Loss of DAXX and ATRX are associated with chromosome instability and reduced survival of patients with pancreatic neuroendocrine tumors

BACKGROUND & AIMS Sporadic pancreatic neuroendocrine tumors (pNETs) are rare and genetically heterogeneous. Chromosome instability (CIN) has been detected in pNETs from patients with poor outcomes, but no specific genetic factors have been associated with CIN. Mutations in death domain-associated protein gene (DAXX) or ATR-X gene (ATRX) (which both encode proteins involved in chromatin remodeling) have been detected in 40% of pNETs, in association with activation of alternative lengthening of telomeres. We investigated whether loss of DAXX or ATRX, and consequent alternative lengthening of telomeres, are related to CIN in pNETs. We also assessed whether loss of DAXX or ATRX is associated with specific phenotypes of pNETs. METHODS We collected well-differentiated primary pNET samples from 142 patients at the University Hospital Zurich and from 101 patients at the University Hospital Bern (both located in Switzerland). Clinical follow-up data were obtained for 149 patients from general practitioners and tumor registries. The tumors were reclassified into 3 groups according to the 2010 World Health Organization classification. Samples were analyzed by immunohistochemistry and telomeric fluorescence in situ hybridization. We correlated loss of DAXX, or ATRX, expression, and activation of alternative lengthening of telomeres with data from comparative genomic hybridization array studies, as well as with clinical and pathological features of the tumors and relapse and survival data. RESULTS Loss of DAXX or ATRX protein and alternative lengthening of telomeres were associated with CIN in pNETs. Furthermore, loss of DAXX or ATRX correlated with tumor stage and metastasis, reduced time of relapse-free survival, and decreased time of tumor-associated survival. CONCLUSIONS Loss of DAXX or ATRX is associated with CIN in pNETs and shorter survival times of patients. These results support the hypothesis that DAXX- and ATRX-negative tumors are a more aggressive subtype of pNET, and could lead to identification of strategies to target CIN in pancreatic tumors.

Long range physical map of the Wilms' tumor - aniridia region on human chromosome 11

Nephroblastoma or Wilms' tumor is a pediatric renal malignancy that is the most frequently occurring childhood solid tumor. Approximately 1-2% of children with Wilms' tumor also present with aniridia, a congenital absence of all or part of the iris of the eye. These children also have high rates of genitourinary anomalies and mental retardation resulting in what is called the WAGR (Wilms' tumor, aniridia, genitourinary anomaly, mental retardation) syndrome. Cytogenetic analysis of metaphase chromosomes from these patients revealed a consistent deletion of band P13 on chromosome 11. These observations suggest close physical linkage between the disease-related loci, and further imply that development of each phenotype results from the loss of normal gene function.^ The objective of this work is to understand the molecular events at chromosome band 11p13 that are essential to the development of sporadic Wilms' tumor and sporadic aniridia. Two human/hamster somatic cell hybrids have been used to identify sixteen independent DNA probes that map to this segment of the human genome. These newly identified DNA probes and four previously reported probes (CAT, FSHB, D11S16, and HBVIS) have been used to subdivide 11p13 into five intervals defined by overlapping constitutional deletions from several WAGR patients. A long-range physical map of 11p13 has been constructed using each of these probes in Southern blot analysis of genomic DNA after digestion with infrequently cutting restriction enzymes and pulse-field gel electrophoresis. This map, established primarily with MluI and NotI, spans approximately 13 $\times$ 10$\sp{6}$ bp and encompasses deletion and translocation breakpoints associated with genitourinary anomalies, aniridia, and sporadic Wilms' tumor. This complete physical map of human chromosome band 11p13 enables us to localize the genes for sporadic Wilms' tumor and sporadic aniridia to a small number of specific NotI fragments. ^

Defining the regions of homology between human chromosome 16p12--13 and mouse chromosomes

In this study, the evolutionary relationship between human chromosome 16p12-p13 and mouse chromosomes was investigated by determining the order of marker loci in the region and then identifying the chromosomal locations of the homologous loci in mice. Eighteen genes from human 16 were mapped to fifteen subchromosomal regions by a variety of mapping approaches.^ Thirteen of the genes were mapped in the mouse. Linkage analysis with backcross mice and segregation analysis in a mouse - Chinese Hamster Ovary (CHO) somatic cell hybrid panel informative for different regions of mouse genome were used. The results assigned the thirteen genes to three different mouse chromosomes.^ A group of six genes on mouse 16 was found to be closely linked to Scid. The order of Myh11 and Mrp remains ambiguous since no recombination was detected in backcross analysis. Their relative position in human is also uncertain since they were shown to be very close to each other. For the other mouse loci, an unambiguous gene order could be determined and was found to be identical to that in human. Therefore, they comprise a new conserved linkage group between the two species. The orientation of the group was inverted relative to the centromeres, i.e. the proximal loci in one species become distal in another. The size of the group was estimated to be from 4.4 to 8 Mb and 10 to 32 cM in human. In mouse, it was about 21 cM in the backcross analysis. The two boundaries of the conserved linkage were defined within a 1 Mb range. It is now possible to predict the locations of mouse homologs for some human disease genes based on their locations on human 16p.^ The six human 16p genes that map to MMU7 showed a different gene order in mouse than in human. No recombination was found between Crym and Umod while Crym was distal to D16S79A and proximal to D16S92. The location of Stp and Cdr2 with respect to the above four loci was not determined since they were not mapped in the same set of backcross mice. These genes greatly expanded an existing conserved synteny group between the human 16p12-p13 region and the MMU7. It now consists of eleven loci that span a region of probably more than 10 Mb in human. The gene order derived from this study provided further evidence for chromosomal rearrangements within the conserved synteny. (Abstract shortened by UMI.) ^

Characterization of new staphylococcal cassette chromosome mec (SCCmec) and topoisomerase genes in fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius

Fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius isolates harbor two new staphylococcal cassette chromosome mec (SCCmec) elements that belong to class A, allotype 3 (SCCmec II-III), and to the new allotype 5 (SCCmec VII). Analysis of the complete nucleotide sequences of the topoisomerase loci gyrB/gyrA and grlB/grlA revealed mutations involved in fluoroquinolone resistance.

Two Loci on Chromosome 5 Are Associated with Serum IgE Levels in Labrador Retrievers

Crosslinking of immunoglobulin E antibodies (IgE) bound at the surface of mast cells and subsequent mediator release is considered the most important trigger for allergic reactions. Therefore, the genetic control of IgE levels is studied in the context of allergic diseases, such as asthma, atopic rhinitis, or atopic dermatitis (AD). We performed genome-wide association studies in 161 Labrador Retrievers with regard to total and allergen-specific immunoglobulin E (IgE) levels. We identified a genome-wide significant association on CFA 5 with the antigen-specific IgE responsiveness to Acarus siro. We detected a second genome-wide significant association with respect to the antigen-specific IgE responsiveness to Tyrophagus putrescentiae at a different locus on chromosome 5. A. siro and T. putrescentiae both belong to the family Acaridae and represent so-called storage or forage mites. These forage mites are discussed as major allergen sources in canine AD. No obvious candidate gene for the regulation of IgE levels is located under the two association signals. Therefore our studies offer a chance of identifying a novel mechanism controlling the host's IgE response.

The brown coat colour of Coppernecked goats is associated with a non-synonymous variant at the TYRP1 locus on chromosome 8

The recent development of a goat SNP genotyping microarray enables genome-wide association studies in this important livestock species. We investigated the genetic basis of the black and brown coat colour in Valais Blacknecked and Coppernecked goats. A genome-wide association analysis using goat SNP50 BeadChip genotypes of 22 cases and 23 controls allowed us to map the locus for the brown coat colour to goat chromosome 8. The TYRP1 gene is located within the associated chromosomal region, and TYRP1 variants cause similar coat colour phenotypes in different species. We thus considered TYRP1 as a strong positional and functional candidate. We resequenced the caprine TYRP1 gene by Sanger and Illumina sequencing and identified two non-synonymous variants, p.Ile478Thr and p.Gly496Asp, that might have a functional impact on the TYRP1 protein. However, based on the obtained pedigree and genotype data, the brown coat colour in these goats is not due to a single recessive loss-of-function allele. Surprisingly, the genotype distribution and the pedigree data suggest that the (496) Asp allele might possibly act in a dominant manner. The (496) Asp allele was present in 77 of 81 investigated Coppernecked goats and did not occur in black goats. This strongly suggests heterogeneity underlying the brown coat colour in Coppernecked goats. Functional experiments or targeted matings will be required to verify the unexpected preliminary findings.

Assignment of the porcine tumour necrosis factor alpha and beta genes to the chromosome region 7p11-q11 by in situ hybridization

The loci of the porcine tumour necrosis factor genes, alpha (TNFA) and beta (TNFB), have been chromosomally assigned by radioactive in situ hybridization. The genomic probes for TNFA and TNFB yielded signals above 7p11-q11, a region that has been shown earlier to carry the porcine major histocompatibility locus (SLA). These mapping data along with preliminary molecular studies suggest a genomic organization of the SLA that is similar to that of human and murine major histocompatibility complexes.

Differential spatial and structural organization of the X chromosome underlies dosage compensation in C. elegans.

The adjustment of X-linked gene expression to the X chromosome copy number (dosage compensation [DC]) has been widely studied as a model of chromosome-wide gene regulation. In Caenorhabditis elegans, DC is achieved by twofold down-regulation of gene expression from both Xs in hermaphrodites. We show that in males, the single X chromosome interacts with nuclear pore proteins, while in hermaphrodites, the DC complex (DCC) impairs this interaction and alters X localization. Our results put forward a structural model of DC in which X-specific sequences locate the X chromosome in transcriptionally active domains in males, while the DCC prevents this in hermaphrodites.

An equine chromosome 3 inversion is associated with the tobiano spotting pattern in German horse breeds

The tobiano white-spotting pattern is one of several known depigmentation phenotypes in horses and is desired by many horse breeders and owners. The tobiano spotting phenotype is inherited as an autosomal dominant trait. Horses that are heterozygous or homozygous for the tobiano allele (To) are phenotypically indistinguishable. A SNP associated with To had previously been identified in intron 13 of the equine KIT gene and was used for an indirect gene test. The test was useful in several horse breeds. However, genotyping this sequence variant in the Lewitzer horse breed revealed that 14% of horses with the tobiano pattern did not show the polymorphism in intron 13 and consequently the test was not useful to identify putative homozygotes for To within this breed. Speculations were raised that an independent mutation might cause the tobiano spotting pattern in this breed. Recently, the putative causative mutation for To was described as a large chromosomal inversion on equine chromosome 3. One of the inversion breakpoints is approximately 70 kb downstream of the KIT gene and probably disrupts a regulatory element of the KIT gene. We obtained genotypes for the intron 13 SNP and the chromosomal inversion for 204 tobiano spotted horses and 24 control animals of several breeds. The genotyping data confirmed that the chromosomal inversion was perfectly associated with the To allele in all investigated horses. Therefore, the new test is suitable to discriminate heterozygous To/+ and homozygous To/To horses in the investigated breeds.

A 4 Mb high resolution BAC contig on bovine chromosome 1q12 and comparative analysis with human chromosome 21q22

The bovine RPCI-42 BAC library was screened to construct a sequence-ready ~4 Mb single contig of 92 BAC clones on BTA 1q12. The contig covers the region between the genes KRTAP8P1 and CLIC6. This genomic segment in cattle is of special interest as it contains the dominant gene responsible for the hornless or polled phenotype in cattle. The construction of the BAC contig was initiated by screening the bovine BAC library with heterologous cDNA probes derived from 12 human genes of the syntenic region on HSA 21q22. Contig building was facilitated by BAC end sequencing and chromosome walking. During the construction of the contig, 165 BAC end sequences and 109 single-copy STS markers were generated. For comparative mapping of 25 HSA 21q22 genes, genomic PCR primers were designed from bovine EST sequences and the gene-associated STSs mapped on the contig. Furthermore, bovine BAC end sequence comparisons against the human genome sequence revealed significant matches to HSA 21q22 and allowed the in silico mapping of two new genes in cattle. In total, 31 orthologues of human genes located on HSA 21q22 were directly mapped within the bovine BAC contig, of which 16 genes have been cloned and mapped for the first time in cattle. In contrast to the existing comparative bovine-human RH maps of this region, these results provide a better alignment and reveal a completely conserved gene order in this 4 Mb segment between cattle, human and mouse. The mapping of known polled linked BTA 1q12 microsatellite markers allowed the integration of the physical contig map with existing linkage maps of this region and also determined the exact order of these markers for the first time. Our physical map and transcript map may be useful for positional cloning of the putative polled gene in cattle.

Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1-q12.2

The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report.

The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein.

The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S. cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene.