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The ontogeny of carp (Cyprinus carpio L.) immune cells was studied in mucosal organs (intestine, gills and skin) using the monoclonal antibodies WCL38 (intraepithelial lymphocytes), WCL15 (monocytes/macrophages) and WCI12 (B cells). In addition, recombination activating gene 1 expression was examined in the intestine with real time quantitative PCR and in situ hybridization to investigate extrathymic generation of lymphocytes. WCL38 + intraepithelial lymphocytes (putative T cells) appeared in the intestine at 3 days postfertilization (dpf), which is shortly after hatching but before feeding, implying an important function at early age. These lymphoid cells appear in the intestine before the observation of the first thymocytes at 3-4 dpf, and together with the expression of recombination activating gene 1 in the intestine, suggests that similar to mammals at least part of these cells are generated in the intestine. WCL15 + monocytes/macrophages appeared in the lamina propria of the intestine at 7 dpf, but considerably later in the epithelium, while WCI12 + (B) cells appeared in intestine and gills at 6-7 weeks. From these results it can be concluded that putative T cells occur much earlier than B cells, and that B cells appear much later in the mucosae than in other internal lymphoid organs (2 wpf).

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Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1.

Summary Basal cell carcinomas (BCCs or “rodent ulcers”) are the most common type of skin cancer and generally do not spread elsewhere on the body. They are not unusual in older people, but someone who develops lots of BCCs from an early age may have the rare genetic condition Basal Cell Naevus Syndrome (BCNS or “Gorlin syndrome”). Other clues to BCNS include an unusually shaped face, cysts in the jaw, and tiny irregularities on the palms and soles called palmar pits. The diagnosis is confirmed by testing DNA extracted from a blood sample. BCNS is caused by a mistake (mutation) in a gene called PTCH1 which is usually inherited from a parent, but occasionally develops for the first time in the embryo, in which case, the patient is described as mosaic because some body cells contain the mistake and some do not. Mosaic patients can be hard to diagnose because they tend to have a milder form of the condition (sometimes the only sign is a BCC developing in childhood) and the blood test may be negative for PTCH1 mutations. This paper, from Belgium and the Netherlands, describes two such patients: a 41 year old woman with BCCs on just one side of her body and no other features of BCNS and a 36 year old woman with BCCs, jaw cysts and palmar pits. In both patients the blood test was negative but PTCH1 mutations were found in all the BCCs tested. Confirming the diagnosis enabled the doctors to counsel these patients about possible complications of BCNS and the risk of passing it on to the next generation.

Summary Basal cell naevus syndrome (BCNS) is associated with germline mutations in the PTCH1 gene. Postzygotic mosaicism can also cause BCNS. Here we describe two patients, one with multiple basal cell carcinomas (BCCs) and one with clinical BCNS, who had no PTCH1 mutation in DNA extracted from blood. In both patients, we performed genetic analysis on different BCCs, revealing the presence of a shared PTCH1 mutation in all tumours. Our findings show that in patients with symptoms of BCNS and initial absence of a PTCH1 mutation in blood, genetic profiling of BCCs can detect postzygotic mosaicism. What's already known about this topic? Basal cell naevus syndrome (BCNS) is associated with germline mutations in the PTCH1 gene, but it can also be caused by low‐grade postzygotic mosaicism in PTCH1. What does this study add? In patients suspected of having BCNS or patients with multiple basal cell carcinomas (BCCs) with a special distribution on the body and no mutation detected in blood, it is worthwhile to search for a shared PTCH1 mutation in their BCCs as this can detect postzygotic mosaicism. This information is important to ensure proper surveillance programmes, choose the right therapy and provide adequate genetic counselling.

PurposeNeurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. A targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants.MethodsRNA was extracted from lymphocytes, followed by targeted NF1 RNAseq. An in-house developed tool (QURNAS) was used to calculate the enrichment score (ERS) for each splicing event.ResultsThis method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%).ConclusionTargeted NF1 RNAseq can be successfully used to detect pathogenic RNA splicing variants, complementary to DNA based diagnostics.

206Nanomechanical properties of collagen VII anchoring fibrils in recessive dystrophic epidermolysis bullosa patients B Mayer, P Silló , M Mazán, D Haluszka, MZ Kellermayer and S Kárpáti Semmelweis University, Budapest, Hungary Our aim was to apply novel nanomechanical tests to investigate the biophysical properties of collagen VII anchoring fibrils in skin sections in a healthy control and in two recessive dystrophic epidermolysis bullosa (RDEB) patients. One patient with localized RDEB was a compound heterozygote with a consequent R1957W change within the hinge region and a premature termination (Y2819*) within the acidic region of collagen VII a1 chain. The other was diagnosed with generalized severe RDEB, was homozygous and had truncated collagen VII a1 chains in the NC-1 domain (K142R). Sections of skin biopsies from patient and control were investigated with atomic force microscopy (AFM) combined with fluorescence imaging. In the localized RDEB patient and healthy control, the anchoring fibrils were first immunostained, identified and scanned using AFM. In the generalized severe RDEB patient (no collagen VII staining), the dermal surface of the basement membrane zone (BMZ) adjacent to bulla formation was scanned. Nanoindentation was used to obtain force-volume maps and to characterize local viscoelasticity. Force-displacement curves were fitted with the Hertz model to calculate the local Young modulus. The control skin specimen could be partitioned into high-, intermediate-and low-Young-modulus areas that correspond to the dermis, BMZ and the epidermis, respectively. Remarkably, the dermis was stiffer than the epidermis by an order of magnitude. The region with intermediate Young modulus could not be clearly identified in the RDEB samples. The healthy skin may thus be seen as a highly compliant epidermis woven onto a stiff dermis. Hence, collagen VII plays an important role in coupling the mechanically distinct epidermis and dermis. The applied biophysical measurements enabled us to obtain clinically relevant data and may be used in the future to evaluate protein or gene therapies in RDEB patients. Molecular diagnostics with conventional Sanger sequencing for ichthyosis vulgaris (IV) has been hampered by the notoriously difficult to analyse filaggrin (FLG) gene, caused by its homologous and polymorphic repeated units. By implementation of single molecule molecular inversion probes (smMIPs) and next generation sequencing (NGS), an alternative screening strategy for analysis of the entire coding region of the FLG gene becomes feasible. Genetic analysis of the whole gene instead of screening for only population-specific mutations, would improve diagnostic yield by scrutinizing also for rare family-specific mutations. The smMIP-NGS strategy is easy to implement, affordable and since exclusion of NGSduplicate-reads is possible, mutation-percentages can be related and assigned to polymorphic duplicated filaggrin-repeat-unit 8 and 10. In a cohort of previously screened Dutch patients (N¼70) for only the population...