increased uptake of propidium iodide occurs when membranes become permeable, the surface membrane could also be a pathway of transport of the membrane-impermeant molecules into the schistosomulum.

Schistosoma mansoni cercariae experience influx of macromolecules during skin penetration.

We have observed that when cercariae penetrate the skin of mice, there is influx into their tissues of Lucifer Yellow and certain labelled molecules of up to 20 kDa molecular weight. This observation was made using a variety of fluorescent membrane-impermeant compounds injected into the skin before the application of cercariae. This unexpected phenomenon was investigated further by transforming cercariae in vitro in the presence of the membrane-impermeant compounds and examining the distribution by microscopy. In schistosomula derived from this procedure, the nephridiopore and surface membrane were labelled while the pre- and post-acetabular glands were not labelled. The region associated with the oesophagus within the pharyngeal muscle clearly contained the fluorescent molecules, as did the region adjacent to the excretory tubules and the germinal mass. We used cercariae stained with carmine to aid identification of regions labelled with Lucifer Yellow. Although the mechanism of this influx is unclear, the observation is significant. From it, we can suggest an hypothesis that, during skin penetration, exposure of internal tissues of the parasite to external macromolecules represents a novel host-parasite interface.

The schistosome in the mammalian host: understanding the mechanisms of adaptation.

In this review, we envisage the host environment, not as a hostile one, since the schistosome thrives there, but as one in which the relationship between the two organisms consists of constant communication, through signalling mechanisms involving sense organs, surface glycocalyx, surface membrane and internal organs of the parasite, with host fluids and cells. The surface and secretions of the schistosome egg have very different properties from those of other parasite stages, but adapted for the dispersal of the eggs and for the preservation of host liver function. We draw from studies of mammalian cells and other organisms to indicate how further work might be carried out on the signalling function of the surface glycocalyx, the raft structure of the surface and existence of pores in the surface membrane, the repair of the surface membrane, the role of the membrane structure in ion channel function (including recent work on the actin cytoskeleton and calcium channels) and the possible role of P-glycoproteins in the adaptation of the parasite to its environment. We are speculative in some areas, such as the suggestions that variability in surface properties of schistosomes may relate to the existence of membrane rafts and that parasite communities may exhibit quorum sensing. This speculative approach is adopted with the hope that future work on the whole organisms and their interactions will be encouraged.

Evaluation of the effect of oxamniquine, praziquantel and a combination of both drugs on the intramolluscan phase of Schistosoma mansoni.

The activity of oxamniquine (OXA), praziquantel (PZQ), and a combination of both drugs was evaluated at the intramolluscan phase of Schistosoma mansoni. Biomphalaria glabrata snails infected with S. mansoni were treated with 500 mg/kg OXA, 1000 mg/kg PZQ or with 250 mg/kg OXA and 500 mg/kg PZQ, in association, at the pre-patent and patent phases of infection. The results showed that either treatments with OXA or PZQ, alone, at the pre-patent period, delayed the parasite's development, increasing the pre-patent period by approximately 10 days. At the same pre-patent period, treatment with a combination of OXA/PZQ delayed the parasite's development even more, extending the pre-patent period up to 56 days. At the patent period, treatment with OXA and PZQ, alone, interrupted cercarial shedding. When the snails were treated with 1000 mg/kg PZQ, the cercarial production was re-established 15 days after treatment, but in lower numbers than those obtained before treatment, whereas the snails treated with 500 mg/kg OXA were able to shed cercariae in similar quantities to those observed before treatment. The association 250 mg/kg OXA+500 mg/kg PZQ, at the patent period, not only discontinued cercarial shedding, but also led to the "cure" of the snails, showing a synergistic effect of this combination of drugs. These results suggest that this model will be useful for selection of resistant parasites, as well as for screening new antischistosomal drugs.

Detection of UV-induced thymine dimers in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy.

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ x cm-2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4',6'-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ x cm-2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ x cm-2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ x cm-2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.

Activity of praziquantel on in vitro transformed Schistosoma mansoni sporocysts

Praziquantel (PZQ) is effective against all the evolutive phases of Schistosoma mansoni. Infected Biomphalaria glabrata snails have their cercarial shedding interrupted when exposed to PZQ. Using primary in vitro transformed sporocysts, labeled with the probe Hoechst 33258 (indicator of membrane integrity), and lectin of Glycine max (specific for carbohydrate of N-acetylgalactosamine membrane), we evaluated the presence of lysosomes at this evolutive phase of S. mansoni, as well as the influence of PZQ on these acidic organelles and on the tegument of the sporocyst. Although the sporocyst remained alive, it was observed that there was a marked contraction of its musculature, and there occurred a change in the parasite's structure. Also, the acidic vesicles found in the sporocysts showed a larger delimited area after contact of the parasites with PZQ. Damages to the tegument was also observed, as show a well-marked labeling either with Hoechst 33258 or with lectin of Glycine max after contact of sporocysts with the drug. These results could partially explain the interruption/reduction mechanism of cercarial shedding in snails exposed to PZQ.