De-Novo Transcriptome Sequencing of a Normalized cDNA Pool from Influenza Infected Ferrets

The ferret is commonly used as a model for studies of infectious diseases. The genomic sequence of this animal model is not yet characterized, and only a limited number of fully annotated cDNAs are currently available in GenBank. The majority of genes involved in innate or adaptive immune response are still lacking, restricting molecular genetic analysis of host response in the ferret model. To enable de novo identification of transcriptionally active ferret genes in response to infection, we performed de-novo transcriptome sequencing of animals infected with H1N1 A/California/07/2009. We also included splenocytes induced with bacterial lipopolysaccharide to allow for identification of transcripts specifically induced by Gram-negative bacteria. We pooled and normalized the cDNA library in order to delimit the risk of sequencing only highly expressed genes. While normalization of the cDNA library removes the possibility of assessing expression changes between individual animals, it has been shown to increase identification of low abundant transcripts. In this study, we identified more than 19000 partial ferret transcripts, including more than 1000 gene orthologs known to be involved in the innate and the adaptive immune response

Genetic analysis of neurofibromatosis type 2 (NF2) patients and NF2-associated tumors with emphasis on chromosome 22 deletions

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease with the hallmark of bilateral vestibular schwannomas. NF2 patients may also develop schwannomas at other locations as well as meningiomas, neurofibromas and ependymomas. NF2 shows a distinct clinical variability ranging from very mild to severe forms. Since identification of the NF2 gene in 1993, mutation-screening analyses have been performed. Mutations were, however, not found in numerous cases and the mechanism behind tumor development in NF2 is not fully understood. The correlation between type of NF2 gene mutation and clinical phenotype of patients does not provide a clear-cut explanation of the clinical variability. Also, absence of NF2 gene mutations in constitutional tissue from a related disorder, such as schwannomatosis, points towards a possible existence of an additional NF2-related locus from chromosome 22, which may modify the disease phenotype. This implies the necessity of further clarification of the genetic factors involved in NF2. Deletions on chromosome 22 and the inactivation of NF2 tumor suppressor gene are critical steps for meningioma formation. However, 40% of the tumors do not show aberrations of chromosome 22 or mutations in the NF2 gene. This suggest that alternative mechanisms are responsible for the development of a large fraction of meningiomas. We analyzed 25 meningiomas, which do not display chromosome 22 deletions, for genetic abnormalities by CGH (comparative genomic hybridization) to metaphase chromosomes. Two tumors showed l oss of chromosome 1p and 3p which suggest that deletions of both lp and 3p may contribute to meningioma turnorigenesis (paper I). In schwannomas, detailed mutation analyses of the NF2 gene showed that 60% of the tumors carry inactivating mutations. Thus, the mechanism behind the development of 40% of schwannomas is unknown. We studied 50 sporadic and NF2-associated schwannomas by high resolution LOH (loss-of-heterozygosity) on chromosome 22 and other chromosomes. Chromosome 22 deletions were detected in over 80% of the cases. Four tumors showed LOH not involving the NF2 locus. All exons of the NF2 gene were sequenced in these tumors and mutations were detected only in one case. Thus, additional regions chromosome 22 may harbor mutations possibly involved in schwannoma turnorigenesis (paper II). We also identified an early-onset NF2 patient with a large constitutional deletion on chromosome 22. Constitutional deletions of the entire NF2 gene were previously described in NF2 patients; two large deletions encompassing 700-800 kb have been reported in mildly affected subjects. Our severely affected case showed a much larger deletion stretching approximately 5 Mb towards the telomere (paper III). It may therefore be hypothesized that the severe phenotype in this patient is a result of a combined mutation in NF2 gene and in a putative modifier gene. Further mapping of deletions in this candidate region was performed by analysis of 116 NF2 patients for deletions on 22q. Analysis was carried out using high-resolution microarray-CGH on a genomic array covering at least 90% of a 7.4 Mb interval of 22q, around and distal to the NF2 locus. This is a novel approach for high-resolution detection of chromosomal abnormalities, both in constitutional and tumor-derived DNA. Deletions were detected in nine severe, nine moderate and six mild patients. Deletions in severely and moderately affected patients varied in size, whereas cases with mild NF2 displayed deletions affecting the NF2 locus only (paper IV). This result indicates that the geneotype/phenotype correlation is unlikely to exist and supports the notion that the NF2 modifier gene may exist in the vicinity of the NF2 locus. Finally, the tumor suppressor gene SMARCB1 was tested for mutations in meningioma and schwannoma. SMARCB1 is mutated in malignant rhabdoid tumors and is located on 22q11.2, a region frequently deleted in meningiomas. Fourty-three meningiomas and twenty-one schwannomas were tested for mutations in exons 2 through 8. However, no mutations were detected, suggesting that the SMARCB1 gene is not frequently involved in the pathogenesis of these tumors. We also identified the mouse ortholog and characterized different splice forms of this gene, both in human and in mouse (paper V)

Growth hormone overexpression in the central nervous system results in hyperphagia-induced obesity associated with insulin resistance and dyslipidemia

It is well known that peripherally administered growth hormone (GH) results in decreased body fat mass. However, GH-deficient patients increase their food intake when substituted with GH, suggesting that GH also has an appetite stimulating effect. Transgenic mice with an overexpression of bovine GH in the central nervous system (CNS) were created to investigate the role of GH in CNS. This study shows that overexpression of GH in the CNS differentiates the effect of GH on body fat mass from that on appetite. The transgenic mice were not GH-deficient but were obese and showed increased food intake as well as increased hypothalamic expression of agouti-related protein and neuropeptide Y. GH also had an acute effect on food intake following intra-cerebroventricular injection of C57BL/6 mice. The transgenic mice were severely hyperinsulinemic and showed a marked hyperplasia of the islets of Langerhans. In addition, the transgenic mice displayed alterations in serum lipid and lipoprotein levels and hepatic gene expression. In conclusion, GH overexpression in the CNS results in hyperphagia-induced obesity indicating a dual effect of GH with a central stimulation of appetite and a peripheral lipolytic effect

A full-coverage, high-resolution human chromosome 22 genomic microarray for clinical and research applications.

We have constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number variation. This chromosome 22 array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb. To demonstrate the utility of the array, we have applied it to profile acral melanoma, dermatofibrosarcoma, DiGeorge syndrome and neurofibromatosis 2. We accurately diagnosed homozygous/heterozygous deletions, amplifications/gains, IGLV/IGLC locus instability, and breakpoints of an imbalanced translocation. We further identified the 14-3-3 eta isoform as a candidate tumor suppressor in glioblastoma. Two significant methodological advances in array construction were also developed and validated. These include a strictly sequence defined, repeat-free, and non-redundant strategy for array preparation. This approach allows an increase in array resolution and analysis of any locus; disregarding common repeats, genomic clone availability and sequence redundancy. In addition, we report that the application of phi29 DNA polymerase is advantageous in microarray preparation. A broad spectrum of issues in medical research and diagnostics can be approached using the array. This well annotated and gene-rich autosome contains numerous uncharacterized disease genes. It is therefore crucial to associate these genes to specific 22q-related conditions and this array will be instrumental towards this goal. Furthermore, comprehensive epigenetic profiling of 22q-located genes and high-resolution analysis of replication timing across the entire chromosome can be studied using our array

High resolution deletion analysis of constitutional DNA from neurofibromatosis type 2 (NF2) patients using microarray-CGH

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder whose hallmark is bilateral vestibular schwannoma. It displays a pronounced clinical heterogeneity with mild to severe forms. The NF2 tumor suppressor (merlin/schwannomin) has been cloned and extensively analyzed for mutations in patients with different clinical variants of the disease. Correlation between the type of the NF2 gene mutation and the patient phenotype has been suggested to exist. However, several independent studies have shown that a fraction of NF2 patients with various phenotypes have constitutional deletions that partly or entirely remove one copy of the NF2 gene. The purpose of this study was to examine a 7 Mb interval in the vicinity of the NF2 gene in a large series of NF2 patients in order to determine the frequency and extent of deletions. A total of 116 NF2 patients were analyzed using high-resolution array-comparative genomic hybridization (CGH) on an array covering at least 90% of this region of 22q around the NF2 locus. Deletions, which remove one copy of the entire gene or are predicted to truncate the schwannomin protein, were detected in 8 severe, 10 moderate and 6 mild patients. This result does not support the correlation between the type of mutation affecting the NF2 gene and the disease phenotype. This work also demonstrates the general usefulness of the array-CGH methodology for rapid and comprehensive detection of small (down to 40 kb) heterozygous and/or homozygous deletions occurring in constitutional or tumor-derived DN

High resolution deletion analysis of constitutional DNA from neurofibromatosis type 2 (NF2) patients using microarray-CGH

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder whose hallmark is bilateral vestibular schwannoma. It displays a pronounced clinical heterogeneity with mild to severe forms. The NF2 tumor suppressor (merlin/schwannomin) has been cloned and extensively analyzed for mutations in patients with different clinical variants of the disease. Correlation between the type of the NF2 gene mutation and the patient phenotype has been suggested to exist. However, several independent studies have shown that a fraction of NF2 patients with various phenotypes have constitutional deletions that partly or entirely remove one copy of the NF2 gene. The purpose of this study was to examine a 7 Mb interval in the vicinity of the NF2 gene in a large series of NF2 patients in order to determine the frequency and extent of deletions. A total of 116 NF2 patients were analyzed using high-resolution array-comparative genomic hybridization (CGH) on an array covering at least 90% of this region of 22q around the NF2 locus. Deletions, which remove one copy of the entire gene or are predicted to truncate the schwannomin protein, were detected in 8 severe, 10 moderate and 6 mild patients. This result does not support the correlation between the type of mutation affecting the NF2 gene and the disease phenotype. This work also demonstrates the general usefulness of the array-CGH methodology for rapid and comprehensive detection of small (down to 40 kb) heterozygous and/or homozygous deletions occurring in constitutional or tumor-derived DN