65 research outputs found
Left ventricular hypertrabeculation/noncompaction with epilepsy, other heart defects, minor facial anomalies and new copy number variants
BACKGROUND: Left ventricular hypertrabeculation/noncompaction (LVHT) is a cardiac abnormality of unknown etiology which has been described in children as well as in adults with and without chromosomal aberrations. LVHT has been reported in association with various cardiac and extracardiac abnormalities like epilepsy and facial dysmorphism. CASE PRESENTATION: A unique combination of LVHT, atrial septal defect, pulmonary valve stenosis, aortic stenosis, epilepsy and minor facial anomalies is presented in a 5.5 years old girl. Microarray-based genomic hybridization (array-CGH) detected six previously not described copy number variants (CNVs) inherited from a clinically unaffected father and minimally affected mother, thus, most likely, not clinically significant but rare benign variants. CONCLUSIONS: Despite this complex phenotype de novo microdeletions or microduplications were not detected by array CGH. Further investigations, such as whole exome sequencing, could reveal point mutations and small indels as the possible cause
Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients
BACKGROUND: A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction. METHODS: The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed. RESULTS: CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84). CONCLUSION: Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined
array CGH screening of 134 unrelated families
Background A growing number of non-coding regulatory mutations are being
identified in congenital disease. Very recently also some exons of protein
coding genes have been identified to act as tissue specific enhancer elements
and were therefore termed exonic enhancers or “eExons”. Methods We screened a
cohort of 134 unrelated families with split-hand/split-foot malformation
(SHFM) with high resolution array CGH for CNVs with regulatory potential.
Results In three families with an autosomal dominant non-syndromic SHFM
phenotype we detected microdeletions encompassing the exonic enhancer (eExons)
15 and 17 of DYNC1I1. In a fourth family, who had hearing loss in addition to
SHFM, we found a larger deletion of 510 kb including the eExons of DYNC1I1
and, in addition, the human brain enhancer hs1642. Exons 15 and 17 of DYNC1I1
are known to act as tissue specific limb enhancers of DLX5/6, two genes that
have been shown to be associated with SHFM in mice. In our cohort of 134
unrelated families with SHFM, deletions of the eExons of DYNC1I1 account for
approximately 3% of the cases, while 17p13.3 duplications were identified in
13% of the families, 10q24 duplications in 12%, and TP63 mutations were
detected in 4%. Conclusions We reduce the minimal critical region for SHFM1 to
78 kb. Hearing loss, however, appears to be associated with deletions of a
more telomeric region encompassing the brain enhancer element hs1642. Thus,
SHFM1 as well as hearing loss at the same locus are caused by deletion of
regulatory elements. Deletions of the exons with regulatory potential of
DYNC1I1 are an example of the emerging role of exonic enhancer elements and
their implications in congenital malformation syndromes
Особенности транспортной логистики сборных грузов в сфере ВЭД
Перемещение товаров в составе сборного груза может быть выгодным совершенно разным субъектам. Для крупных компаний это удобно для доставки разнородных товаров и пробных образцов, а для более мелких позволяет оптимизировать оборотные средства. Также огромную часть клиентов транспортных компаний составляют ретейловые компании и индивидуальные предприниматели, объемы поставок которых не позволяют экономически выгодно перемещать свои товары иначе.
Движение товара в составе сборного груза имеет ряд неоспоримых преимуществ, такие как: упрощение отслеживания товаров и сокращение транспортных издержек, благодаря которым всё больше компаний и физических лиц обращаются к данному способу перемещения товаров, хоть и время доставки увеличивается из-за дополнительных этапов в перемещении товара.The movement of goods as part of a consolidated cargo can be beneficial to completely different entities. For large companies, it is convenient for the delivery of heterogeneous goods and test samples, and for smaller ones, it allows optimizing working capital. Also, a huge part of the customers of transport companies are retail companies and individual entrepreneurs, the volume of supply of which does not allow economically move their goods otherwise.
The movement of goods as part of the consolidated cargo has a number of undeniable advantages, such as: simplification of tracking of goods and reduction of transport costs, thanks to which more and more companies and individuals are turning to this method of movement of goods, although the delivery time is increased due to additional step
Phenotypic overlap in the contribution of individual genes to CNV pathogenicity revealed by cross-species computational analysis of single-gene mutations in humans, mice and zebrafish
SUMMARY
Numerous disease syndromes are associated with regions of copy number variation (CNV) in the human genome and, in most cases, the pathogenicity of the CNV is thought to be related to altered dosage of the genes contained within the affected segment. However, establishing the contribution of individual genes to the overall pathogenicity of CNV syndromes is difficult and often relies on the identification of potential candidates through manual searches of the literature and online resources. We describe here the development of a computational framework to comprehensively search phenotypic information from model organisms and single-gene human hereditary disorders, and thus speed the interpretation of the complex phenotypes of CNV disorders. There are currently more than 5000 human genes about which nothing is known phenotypically but for which detailed phenotypic information for the mouse and/or zebrafish orthologs is available. Here, we present an ontology-based approach to identify similarities between human disease manifestations and the mutational phenotypes in characterized model organism genes; this approach can therefore be used even in cases where there is little or no information about the function of the human genes. We applied this algorithm to detect candidate genes for 27 recurrent CNV disorders and identified 802 gene-phenotype associations, approximately half of which involved genes that were previously reported to be associated with individual phenotypic features and half of which were novel candidates. A total of 431 associations were made solely on the basis of model organism phenotype data. Additionally, we observed a striking, statistically significant tendency for individual disease phenotypes to be associated with multiple genes located within a single CNV region, a phenomenon that we denote as pheno-clustering. Many of the clusters also display statistically significant similarities in protein function or vicinity within the protein-protein interaction network. Our results provide a basis for understanding previously un-interpretable genotype-phenotype correlations in pathogenic CNVs and for mobilizing the large amount of model organism phenotype data to provide insights into human genetic disorders
Molecular mechanism of CHRDL1-mediated X-linked megalocornea in humans and in Xenopus model
Chordin-Like 1 (CHRDL1) mutations cause non-syndromic X-linked megalocornea (XMC) characterized by enlarged anterior eye segments. Mosaic corneal degeneration, presenile cataract and secondary glaucoma are associated with XMC. Beside that CHRDL1 encodes Ventroptin, a secreted bone morphogenetic protein (BMP) antagonist, the molecular mechanism of XMC is not well understood yet. In a family with broad phenotypic variability of XMC, we identified the novel CHRDL1 frameshift mutation c.807_808delTC [p.H270Wfs*22] presumably causing CHRDL1 loss of function. Using Xenopus laevis as model organism, we demonstrate that chrdl1 is specifically expressed in the ocular tissue at late developmental stages. The chrdl1 knockdown directly resembles the human XMC phenotype and confirms CHRDL1 deficiency to cause XMC. Interestingly, secondary to this bmp4 is down-regulated in the Xenopus eyes. Moreover, phospho-SMAD1/5 is altered and BMP receptor 1A is reduced in a XMC patient. Together, we classify these observations as negative-feedback regulation due to the deficient BMP antagonism in XMC. As CHRDL1 is preferentially expressed in the limbal stem cell niche of adult human cornea, we assume that CHRDL1 plays a key role in cornea homeostasis. In conclusion, we provide novel insights into the molecular mechanism of XMC as well as into the specific role of CHRDL1 during cornea organogenesis, among others by the establishment of the first XMC in vivo model. We show that unravelling monogenic cornea disorders like XMC—with presumably disturbed cornea growth and differentiation—contribute to the identification of potential limbal stem cell niche factors that are promising targets for regenerative therapies of corneal injurie
Identification of FOXP1 Deletions in Three Unrelated Patients with Mental Retardation and Significant Speech and Language Deficits
Mental retardation affects 2-3% of the population and shows a high heritability. Neurodevelopmental disorders that include pronounced impairment in language and speech skills occur less frequently. For most cases, the molecular basis of mental retardation with or without speech and language disorder is unknown due to the heterogeneity of underlying genetic factors. We have used molecular karyotyping on 1523 patients with mental retardation to detect copy number variations (CNVs) including deletions or duplications. These studies revealed three heterozygous overlapping deletions solely affecting the forkhead box P1 (FOXP1) gene. All three patients had moderate mental retardation and significant language and speech deficits. Since our results are consistent with a de novo occurrence of these deletions, we considered them as causal although we detected a single large deletion including FOXP1 and additional genes in 4104 ancestrally matched controls. These findings are of interest with regard to the structural and functional relationship between FOXP1 and FOXP2. Mutations in FOXP2 have been previously related to monogenic cases of developmental verbal dyspraxia. Both FOXP1 and FOXP2 are expressed in songbird and human brain regions that are important for the developmental processes that culminate in speech and language. ©2010 Wiley-Liss, Inc
Fine Mapping of the 1p36 Deletion Syndrome Identifies Mutation of PRDM16 as a Cause of Cardiomyopathy
Deletion 1p36 syndrome is recognized as the most common terminal deletion syndrome. Here, we describe the loss of a gene within the deletion that is responsible for the cardiomyopathy associated with monosomy 1p36, and we confirm its role in nonsyndromic left ventricular noncompaction cardiomyopathy (LVNC) and dilated cardiomyopathy (DCM). With our own data and publically available data from array comparative genomic hybridization (aCGH), we identified a minimal deletion for the cardiomyopathy associated with 1p36del syndrome that included only the terminal 14 exons of the transcription factor PRDM16 (PR domain containing 16), a gene that had previously been shown to direct brown fat determination and differentiation. Resequencing of PRDM16 in a cohort of 75 nonsyndromic individuals with LVNC detected three mutations, including one truncation mutant, one frameshift null mutation, and a single missense mutant. In addition, in a series of cardiac biopsies from 131 individuals with DCM, we found 5 individuals with 4 previously unreported nonsynonymous variants in the coding region of PRDM16. None of the PRDM16 mutations identified were observed in more than 6,400 controls. PRDM16 has not previously been associated with cardiac disease but is localized in the nuclei of cardiomyocytes throughout murine and human development and in the adult heart. Modeling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish resulted in both contractile dysfunction and partial uncoupling of cardiomyocytes and also revealed evidence of impaired cardiomyocyte proliferative capacity. In conclusion, mutation of PRDM16 causes the cardiomyopathy in 1p36 deletion syndrome as well as a proportion of nonsyndromic LVNC and DCM
Application of a functional model for the modernization of devices designed to eliminate emergency oil spills
The article describes the stages of creation and composition of the functional model, which can be used for the design of oil spill response devices. The principle of operation of the functional model given in the article and it's graphical scheme are show
Expression analysis and functional characterization of two novel tumorsuppressor candidate genes in mammary carcinoma
Titelblatt Inhaltsverzeichnis, Danksagung
1\. Einleitung 1
1.1 Epidemiologie des Mammakarzinoms
1.2 Tumorgenese 3
1.3 Strategie zur Validierung von Kandidatengenen 7
1.4 Epidemiologie des Mammakarzinoms 8
1.5 Zell-Matrix-Verbindungen 9
1.6 Wnt-Signaltransduktionsweg11
2\. Zielsetzung 14
3\. Material und Methoden 16
3.1 Puffer und Lösungen 16
3.2 Zelllinien 18
3.3 Plasmid-Isolierung 18
3.4 Sequenzierung 19
3.5 LoH-Analyse 21
3.6 Mutationsanalyse 21
3.7 Northern Hybridisierung 25
3.8 RNA in situ Hybridisierung 27
3.9 Poly-A+-ENA-Präparation 32
3.10 cDNA-Synthese 33
3.11 Quantitative PCR (TaqManTM 35
3.12 DANN-Präparation aus Gewebeproben 38
3.13 Proteinchemische Methoden 39
3.14 Zellbiologische Methoden 51
3.15 Statistische Methoden 58
4\. Ergebnisse
4.1 Auswahl und Bestätigung der differentiellen Expression der Kandidatengene
auf RNA-Ebene 59
4.2 Prognostische Relevanz von SFRP1
4.3 Funktionelle Analyse von SFRP1 83
5\. Diskussion 90
5.1 Tensin 90
5.2 Secreted frizzled-related protein 1 (SFRP1) 95
6\. Zusammenfassung 108
7\. Summary 110
8\. Literaturverzeichnis 110
9\. Abbildungsverzeichnis 124
10\. Tabellenverzeichnis 125
11\. Lebenslauf 126
12\. Publikationsliste 127
13\. Anhang 129
13.1 Abkürzungsverzeichnis 129
13.2 Histologie und klinisch-pathologische Eigenschaften der verwendeten
Gewebeproben 131
13.3 Primer für Mutationsanalyse 132
13.4 Erklärung 135600 Kandidatengene, die möglicherweise an der Entwicklung von gynäkologischen
Tumoren beteiligt sind, wurden mit Hilfe eines in silico Ansatzes (eNorthern)
identifiziert. Ausgehend von diesen Kandidatengenen wurden 40 Kandidaten für
die weitere Validierung ausgewählt und im Rahmen der vorliegenden Dissertation
wurden zwei dieser 40 Kandidatengene, Tensin und SFRP1, untersucht. Für beide
Kandidatengene konnten die in silico Daten auf RNA-Ebene mit mehreren
unabhängigen Methoden (Northern Blot, quantitative PCR, RNA in situ
Hybridisierung) bestätigt werden. Beide Gene zeigen in den Tumorzellen von
gynäkologischen Karzinomen eine starke Expressionsreduktion im Vergleich zum
entsprechenden Normalepithel. Da mit Mutationsanalysen keine Veränderungen der
kodierenden Gensequenz nachgewiesen werden konnten, wird postuliert, dass
beide Kandidatengene zur Tumorsuppressorgen Klasse II zählen. Als Komponente
der Fokalkontakte ist Tensin sowohl an der Zell-Matrix-Adhäsion, als auch an
der Signaltransduktion beteiligt. Die Analyse des Tensin-Expressionsmusters in
Brustgewebe mit Hilfe der RNA in situ Hybridisierung zeigte eine starke
Expression vor allem in Epithelzellen des normalen Brustgewebes. In
Brusttumorzellen war die Tensin-Expression hingegen in ca. 50% der
untersuchten Fälle reduziert oder vollständig verloren. Diese RNA-Daten
stützen die auf den Daten des eNortherns basierende Hypothese, dass Tensin
möglicherweise Tumorsuppressorfunktion besitzt. Es konnten keine
Sequenzveränderungen in der kodierenden Sequenz des Tensin-Gens in genomischer
DNA aus Brusttumorgewebe nachgewiesen werden. Das Tensin-Gen könnte während
der Tumorentstehung stattdessen durch epigenetische Mechanismen wie Promotor-
Hypermethylierung inaktiviert werden. Ob der Verlust der Tensin-Expression
direkte Relevanz für die Tumorentstehung hat oder ob das Kandidatengen in der
Tumordiagnostik als Marker verwendet werden kann, muss durch weitere
Untersuchungen in Zellkultur-Modellen und auf Proteinebene, z.B. an �Tissue
Microarrays�, analysiert werden. Das zweite in dieser Arbeit untersuchte Gen,
SFRP1, ist ein negativer Regulator des Wnt-Signaltransduktionsweges, der in
der Entstehung von soliden Tumoren, z.B. Brust- und Kolontumoren eine wichtige
Rolle spielt. Ein selbst-generierter, polyklonaler SFRP1-Antikörper wurde
charakterisiert, um die SFRP1 Expression auf Proteinebene zu analysieren und
die Assoziation mit klinischen Parametern und tumorspezifischem Überleben zu
untersuchen. Die Analyse von 56 in situ Karzinomen und mehr als 2000 invasiven
Karzinomen ergab, dass SFRP1 in diesen Karzinomen im gleichen Maß (43% bzw.
46%) vollständig verloren ist. Das deutet darauf hin, dass der Verlust von
SFRP1 ein frühes Ereignis in der Tumorentstehung darstellt. Die SFRP1
Expression ist revers mit der Tumorgröße (pT) korreliert (p < 0,001), aber es
wurde keine Korrelation mit anderen klinischen Parametern wie Tumorgrad oder
Lymphknotenstatus beobachtet. Dies konnte in einer multivariaten Analyse
bezüglich der Assoziation von SFRP1 Expression und Tumorgröße bestätigt werden
(p = 0,029). Bei der Korrelation der Überlebensdaten mit der SFRP1 Expression
konnte eine schlechtere Prognose für Patientinnen mit SFRP1-negativen kleinen
Tumoren (pT1) beobachtet werden (p = 0,04). Die funktionellen Analysen in
Brusttumor-Zelllinien ergaben eine mögliche Funktion von SFRP1 in der
Regulation der Zelladhäsion. In den hier verwendeten Modellen konnte keine
Bedeutung von SFRP1 für die Kontrolle der Invasivität von Tumorzellen
beobachtet werden. Ein Einfluss auf die Proliferation von Tumorzelllinien war
zu bestimmten Zeitpunkten (24h und 48h) nachweisbar. Folglich ist der Verlust
von SFRP1 wahrscheinlich nicht als initialer Faktor für die Tumorentstehung
relevant, sondern in Verbindung mit anderen genetischen Veränderungen. SFRP1
könnte aber ein neuer prognostischer Marker in der Brustkrebs-
Diagnostik/-Therapie bei frühen Tumoren sein.600 candidate genes which are possibly involved in the development of
gynecological tumors were identified using an in silico approach (eNorthern).
Fourty of these candidate genes were selected for further validation within a
German research consortium. This dissertation project deals with the
validation of two candidate genes, Tensin and SFRP1. The in silico data for
Tensin and SFRP1 were confirmed on the RNA level by three independent methods
(Northern Blot, quantitative PCR and RNA in situ hybridization). Both genes
show strongly reduced expression in gynecological tumors compared to normal
tissue. Since no aberrations were detected in the coding sequence of both
genes, I postulate that the two candidate genes belong to the group of class
II tumor suppressor genes. Tensin is part of focal adhesion complexes, thus
playing a role in cell-matrix adhesion as well as in signal transduction
processes. The analysis of the Tensin expression pattern in mammary gland
tissue using RNA in situ hybridization revealed an abundant expression in
normal breast epithelial cells. Breast tumor cells exhibited a reduced
expression or complete loss of Tensin in ~50% of all cases investigated in
this study. The RNA expression data support the possible involvement of Tensin
as a tumor suppressor gene in breast cancer development. No mutations were
identified in the coding sequence of Tensin in genomic DNA isolated from
breast tumor tissues. It is conceivable that the Tensin inactivation during
tumor development is due to epigenetic mechanisms, i.e. promoter
hypermethylation. If the loss of Tensin expression is relevant for
tumorigenesis or if Tensin can be used as a novel marker in cancer
diagnostics, has to be shown by further studies on protein level as well as in
cell culture experiments. The second gene investigated in this thesis,
secreted Frizzled-related protein 1 (SFRP1), is a negative regulator of the
Wnt pathway. An SFRP1 specific antibody was generated and characterized to
analyze SFRP1 expression on protein level and to investigate the association
of SFRP1 expression with clinicopathological parameters and patient survival.
The analysis of >2000 invasive breast tumors and 56 carcinoma in situ revealed
similar frequencies of SFRP1 loss in these tumors (46% and 43% respectively).
Therefore, I propose that loss of SFRP1 expression is an early event in breast
tumorigenesis. SFRP1 expression was inversely correlated with tumor stage
(p<0.001) but not with other prognostic parameters like tumor grade or lymph
node status. Performing a multivariate analysis we could confirm the
association between tumor stage and SFRP1 expression (p=0.029). In particular,
loss of SFRP1 expression in early stage breast tumors (pT1) was associated
with poor prognosis (p=0.04). Functional analyses revealed a possible
influence of SFRP1 on the regulation of cell adhesion to the ECM whereas no
effect on invasiveness of tumor cell lines was observed in the cell culture
model. A possible involvement in proliferation of tumor cell was detectable at
certain time points (24h and 48h). In conclusion, loss of SFRP1 is most likely
not the initial event directly leading to breast tumorigenesis but
facilitating breast cancer development if other genetic changes occur in the
cell. Still, SFRP1 expression might be useful as a novel prognostic marker in
early stage breast cancer
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