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Neurofibromatosis type 1 results from loss-of-function NF1 pathogenic variants (PVs). Up to 30% of all NF1 PVs disrupt mRNA splicing, including deep intronic variants. Here, we retrospectively investigated the spectrum of NF1 deep intronic PVs in a cohort of 8,090 unrelated individuals from the University of Alabama at Birmingham (UAB) dataset with a molecularly confirmed neurofibromatosis type 1. All variants were identified through their effect on the NF1 transcript, followed by variant characterization at the DNA-level. A total of 68 distinct variants, which were ≥ 20 nucleotides away from the closest exon–intron junction, were identified in 2.5% unrelated individuals with NF1 (200/8,090). Nine different pathogenic splice variants, identified in 20 probands, led to exonization of different parts of intron 30 [23.2] or 31 [23a]. The two major NF1 transcript isoforms, distinguished by the absence (type I) or presence (type II) of the alternatively spliced cassette exon 31 [23a], are equally expressed in blood in control individuals without NF1 or NF1-affected individuals carrying their PV not in the introns flanking exon 31 [23a]. By fragment and cloning analysis we demonstrated that the exonization of intron 31 [23a] sequences due to deep intronic PV predominantly affects the NF1 isoform II. Seven additional (likely) pathogenic NF1 deep intronic variants not observed in the UAB dataset were found by classification of 36 variants identified by a literature search. Hence, the unique list of these 75 deep intronic (likely) PVs should be included in any comprehensive NF1 testing strategy.

ZusammenfassungDie Möglichkeit einer Tumorerkrankung auf Basis eines familiären Tumorprädispositionssyndroms muss bei jeder Krebsdiagnose in Betracht gezogen werden. Die Erfassung erkrankter „Index“-PatientInnen ist entscheidend für die Ermittlung des Risikos für Neu- oder Wiedererkrankungen bei den Betroffenen wie auch für das Auftreten von Tumoren bei bisher gesunden Verwandten. Die Erfassung von PatientInnen mit familiärer Tumorprädisposition erlaubt es, Betroffene in Vorsorgeprogramme zur Senkung von Morbidität und Letalität aufzunehmen. Für das erbliche Brust- und Eierstockkrebssyndrom besteht in Österreich ein breites Bewusstsein. Dadurch wird eine zufriedenstellende Erfassung der PatientInnen erreicht. Das ist für das Lynch-Syndrom, welches bei 2–3 % aller Kolorektal- und Endometriumkarzinome vorliegt, leider nicht der Fall. Um die Identifizierung von Lynch-Syndrom-PatientInnen zu verbessern, empfiehlt die Österreichische Arbeitsgemeinschaft Pathologie-Humangenetik (die Österreichische Arbeitsgemeinschaft Pathologie-Humangenetik setzt sich aus jeweils fünf Delegierten der Österreichischen Gesellschaft für Klinische Pathologie und Molekularpathologie und der Österreichischen Gesellschaft für Humangenetik zusammen) in diesem Konsensus-Statement eine diagnostische Strategie, die möglichst alle Lynch-Syndrom-assoziierten Kolorektal- und Endometriumkarzinome im Rahmen der pathologischen Tumorbeurteilung erfasst. Durch eine darauf basierende systematische Zuweisung von PatientInnen mit Verdacht auf Lynch-Syndrom an ein Zentrum für medizinische Genetik zur genetischen Beratung und weiterführenden genetischen Diagnostik wird sichergestellt, dass auch Familienangehörige mit Lynch-Syndrom erfasst werden.

Constitutional mismatch repair deficiency (CMMRD) is an autosomal recessively inherited childhood cancer susceptibility syndrome caused by biallelic germline mutations in one of the mismatch repair (MMR) genes. The spectrum of CMMRD-associated tumours is very broad and many CMMRD patients additionally display signposting non-neoplastic features, most frequently café-au-lait macules and other pigmentation alterations. We report on a 13-month-old girl suspected of having CMMRD due to a desmoplastic medulloblastoma and a striking skin pigmentation that included multiple café-au-lait macules, hypopigmented areas and Mongolian spots. Whole-exome sequencing revealed homozygosity for MSH2 variant p.(Leu92Val) and MSH6 variant p.(Val809del), both variants of uncertain significance (VUS). Immunohistochemical analysis of the tumour tissue showed expression of all four MMR proteins and gMSI testing was negative. However, functional assays demonstrated that the cells of the patient displayed methylation tolerance and ex vivo microsatellite instability, which unequivocally confirmed the diagnosis of CMMRD. Taken together, the results render the MSH2 variant unlikely to be responsible for the phenotype, while they are compatible with MSH6-associated CMMRD. This case illustrates the diagnostic strategy of confirming CMMRD syndrome in patients with VUS.

In a 14-year-old boy with polyposis and rectosigmoid carcinoma, we identified a novel POLE germline mutation, p.(Val411Leu), previously found as recurrent somatic mutation in 'ultramutated' sporadic cancers. This is the youngest reported cancer patient with polymerase proofreading-associated polyposis indicating that POLE mutation p.(Val411Leu) may confer a more severe phenotype than previously reported POLE and POLD1 germline mutations. The patient had multiple café-au-lait macules and a pilomatricoma mimicking the clinical phenotype of constitutional mismatch repair deficiency. We hypothesize that these skin features may be common to different types of constitutional DNA repair defects associated with polyposis and early-onset cancer.

Biallelic PMS2 mutations are responsible for more than half of all cases of constitutional mismatch repair deficiency (CMMRD), a recessively inherited childhood cancer predisposition syndrome. The mismatch repair gene PMS2 is partly embedded within one copy of an inverted 100-kb low-copy repeat (LCR) on 7p22.1. In an individual with CMMRD syndrome, PMS2 was found to be homozygously inactivated by a complex chromosomal rearrangement, which separates the 5'-part from the 3'-part of the gene. The rearrangement involves sequences of the inverted 100-kb LCR and a human endogenous retrovirus element and may be associated with an inversion that is indistinguishable from the known inversion polymorphism affecting the ~0.7-Mb sequence intervening the LCR. Its formation is best explained by a replication-based mechanism (RBM) such as fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR). This finding supports the hypothesis that the inverted LCR can not only facilitate the formation of the non-allelic homologous recombination-mediated inversion polymorphism but it also promotes the occurrence of more complex rearrangements that can be associated with a large inversion, as well, but are mediated by a RBM. This further suggests that among the inversion polymorphism on 7p22.1, more complex rearrangements might be hidden. Furthermore, as the locus is embedded in a common fragile site (CFS) region, this rearrangement also supports the recently raised hypothesis that CFS sequence motifs may facilitate replication-based rearrangement mechanisms.

Die Neurofibromatose Typ 1 (NF1) ist eine der haufigsten autosomal dominanten genetischen Krankheiten und wahrscheinlich die haufigste, die mit einer Tumordisposition einhergeht. Sie tritt bei einer von 3000–4000 Personen auf, wobei etwa die Halfte aller Patienten ihre NF1-Genmutation von einem Elternteil ererbt hat, und in der anderen Halfte die Mutation neu (spontan) entstanden ist. Die typischen klinischen Merkmale der Krankheit, die bei uber 90 % aller Patienten beobachtet werden, sind Cafe-au-lait-Flecken und dermale Neurofibrome. Die Krankheit sollte aber nicht als „rein kosmetisches Problem“ unterschatzt werden. NF1-Patienten haben ein erhohtes Risiko fur eine Reihe von auch malignen Tumoren, insbesondere malignen peripheren Nervenscheidentumoren (MPNST), juvenilen myelomonozytaren Leukamien (JMML), Optikusgliomen und Phaochromozytomen. Renovaskulare Erkrankungen stellen ein weiteres Risiko fur NF1-Patienten dar. Das NF1-Gen ist ein klassisches Beispiel eines Tumorsuppressorgens und seine Funktion als negativer Regulator des Ras-Protoonkogens kann seine Rolle bei der Tumorentstehung gut erklaren. Trotz der enormen Fortschritte, die in den letzten 15 Jahren seit der Klonierung des Gens im Verstandnis der Pathogenese dieser Erkrankung gemacht wurden, ist derzeit keine systemische Therapie, die die Krankheit heilen kann, in Sichtweite. In der Behandlung einzelner Komplikationen, wie zum Beispiel Knochendysplasien und Optikusgliomen, konnten in den letzten Jahren aber sehr grose Fortschritte erzielt werden. Die technischen Entwicklungen der jungsten Jahre erlauben heutzutage auch neben der klinischen Diagnose, die besonders bei Kleinkindern und atypischen Patienten haufig nicht eindeutig gestellt werden kann, eine molekular-genetische Abklarung der NF1 und NF2, die in vielen Fallen wunschenswert und hilfreich ist. Ein groseres Bewusstsein fur die Komplikationen und Auspragungsformen der NF1 und NF2 aller behandelnden Arzte ist wichtig, um moglichst allen NF1-und NF2-Patienten eine fruhzeitige und adaquate Beratung und Behandlung zukommen zu lassen. Ein wichtiger Schritt zur besseren Behandlung der Patienten konnte der Aufbau eines multidisziplinaren Zentrums zur Beratung und Behandlung von Neurofibromatose-Patienten sein.

Childhood T-cell non-Hodgkin's lymphoma, colorectal carcinoma and brain tumor in association with cafe-au-lait spots caused by a novel homozygous PMS2 mutation

During experiments investigating the purification of waste gas a bacterium capable of using carbon disulphide (CS2) als sole energy source was isolated. It could be identified as a Thiobacillus sp.; however, the species remains unclear. Both the properties of T. thioparus and T. thiooxidans have been observed. Since the organism could be used for removing CS2 in the environment, the degradation kinetics have been investigated by different methods. Substrate concentrations of up to 100 mg CS2·l−1 were oxidized at maximum rates of 2.5 mg CS2·g−1 protein·min−1 at pH 7.0 and at 30°C. CS2 levels above 150 mg CS2·l−1 caused termination of degradative activity.

A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-term in vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extended in vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years. Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.

Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 10(8) cells/ml a stable growth rate of 0.004 h-1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.

Neurofibromatosis type 1 (NF1) is a common inherited disease affecting one in 3,500 individuals. The mutation rate in the NF1 gene is one of the highest known for human genes. Compared to other methods, the protein truncation test (PTT) and subsequent sequence analysis of cloned cDNA provides improved efficiency in detecting NF1 mutations that are dispersed throughout the gene spanning 350 kb of genomic DNA. Sequencing of cDNA of patients affected with NF1 mutations revealed multiple splicing errors. Since similar missplicings were also found in "aged" blood of healthy individuals, they are most likely attributable to a general decrease in splice site selection in aged blood. We show that restoring viability of lymphocytes before RNA extraction by cultivation and PHA stimulation diminishes aberrant splicing in aged blood and is thus useful to circumvent splicing alterations which are frequently compromising mutation detection in patient samples and mimic mutation-induced alterations of mRNA.

Culture conditions were evaluated for their relevance to chemical and biological oxidation of sulphide in aqueous solution. With a mixed culture of colourless sulphur bacteria sulphide oxidation was investigated over a concentration range of 0.005 to 3.5 mm S2−, a pH range from 1.0 to 9.0 and temperatures from 10 to 40° C. Biological sulphide oxidation quickly decreased when fermentation conditions were suboptimal and the proportion of chemical oxidation increased with high pH values and sulphide concentration and increasing temperature. The optimal conditions for biological activity were found to be 3 mm S2−, pH 7.0, and a mesophilic temperature (40° C).