Reduced fractional anisotropy and axial diffusivity in white matter in 22q11.2 deletion syndrome: a pilot study.

Individuals with 22q11.2 deletion syndrome (22q11.2DS) evince a 30% incidence of schizophrenia. We compared the white matter (WM) of 22q11.2DS patients without schizophrenia to a group of matched healthy controls using Tract-Based-Spatial-Statistics (TBSS). We found localized reduction of Fractional Anisotropy (FA) and Axial Diffusivity (AD; measure of axonal integrity) in WM underlying the left parietal lobe. No changes in Radial Diffusivity (RD; measure of myelin integrity) were observed. Of note, studies in chronic schizophrenia patients report reduced FA, no changes in AD, and increases in RD in WM. Our findings suggest different WM microstructural pathology in 22q11.2DS than in patients with schizophrenia.

Obesity enhances gastrointestinal tumorigenesis in Apc-mutant mice.

Epidemiological evidence indicates a link between obesity and human colon cancer. A putative association between obesity and colon tumorigenesis has been explored experimentally using chemical carcinogens administered to obese rodents. The main objective of this study was to generate a new mouse line that displays both obesity and intestinal tumorigenesis. To this end, we have generated C57BLKS-mLepr(db/db); Apc(1638N/+) mice combining both db and Apc mutations. The db mutation results in obesity and type 2 diabetes, the Apc mutation is a key initiating event of intestinal neoplasia. All mice were euthanized at 6 months of age and all regions of the gastrointestinal tract examined for tumors. The results show that the combination of Apc(1638N/+) and db mutations not only enhanced mutant Apc-driven small intestinal tumorigenesis but also induced gastric and colonic tumors. Homozygous db mice did not develop gastrointestinal neoplasia. These findings indicate that obesity associated with type 2 diabetes promotes gastrointestinal tumorigenesis in Apc-deficient mice and provides evidence of a mechanistic link between obesity and colorectal neoplasia.

Converging evidence for a pseudoautosomal cytokine receptor gene locus in schizophrenia.

Schizophrenia is a strongly heritable disorder, and identification of potential candidate genes has accelerated in recent years. Genomewide scans have identified multiple large linkage regions across the genome, with fine-mapping studies and other investigations of biologically plausible targets demonstrating several promising candidate genes of modest effect. The recent introduction of technological platforms for whole-genome association (WGA) studies can provide an opportunity to rapidly identify novel targets, although no WGA studies have been reported in the psychiatric literature to date. We report results of a case-control WGA study in schizophrenia, examining approximately 500 000 markers, which revealed a strong effect (P=3.7 x 10(-7)) of a novel locus (rs4129148) near the CSF2RA (colony stimulating factor, receptor 2 alpha) gene in the pseudoautosomal region. Sequencing of CSF2RA and its neighbor, IL3RA (interleukin 3 receptor alpha) in an independent case-control cohort revealed both common intronic haplotypes and several novel, rare missense variants associated with schizophrenia. The presence of cytokine receptor abnormalities in schizophrenia may help explain prior epidemiologic data relating the risk for this illness to altered rates of autoimmune disorders, prenatal infection and familial leukemia.

Tumor progression in Apc(1638N) mice with Exo1 and Fen1 deficiencies.

Flap endonuclease 1 (Fen1) and exonuclease 1 (Exo1) have sequence homology and similar nuclease capabilities. Both function in multiple pathways of DNA metabolism, but appear to have distinct in vivo nucleic acid substrates, and therefore distinct metabolic roles. When combined with Apc(1638N), Fen1 promotes tumor progression. Because of functional similarity to Fen1, and because Exo1 is involved in DNA mismatch repair (MMR) by interaction with Msh2 and Mlh1, genes that cause hereditary nonpolyposis colorectal cancer (HNPCC), we investigated the possibility that Exo1 might also act as a modifier to Apc(1638N). We present evidence that mice with combined mutations in Apc(1638N) and Exo1 and Apc(1638N), Exo1 and Fen1 genes show moderate increased tumor incidence and multiplicity in comparison to Apc(1638N) siblings, implying a low penetrance role for Exo1 in early gastrointestinal (GI) tumorigenesis. Despite a decrease in median survival (10 months) in Apc(1638N) Exo1 mice, their tumors do not progress any more rapidly than those of Apc(1638N). Instead these animals die from infections that are the result of impaired immune response. Apc(1638N) Exo1 Fen1 mice survive longer (18 months), and therefore appear relatively immune competent. They die of invasive GI tumors that display microsatellite instability (MSI). Our results show that Exo1 has a modest tumor suppressor function.

Lung cancer genetics and pharmacogenomics.

Epidermal growth factor receptor (EGFR)-targeted therapies have demonstrated remarkable success in a small subset of non-small cell lung cancer patients. The mechanism of response has been an area of active research, with somatic mutation in a number of genes in the EGFR signal transduction pathway and copy number alterations of genes of the EGFR family as candidates contributing towards response. Continuing studies should help determine an appropriate biomarker or combination of biomarkers that can be used to predict response to this class of therapy.

A new mouse model for evaluating the immunotherapy of human colorectal cancer.

A new murine model of human colorectal cancer was generated by crossing human carcinoembryonic antigen (CEA) transgenic mice (H-2K(b)) with adenomatous polyposis coli (Apc1638N) knockout mice (H-2K(b)). The resulting hybrid mice developed gastrointestinal polyps in 6-8 months that progressed to invasive carcinomas with a similar pattern of dysplasia and CEA expression as observed in human colorectal cancer. These animals exhibited incomplete or partial tolerance to CEA as evidenced by delayed growth of CEA-expressing tumors and the inability to inhibit CEA-specific CTL responses. These results have important implications for understanding the role of CEA-specific immunity in human colon cancer patients and suggest that vaccine strategies targeting CEA may be feasible. This model provides a powerful system for evaluating antigen-specific tumor immunity against spontaneous tumors arising in an orthotopic location and permits evaluation of therapeutic vaccine strategies for human colorectal cancer.

The distinct spectra of tumor-associated Apc mutations in mismatch repair-deficient Apc1638N mice define the roles of MSH3 and MSH6 in DNA repair and intestinal tumorigenesis.

In mammalian cells, mismatch recognition has been attributed to two partially redundant heterodimeric protein complexes of MutS homologues, MSH2-MSH3 and MSH2-MSH6. We have conducted a comparative analysis of Msh3 and Msh6 deficiency in mouse intestinal tumorigenesis by generating Apc1638N mice deficient in Msh3, Msh6 or both. We have found that Apc1638N mice defective in Msh6 show reduced survival and a 6-7-fold increase in intestinal tumor multiplicity. In contrast, Msh3-deficient Apc1638N mice showed no difference in survival and intestinal tumor multiplicity as compared with Apc1638N mice. However, when Msh3 deficiency is combined with Msh6 deficiency (Msh3(-/-)Msh6(-/-)Apc1638N), the survival rate of the mice was further reduced compared to Msh6(-/-)Apc(1638N) mice because of a high multiplicity of intestinal tumors at a younger age. Almost 90% of the intestinal tumors from both Msh6(-/-)Apc1638N and Msh3(-/-)Msh6(-/-)Apc1638N mice contained truncation mutations in the wild-type Apc allele. Apc mutations in Msh6(-/-)Apc1638N mice consisted predominantly of base substitutions (93%) creating stop codons, consistent with a major role for Msh6 in the repair of base-base mismatches. However, in Msh3(-/-)Msh6(-/-)Apc1638N tumors, we observed a mixture of base substitutions (46%) and frameshifts (54%), indicating that in Msh6(-/-)Apc1638N mice frameshift mutations in the Apc gene were suppressed by Msh3. Interestingly, all except one of the Apc mutations detected in mismatch repair-deficient intestinal tumors were located upstream of the third 20-amino acid beta-catenin binding repeat and before all of the Ser-Ala-Met-Pro repeats, suggesting that there is selection for loss of multiple domains involved in beta-catenin regulation. Our analysis therefore has revealed distinct mutational spectra and clarified the roles of Msh3 and Msh6 in DNA repair and intestinal tumorigenesis.

Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome.

Noonan syndrome (MIM 163950) is an autosomal dominant disorder characterized by dysmorphic facial features, proportionate short stature and heart disease (most commonly pulmonic stenosis and hypertrophic cardiomyopathy). Webbed neck, chest deformity, cryptorchidism, mental retardation and bleeding diatheses also are frequently associated with this disease. This syndrome is relatively common, with an estimated incidence of 1 in 1,000-2,500 live births. It has been mapped to a 5-cM region (NS1) [corrected] on chromosome 12q24.1, and genetic heterogeneity has also been documented. Here we show that missense mutations in PTPN11 (MIM 176876)-a gene encoding the nonreceptor protein tyrosine phosphatase SHP-2, which contains two Src homology 2 (SH2) domains-cause Noonan syndrome and account for more than 50% of the cases that we examined. All PTPN11 missense mutations cluster in interacting portions of the amino N-SH2 domain and the phosphotyrosine phosphatase domains, which are involved in switching the protein between its inactive and active conformations. An energetics-based structural analysis of two N-SH2 mutants indicates that in these mutants there may be a significant shift of the equilibrium favoring the active conformation. This implies that they are gain-of-function changes and that the pathogenesis of Noonan syndrome arises from excessive SHP-2 activity.

p21(WAF1/cip1) is an important determinant of intestinal cell response to sulindac in vitro and in vivo.

Sulindac, a nonsteroidal anti-inflammatory drug, inhibits intestinal tumorigenesis in humans and rodents. Sulindac induced complex alterations in gene expression, but only 0.1% of 8063 sequences assayed were altered similarly by the drug in rectal biopsies of patients treated for 1 month and during response of colonic cells in culture. Among these changes was induction of the cyclin-dependent kinase inhibitor, p21(WAF1/cip1). In Apc1638(+/-) mice, targeted inactivation of p21 increased intestinal tumor formation in a gene-dose-dependent manner, but inactivation of p21 completely eliminated the ability of sulindac to both inhibit mitotic activity in the duodenal mucosa and to inhibit Apc-initiated tumor formation. Thus, p21 is essential for tumor inhibition by this drug. The array data can be accessed on the Internet at http://sequence.aecom.yu.edu/genome/.

Mouse models for human familial adenomatous polyposis.

Colorectal cancer (CRC) is the second most frequent type of cancer in the Western hemisphere. In the United States alone, it is estimated that 150 000 new cases are detected every year and more than 65 000 patients die from complications associated with this cancer. Identification of genes implicated in the initiation and progression of colorectal cancer in humans has prompted the generation of mouse models for this cancer. We will provide a brief overview of these mouse models for CRC and what they have contributed to our understanding of the events involved in the initiation and progression of this cancer.

Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts.

A prominent pathway of transforming growth factor (TGF)-beta signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-beta1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-beta1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-beta1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [(3)H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-beta1. Lack of Smad2 or Smad3 expression did not affect TGF-beta1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-beta1. Moreover, TGF-beta1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-beta1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-beta-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element)(4)-Lux reporter by TGF-beta1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-beta1 signaling.

TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome.

Velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a human disorder characterized by a number of phenotypic features including cardiovascular defects. Most VCFS/DGS patients are hemizygous for a 1.5-3.0 Mb region of 22q11. To investigate the etiology of this disorder, we used a cre-loxP strategy to generate mice that are hemizygous for a 1.5 Mb deletion corresponding to that on 22q11. These mice exhibit significant perinatal lethality and have conotruncal and parathyroid defects. The conotruncal defects can be partially rescued by a human BAC containing the TBX1 gene. Mice heterozygous for a null mutation in Tbx1 develop conotruncal defects. These results together with the expression patterns of Tbx1 suggest a major role for this gene in the molecular etiology of VCFS/DGS.

A high-resolution map of human chromosome 12.

Our sequence-tagged site-content map of chromosome 12 is now integrated with the whole-genome fingerprinting effort. It provides accurate and nearly complete bacterial clone coverage of chromosome 12. We propose that this integrated mapping protocol serves as a model for constructing physical maps for entire genomes.

Targeted inactivation of the p21(WAF1/cip1) gene enhances Apc-initiated tumor formation and the tumor-promoting activity of a Western-style high-risk diet by altering cell maturation in the intestinal mucosal.

Elimination of both alleles of the gene that encodes the cyclin kinase inhibitor p21(WAF1/cip1) increases the frequency and size of intestinal tumors in Apc1638+/- mice that inherit a mutant allele of the Apc gene, and intermediate effects are seen if a single p21 allele is inactivated. The increased tumor formation is associated with altered cell maturation in the intestinal mucosa of the p21-deficient mice--increased cell proliferation, and decreased apoptosis, and goblet cell differentiation--that is also a function of p21 gene dosage. Moreover, a Western-style diet that mimics principal risk factors for colon cancer (high fat and phosphate, low calcium and vitamin D) accelerates tumor formation in Apc1638+/- mice, and the loss of a single or both p21 alleles is additive with the tumor-promoting effects of this diet, resulting in more and larger tumors, and a highly significant decrease in survival time. Thus, p21 normally suppresses Apc-initiated tumor formation and is haplo-insufficient in this regard. This is consistent with recent reports that Apc initiates tumor formation by up-regulating c-myc expression through altered beta-catenin-Tcf signaling and that c-myc then up-regulates cdk4, whose activity is inhibited by p21. Decreased expression of p21 is also a marker of poor prognosis in patients, and the data presented suggest that dietary alterations in patients undergoing treatment for colon cancer might be highly effective in improving outcome.

Tumor-associated Apc mutations in Mlh1-/- Apc1638N mice reveal a mutational signature of Mlh1 deficiency.

Apc1638N mice, which are heterozygous for a germline mutation in Apc, typically develop three to five spontaneous intestinal tumors per animal. In most cases this is associated with allelic loss of wildtype Apc. We have previously reported that the multiplicity of intestinal tumors is increased dramatically by crossing Apc1638N with an Mlh1-deficient mouse strain that represents an animal model of hereditary non-polyposis colorectal cancer (HNPCC). The increased tumor multiplicity in these mice was associated with somatic mutations in the Apc tumor suppressor gene. Here, we have examined the nature and distribution of 91 Apc mutations implicated in the development of intestinal tumors in Mlh1-/- Apc1638N animals. Protein truncation mutations were detected in a majority of tumor samples, indicating that the prevailing mechanism of Apc mutation in tumors is altered from allelic loss to intragenic mutation as a result of Mlh1 deficiency. The observed mutations were a mixture of base substitutions (27%) and frameshifts (73%). Most frameshifts were detected within dinucleotide repeats and there were prominent mutational hotspots within sequences of this sort at codons 927-929, 1209-1211 and 1461-1464. The observed Apc mutations caused protein truncation upstream of the third 20 amino acid beta-catenin binding domain and the first Axin-binding SAMP repeat, yielding Apc proteins that are predicted to be deficient in destabilizing beta-catenin. Our results reveal a characteristic mutational signature in Apc that is attributable to Mlh1 deficiency. This demonstrates a direct effect of Mlh1 deficiency in the mutation of Apc in these tumors, and provides data that clarify the role of Mlh1 in mammalian DNA mismatch repair.

A sequence-ready map for human chromosome 12q15-21.

Construction of sequence-ready clone map is an essential step toward sequencing the human genome. We chose a region that is frequently amplified in liposarcoma between D12S350 and D12S106 in chromosome 12q15-21 to build a PAC/BAC clone contig map. This region was spanned by 4 YACs and contained 30 STS on the YAC and radiation hybrid (RH) framework maps, providing an average STS spacing of 160 kb if each YAC is approximately 1.2 Mb in size. To convert a STS-based YAC map to a STS-based contig map of bacterial clones, 22 non-polymorphic STS markers were used as probes to screen the high density gridded arrays of PAC and BAC clones by filter hybridizations, followed by assembly of clones into contigs by marker content. Contigs have been extended and joined by direct end sequencing of appropriate clones, generating new STSs and rescreening the library as necessary. Using these approaches, we have constructed 5 contigs covering the region with the largest single contig being 1.4 Mb and a final size estimation of 3.6 Mb. The map is comprised of 17 YACs, 187 PACs, 160 BACs, and 17 cosmids; onto this, 6 polymorphic, 97 non-polymorphic, 24 ESTs, and 4 gene-based markers are now placed in a unique order, providing an average resolution of approximately 28 kb. Of a total of 131 markers, 97 were developed in the present study. The sequence-ready map should provide a framework to generate complete DNA sequence and ultimately gene map of this segment of chromosome 12.

High-resolution transcript map of the region spanning D12S1629 and D12S312 at chromosome 12q13: triple A syndrome-linked region.

For those searching for human disease-causing genes, information on the position of genes with respect to genetic markers is essential. The physical map composed of ESTs and genetic markers provides the positional information of these markers as well as the starting point of gene identification in the form of genomic clones containing exons. To facilitate the effort of identification of genes in the region spanning D12S1629 and D12S312, we constructed a high-resolution transcript map with PAC/BAC/cosmid clones. The strategy for the construction of such a map involved utilization of STSs for the screening of the large insert bacterial chromosome libraries and a chromosome 12-specific cosmid library by hybridization. The contig was constructed based on the STS contents of the clones. The resulting high-resolution transcript map of the region between P273P14/SP6 and D12S312 spans 4.4 cM from 66.8 to 71.2 cM of the Généthon genetic map and represents approximately 2.4 Mb. It was composed of 81 BAC, 45 PAC, and 91 cosmid clones with a minimal tiling path consisting of 16 BAC and 4 PAC clones. These clones are being used to sequence this part of chromosome 12. We determined the order of 135 STSs including 74 genes and ESTs in the map. Among these, 115 STSs were unambiguously ordered, resulting in one ordered marker per 21 kb. The order of keratin type II locus genes was determined. This map would greatly enhance the positional cloning effort of the responsible genes for those diseases that are linked to this region, including male germ cell tumor as well as palmoplantar keratoderma, Bothnian-type, and triple A syndrome. This transcript map was localized at human chromosome 12q13.

Somatic Apc mutations are selected upon their capacity to inactivate the beta-catenin downregulating activity.

The APC gene, originally identified as the gene for familial adenomatous polyposis (FAP), is now considered as the true "gatekeeper" of colonic epithelial proliferation. Its main tumor suppressing activity seems to reside in the capacity to properly regulate intracellular beta-catenin signaling. Most somatic APC mutations are detected between codons 1286 and 1513, the mutation cluster region (MCR). This clustering can be explained either by the presence of mutation-prone sequences within the MCR, or by the selective advantage provided by the resulting truncated polypeptides. Here, a Msh2-deficient mouse model (Msh2(delta 7N) ) was generated and bred with Apc(1638N) and Apc(Min) that allowed the comparison of the somatic mutation spectra along the Apc gene in the different allelic combinations. Mutations identified in Msh2(delta 7N/delta 7N) tumors are predominantly dinucleotide deletions at simple sequence repeats leading to truncated Apc polypeptides that partially retain the 20 a.a. beta-catenin downregulating motifs. In contrast, the somatic mutations identified in the wild type Apc allele of Msh2(delta 7N/delta 7N) /Apc(+/1638N) and Msh2(delta 7N/delta 7N) /Apc(+/Min) tumors are clustered more to the 5' end, thereby completely inactivating the beta-catenin downregulating activity of APC. These results indicate that somatic Apc mutations are selected during intestinal tumorigenesis and that inactivation of the beta-catenin downregulating function of APC is likely to represent the main selective factor.