Screening for copy number variation in genes associated with the long QT syndrome: clinical relevance.

OBJECTIVES: The aim of this study was to investigate, in a set of 93 mutation-negative long QT syndrome (LQTS) probands, the frequency of copy number variants (CNVs) in LQTS genes. BACKGROUND: LQTS is an inherited cardiac arrhythmia characterized by a prolonged heart rate-corrected QT (QTc) interval associated with sudden cardiac death. Recent studies suggested the involvement of duplications or deletions in the occurrence of LQTS. However, their frequency remains unknown in LQTS patients. METHODS: Point mutations in KCNQ1, KCNH2, and SCN5A genes were excluded by denaturing high-performance liquid chromatography or direct sequencing. We applied Multiplex Ligation-dependent Probe Amplification (MLPA) to detect CNVs in exons of these 3 genes. Abnormal exon copy numbers were confirmed by quantitative multiplex PCR of short fluorescent fragment (QMPSF). Array-based comparative genomic hybridization (array CGH) analysis was performed using Agilent Human Genome 244K Microarrays to further map the genomic rearrangements. RESULTS: We identified 3 different deletions in 3 unrelated families: 1 in KCNQ1 and 2 involving KCNH2. We showed in the largest family that the deletion involving KCNH2 is fully penetrant and segregates with the long QT phenotype in 7 affected members. CONCLUSIONS: Our study demonstrates that CNVs in KCNQ1 and KCNH2 explain around 3% of LQTS in patients with no point mutation in these genes. This percentage is likely higher than the frequency of point mutations in ANKB, KCNE1, KCNE2, KCNJ2, CACNA1C, CAV3, SCN4B, AKAP9, and SNTA1 together. Thus, we propose that CNV screening in KCNQ1 and KCNH2 may be performed routinely in LQTS patients.

Complex constitutional subtelomeric 1p36.3 deletion/duplication in a mentally retarded child with neonatal neuroblastoma.

Monosomy 1p36 is one of the most frequent subtelomeric microdeletion syndromes characterized by distinct craniofacial features and developmental delay/mental retardation. Other common symptoms include hypotonia, seizures, brain abnormalities, visual, auditory and heart defects. Neuroblastoma is a rare feature since to our knowledge only two patients with "pure" 1p36 deletion have been described. We report on a child with developmental delay and facial dysmorphy who developed neuroblastoma at 1 month of age. No primary site outside of the liver could be demonstrated and the tumour regressed spontaneously. Standard karyotyping was normal while subtelomeric screening using Multiplex Ligation-dependent Probe Amplification (MLPA) method revealed a constitutional de novo subtelomeric 1p36 deletion. Subsequent Agilent 244K oligonucleotide array-based comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis showed a complex 1p36.3 deletion/duplication rearrangement. Among the best candidate genes predisposing to the development of neuroblastoma located in 1p36, the AJAP1 gene is the only gene present in the duplication while CHD5, TNFRSF25 and CAMTA1 are located outside of the rearrangement. Therefore, a gene-dosage effect involving a gene located in the duplication including AJAP1 might explain the neuroblastoma observed in our patient. The rearrangement might equally interfere with the expression of a gene located outside of it (including CHD5 located 1Mb away from the rearrangement) playing a role in the tumorigenesis. In conclusion, this study illustrates the complexity of such rearrangement characterized by array CGH and strengthens that constitutional 1p36.3 rearrangement predisposes to the development of neuroblastoma.

Identification of differentially expressed genes in human varicose veins: involvement of matrix gla protein in extracellular matrix remodeling.

This study was designed to identify the global pattern of differentially expressed genes in human varicose veins. Using suppressive subtractive hybridization, we identified overexpression of genes known to be associated with extracellular matrix remodeling, including collagen III, tissue inhibitor of metalloproteinases I, dermatopontin, matrix Gla protein (MGP) and tenascin C. Real-time polymerase chain reaction analysis confirmed the differential expression of these genes. The overexpression of MGP transcript was associated with increased MGP level in varicose veins, in particular the undercarboxylated form of the protein. Smooth muscle cells from varicose veins showed increased proliferation rate and enhanced matrix mineralization. This observation correlated with the presence of ectopic mineralization areas in the varicose vein walls. The use of warfarin, to inhibit MGP activity, or siRNA targeting MGP transcript induced a reduction in the exacerbated proliferation of varicose vein smooth muscle cells. Our results suggest that high expression of MGP in varicose veins may contribute to venous wall remodeling by affecting proliferation and mineralization processes probably through impaired carboxylation of MGP. In addition, suppressive subtractive hybridization results also produce a profile of differentially expressed genes in varicose veins, in particular extracellular matrix components. Further study of these genes will provide insights into their specific roles in the etiology of venous disease.

New indicators of beef sensory quality revealed by expression of specific genes.

To identify new molecular markers of beef sensory quality, the transcriptomes of Longissimus thoracis muscle from 25 Charolais bull calves were analyzed using microarrays and compared between high and low meat quality groups; 215 genes were differentially expressed according to tenderness, juiciness, and/or flavor. Among these, 23 were up-regulated in the tenderest, juiciest, and tastiest meats, and 18 were highly correlated with both flavor and juiciness (e.g., PRKAG1), explaining up to 60% of their variability. Nine were down-regulated in the same meats, but only DNAJA1 [the results relating to DNAJA1 and its relationship with tenderness have been patented (Genomic marker for meat tenderness; Patent EP06300943.5, September 12, 2006)], which encodes a heat shock protein, showed a strong negative correlation with tenderness that alone explained 63% of its variability. This protein, known for its anti-apoptotic role, could be involved in meat aging. Thus, DNAJA1 could constitute a new marker of beef sensory quality.

Involvement of BTBD1 in mesenchymal differentiation.

BTBD1 is a recently cloned BTB-domain-containing protein particularly expressed in skeletal muscle and interacting with DNA topoisomerase 1 (Topo1), a key enzyme of cell survival. We have previously demonstrated that stable overexpression of a N-terminal truncated BTBD1 inhibited ex vivo myogenesis but not adipogenesis of pluripotent C2C12 cells. Here, BTBD1 expression was studied in three models of cellular differentiation: myogenesis (C2C12 cells), adipogenesis (3T3-L1 cells) and osteogenesis (hMADS cells). BTBD1 mRNA was found to be upregulated during myogenesis. At the opposite, we have not observed BTBD1 upregulation in an altered myogenesis cellular model and we observed a downregulation of BTBD1 mRNA expression in adipogenesis. Interestingly, amounts of Topo1 protein, but not Topo1 mRNA, were found to be modulated at the opposite of BTBD1 mRNA. No variation of BTBD1 expression was measured during osteogenesis. Taken together, these results indicate that BTBD1 mRNA is specifically regulated during myogenic and adipogenic differentiation, in relation with Topo1 expression. Moreover, they corroborate observations made previously with truncated BTBD1 and show that BTBD1 is a key protein of balance between adipogenesis and myogenesis. Finally, a transcriptome analysis gave molecular clues to decipher BTBD1 role, with an emphasis on the involvement in ubiquitin/proteasome degradation pathway.

Cellular gene expression survey of PseudoRabies Virus (PRV) infected Human Embryonic Kidney cells (HEK-293).

Pseudorabies virus (PRV) is an alpha herpesvirus that causes Aujezsky disease in the pig. To characterize the impact of PRV infection on cellular expression, we used microarrays consisting of 9850 oligonucleotides corresponding to human genes and examined the expression levels of mRNA isolated 0.5, 3, 6, and 9 h post infection (hpi) from cultures of infected HEK-293 cells. Very few changes were observed during the first 3 h of infection but significant modifications in the cell expression of more than 1000 genes were clearly apparent by 6 hpi. More than 2400 genes were either up- or down-regulated during the 9 h experiment. These results were then analyzed using gene ontology and the MAPP and MAPPFinder software. This comprehensive analysis clearly shows that the down-regulated genes were mainly involved in macromolecular synthesis (DNA, RNA and proteins) and the cell cycle. The up-regulated genes primarily concerned the regulation of DNA transcription, developmental processes (central nervous system development, neurogenesis, angiogenesis), cell adhesion and potassium transport. This study is the first qualitative analysis of a gene expression survey in a human cell line following PRV infection. It demonstrates global changes in the cell expression profile, and identifies the main biological processes that are altered during virus replication.

Characterization of the expression of the hypoxia-induced genes neuritin, TXNIP and IGFBP3 in cancer.

By triggering an adaptive response to hypoxia which is a common feature of tumor microenvironments, endothelial cells contribute to the onset of angiogenic responses involved in tumor growth. Therefore, identifying hypoxic markers represent a challenge for a better understanding of tumor angiogenesis and for the optimization of anti-angiogenic therapeutic strategy. Using representational difference analysis combined with microarray, we here report the identification of 133 hypoxia-induced transcripts in human microendothelial cells (HMEC-1). By Northern blot, we confirm hypoxia-induced expression of insulin-like growth factor binding protein 3 (igfbp3), thioredoxin-interacting protein (txnip), neuritin (nrn1). Finally, by performing in situ hybridization on several types of human tumors, we provide evidence for nrn1 and txnip as hypoxic perinecrotic markers and for igfbp3 as a tumor endothelial marker. We propose these hypoxia-induced genes could represent relevant prognostic tools and targets for therapeutic intervention in cancers.

Hepatocyte iron loading capacity is associated with differentiation and repression of motility in the HepaRG cell line.

High liver iron content is a risk factor for developing hepatocellular carcinoma (HCC). However, HCC cells are always iron-poor. Therefore, an association between hepatocyte iron storage capacity and differentiation is suggested. To characterize biological processes involved in iron loading capacity, we used a cDNA microarray to study the differentiation of the human HepaRG cell line, from undifferentiated proliferative cells to hepatocyte differentiated cells. We were able to identify genes modulated along HepaRG differentiation, leading us to propose new genes not previously associated with HCC. Moreover, using Gene Ontology annotations, we demonstrated that HepaRG hepatocyte iron loading capacity occurred both with the repression of genes involved in cell motility, signal transduction, and biosynthesis and with the appearance of genes linked to lipid metabolism and immune response. These results provide new insights in the understanding of the relationship between iron and hepatocyte differentiation during iron-related hepatic diseases.

Distinct molecular portraits of human failing hearts identified by dedicated cDNA microarrays.

AIMS: This study aimed to investigate whether a molecular profiling approach should be pursued for the classification of heart failure patients. METHODS AND RESULTS: Applying a subtraction strategy we created a cDNA library consisting of cardiac- and heart failure-relevant clones that were used to construct dedicated cDNA microarrays. We measured relative expression levels of the corresponding genes in left ventricle tissue from 17 patients (15 failing hearts and 2 nonfailing hearts). Significance analysis of microarrays was used to select 159 genes that distinguished between all patients. Two-way hierarchical clustering of the 17 patients and the 159 selected genes led to the identification of three major subgroups of patients, each with a specific molecular portrait. The two nonfailing hearts clustered closely together. Interestingly, our classification of patients based on their molecular portraits did not correspond to an identified etiological classification. Remarkably, patients with the highest medical urgency status (United Network for Organ Sharing, Status 1A) clustered together. CONCLUSION: With this pilot feasibility study we demonstrated a novel classification of end-stage heart failure patients, which encourages further development of this approach in prospective studies on heart failure patients at earlier stages of the disease.

Transcriptome variations in human CaCo-2 cells: a model for enterocyte differentiation and its link to iron absorption.

Complete clinical expression of the HFE1 hemochromatosis is very likely modulated by genes linked to duodenal iron absorption, whose level is conditioned by unknown processes taking place during enterocyte differentiation. We carried out a transcriptomic study on CaCo-2 cells used as a model of enterocyte differentiation in vitro. Of the 720 genes on the microarrays, 80, 50, and 56 were significantly down-regulated up-regulated, and invariant during differentiation. With regard to iron metabolism, we showed that HEPH, SLC11A2, SLC11A3, and TF are significantly up-regulated, while ATP7B and SLC39A1 (and SFT) are down-regulated and ACO1, dCYTb, FECH, and FTH1 show constant expression. Ontological annotations highlight the decrease in the expression of cell cycle and DNA metabolism associated genes as well as transcription, protein metabolism, signal transduction, and nucleocytoplasmic transport associated genes, whereas there are increases in the expression of genes linked to cell adhesion, lipid and xenobiotic metabolism, iron transport and homeostasis, and immune response.

Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice.

The mdx mouse is a model for human Duchenne muscular dystrophy (DMD), an X-linked degenerative disease of skeletal muscle tissue characterized by the absence of the dystrophin protein. The mdx mice display a much milder phenotype than DMD patients. After the first week of life when all mdx muscles evolve like muscles of young DMD patients, mdx hindlimb muscles substantially compensate for the lack of dystrophin, whereas mdx diaphragm muscle becomes progressively affected by the disease. We used cDNA microarrays to compare the expression profile of 1,082 genes, previously selected by a subtractive method, in control and mdx hindlimb and diaphragm muscles at 12 time points over the first year of the mouse life. We determined that 1) the dystrophin gene defect induced marked expression remodeling of 112 genes encoding proteins implicated in diverse muscle cell functions and 2) two-thirds of the observed transcriptomal anomalies differed between adult mdx hindlimb and diaphragm muscles. Our results showed that neither mdx diaphram muscle nor mdx hindlimb muscles evolve entirely like the human DMD muscles. This finding should be taken under consideration for the interpretation of future experiments using mdx mice as a model for therapeutic assays.