Breakpoint characterization of large deletions in EXT1 or EXT2 in 10 Multiple Osteochondromas families

<p>Abstract</p> <p>Background</p> <p>Osteochondromas (cartilage-capped bone tumors) are by far the most commonly treated of all primary benign bone tumors (50%). In 15% of cases, these tumors occur in the context of a hereditary syndrome called multiple osteochondromas (MO), an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped bone tumors at children's metaphyses. MO is caused by various mutations in <it>EXT1 </it>or <it>EXT2</it>, whereby large genomic deletions (single-or multi-exonic) are responsible for up to 8% of MO-cases.</p> <p>Methods</p> <p>Here we report on the first molecular characterization of ten large <it>EXT1</it>- and <it>EXT2</it>-deletions in MO-patients. Deletions were initially indentified using MLPA or FISH analysis and were subsequently characterized using an MO-specific tiling path array, allele-specific PCR-amplification and sequencing analysis.</p> <p>Results</p> <p>Within the set of ten large deletions, the deleted regions ranged from 2.7 to 260 kb. One <it>EXT2 </it>exon 8 deletion was found to be recurrent. All breakpoints were located outside the coding exons of <it>EXT1 </it>and <it>EXT2</it>. Non-allelic homologous recombination (NAHR) mediated by <it>Alu</it>-sequences, microhomology mediated replication dependent recombination (MMRDR) and non-homologous end-joining (NHEJ) were hypothesized as the causal mechanisms in different deletions.</p> <p>Conclusions</p> <p>Molecular characterization of <it>EXT1</it>- and <it>EXT2</it>-deletion breakpoints in MO-patients indicates that NAHR between <it>Alu-</it>sequences as well as NHEJ are causal and that the majority of these deletions are nonrecurring. These observations emphasize once more the huge genetic variability which is characteristic for MO. To our knowledge, this is the first study characterizing large genomic deletions in <it>EXT1 </it>and <it>EXT2</it>.</p

Multiple Statistical Analysis Techniques Corroborate Intratumor Heterogeneity in Imaging Mass Spectrometry Datasets of Myxofibrosarcoma

MALDI mass spectrometry can generate profiles that contain hundreds of biomolecular ions directly from tissue. Spatially-correlated analysis, MALDI imaging MS, can simultaneously reveal how each of these biomolecular ions varies in clinical tissue samples. The use of statistical data analysis tools to identify regions containing correlated mass spectrometry profiles is referred to as imaging MS-based molecular histology because of its ability to annotate tissues solely on the basis of the imaging MS data. Several reports have indicated that imaging MS-based molecular histology may be able to complement established histological and histochemical techniques by distinguishing between pathologies with overlapping/identical morphologies and revealing biomolecular intratumor heterogeneity. A data analysis pipeline that identifies regions of imaging MS datasets with correlated mass spectrometry profiles could lead to the development of novel methods for improved diagnosis (differentiating subgroups within distinct histological groups) and annotating the spatio-chemical makeup of tumors. Here it is demonstrated that highlighting the regions within imaging MS datasets whose mass spectrometry profiles were found to be correlated by five independent multivariate methods provides a consistently accurate summary of the spatio-chemical heterogeneity. The corroboration provided by using multiple multivariate methods, efficiently applied in an automated routine, provides assurance that the identified regions are indeed characterized by distinct mass spectrometry profiles, a crucial requirement for its development as a complementary histological tool. When simultaneously applied to imaging MS datasets from multiple patient samples of intermediate-grade myxofibrosarcoma, a heterogeneous soft tissue sarcoma, nodules with mass spectrometry profiles found to be distinct by five different multivariate methods were detected within morphologically identical regions of all patient tissue samples. To aid the further development of imaging MS based molecular histology as a complementary histological tool the Matlab code of the agreement analysis, instructions and a reduced dataset are included as supporting information

HSPG-Deficient Zebrafish Uncovers Dental Aspect of Multiple Osteochondromas

Multiple Osteochondromas (MO; previously known as multiple hereditary exostosis) is an autosomal dominant genetic condition that is characterized by the formation of cartilaginous bone tumours (osteochondromas) at multiple sites in the skeleton, secondary bursa formation and impingement of nerves, tendons and vessels, bone curving, and short stature. MO is also known to be associated with arthritis, general pain, scarring and occasional malignant transformation of osteochondroma into secondary peripheral chondrosarcoma. MO patients present additional complains but the relevance of those in relation to the syndromal background needs validation. Mutations in two enzymes that are required during heparan sulphate synthesis (EXT1 or EXT2) are known to cause MO. Previously, we have used zebrafish which harbour mutations in ext2 as a model for MO and shown that ext2−/− fish have skeletal defects that resemble those seen in osteochondromas. Here we analyse dental defects present in ext2−/− fish. Histological analysis reveals that ext2−/− fish have very severe defects associated with the formation and the morphology of teeth. At 5 days post fertilization 100% of ext2−/− fish have a single tooth at the end of the 5th pharyngeal arch, whereas wild-type fish develop three teeth, located in the middle of the pharyngeal arch. ext2−/− teeth have abnormal morphology (they were shorter and thicker than in the WT) and patchy ossification at the tooth base. Deformities such as split crowns and enamel lesions were found in 20% of ext2+/− adults. The tooth morphology in ext2−/− was partially rescued by FGF8 administered locally (bead implants). Our findings from zebrafish model were validated in a dental survey that was conducted with assistance of the MHE Research Foundation. The presence of the malformed and/or displaced teeth with abnormal enamel was declared by half of the respondents indicating that MO might indeed be also associated with dental problems

Peripheral chondrosarcoma progression is associated with increased type X collagen and vascularisation

Endochondral bone formation requires a cartilage template, known as the growth plate, and vascular invasion, bringing osteoblasts and osteoclasts. Endochondral chondrocytes undergo sequences of cell division, matrix secretion, cell hypertrophy, apoptosis, and matrix calcification/mineralisation. In this study, two critical steps of endochondral bone formation, the deposition of collagen X-rich matrix and blood vessel attraction/invasion, were investigated by immunohistochemistry. Fourteen multiple osteochondromas and six secondary peripheral chondrosarcomas occurring in patients with multiple osteochondromas were studied and compared to epiphyseal growth plate samples. Mutation analysis showed all studied patients (expect one) to harbour a germ-line mutations in either EXT1 or EXT2. Here, we described that homozygous mutations in EXT1/EXT2, which are causative for osteochondroma formation, are likely to affect terminal chondrocyte differentiation and vascularisation in the osteocartilaginous interface. Contrastingly, terminal chondrocyte differentiation and vascularisation seem to be unaffected in secondary peripheral chondrosarcoma. In addition, osteochondromas with high vascular density displayed a higher proliferation rate. A similar apoptotic rate was observed in osteochondromas and secondary peripheral chondrosarcomas. Recently, it has been shown that cells with functional EXT1 and EXT2 are outnumbering EXT1/EXT2 mutated cells in secondary peripheral chondrosarcomas. This might explain the increased type X collagen production and blood vessel attraction in these malignant tumours

Intermediate grade osteosarcoma and chondrosarcoma arising in an osteochondroma. A case report of a patient with hereditary multiple exostoses

A 40 year old man with hereditary multiple exostoses (HME), affecting predominantly his left proximal tibia, distal femur, and proximal femur, underwent resection of an osteochondroma near the trochanter major of his left proximal femur because of malignant transformation of the cartilaginous cap towards secondary peripheral chondrosarcoma. The patient had a history of a papillary thyroid carcinoma four years previously. At examination of the resected specimen, a third malignant tumour, an intermediate grade osteosarcoma (grade II/IV), was found in the osseous stalk of the osteochondroma. Although no mutations were found in the EXT1 and EXT2 genes, the genes involved in HME, or in exons 5–8 of the p53 gene, the development of three malignancies before the age of 40 suggests that this patient is genetically prone to malignant transformation

Effects of nimodipine on EEG and 31P-NMR spectra during and after incomplete forebrain ischemia in the rat

The effect of nimodipine was studied on EEG power density spectra as well as on 31P-NMR spectra of the brain before, during and after four-vessel occlusion (FVO) in the restrained conscious rat. EEG-spectral analysis: eight rats were submitted to 5 min FVO and four of them received nimodipine (1 mg/kg orally) 75 min before FVO. In the nimodipine-treated rats the post-ischemic overshoot of the EEG power density was significantly lower for the theta, alpha and beta band activity. 31P-NMR spectroscopy: the relative concentrations of phosphocreatine (PCr), ATP and Pi as well as intracellular pH were measured at different intervals after 10 min FVO (n = 10). All values normalised within 15-30 min after restoration of cerebral blood flow without significant differences between nimodipine-treated and control rats. It is concluded that pretreatment with nimodipine has a decreasing effect on the overshoot in EEG power density after transient incomplete forebrain ischemia. This effect was not correlated to significant changes in cerebral 'energy state', as measured by 31P-NMR spectroscop

Kardiak resenkronizasyon tedavisi uygulanan hastaların pil optimizasyonunda invaziv yöntem ve ekokardiyografik yöntemin karşılaştırılması

Kardiyak resenkronizasyon tedavisi (KRT), kalp yetersizliği (KY) tedavisi için yeni bir tedavi yöntemidir. KRT’ ye yeterli yanıt alınamayan olgularda kalp pili optimizasyonu olumlu etkiler sağlamaktadır. Bu çalışmada KRT sonrası invazif olarak ve ekokardiyografi ile yapılan optimizasyonun, hemodinami ve hacim cevabı üzerindeki akut ve orta vadedeki etkilerinin karşılaştırılması hedeflenmiştir. Atriyoventriküler (AV) ve ventriküloventriküler (VV) gecikmenin programlamasında invazif yöntem ve ekokardiyografiyi karşılşatıran bu prospektif, klinik çalışmada olgular ekokardiyografi grubu (s=20) ve invazif grup (s=20) olarak ayrılmıştır. Başlangıçta tüm olgularda, her iki metotla, AV gecikme için 60’ tan 160 msn’ ye, VV gecikme için ise -60’ dan, +60 msn’ ye kadar tüm aralıklar test edilmiş ve sonrasında olguların yarısı invazif grup, yarısı ekokardiyografi grubu olarak randomize edilmiştir. Optimal AV ve VV gecikmeler, ekokardiyografi ile en iyi sol ventriküler çıkım yolu hız zaman integralini (SlVÇY-HZİ) ve en uygun diyastolik doluş zamanını (DDZ), invazif yöntemle ise en yüksek sol ventrikül dP/dtmax’ını sağlayan değerler olarak tanımlanmıştır. Altı ay sonunda, sistol sonu hacminde ≥%15 azalma, ve ejeksiyon fraksiyonunda (EF) >%5 mutlak artış anlamlı hacim cevabı, New York Kalp Birliği (NYHA) sınıfında ≥1 artış ise klinik cevap olarak değerlendirilmiştir. Optimal AV gecikmeler, %57.5 olguda, en fazla ±10ms farkla, optimal VV gecikmeler ise %65 olguda, en fazla ±20ms farkla uyumlu olarak saptanmıştır. Ekokardiyografi ile (DDZ:360±123 msn’ den, 467±137 msn’ ye; p<0.001, SlVÇY-HZİ: 13.5±4 cm’ den, 16±4.4 cm’ ye; p<0.001) ve invazif yöntemle (SlV dP/dtmax: 1088±327dynes/s’ den, 1336±327dynes/s’ ye; p=<0.001) yapılan optimizasyonlarda, akut hemodinamik cevapta istatistiksel olarak anlamlı bir düzelme olduğu görülmüştür. Altı ay sonunda, invazif yöntemle optimizasyon uygulanan hastaların %70’ inde klinik cevap, %40’ ında hacim cevabı, %70’ inde EF cevabı, ekokardiyografi ile optimizasyon uygulanan hastaların ise %45’ inde klinik cevap, %60’ ında hacim cevabı, %60’ ınde EF cevabı izlenmiştir (p=AD). Optimizasyon metodu, 6 ay sonunda klinik cevap ve hacim cevabının öngördürücüsü olarak bulunmamıştır. v Sonuç olarak, KRT optimizasyonunda hem invazif hemodinamik, hem de ekokardiyografi Doppler metodlarının, uygulanabilir ve etkili yöntemler olduğu saptanmıştır