Multi-omics analysis reveals multiple mechanisms causing Prader-Willi like syndrome in a family with a X;15 translocation.

Prader-Willi syndrome (PWS; MIM# 176270) is a neurodevelopmental disorder caused by the loss of expression of paternally imprinted genes within the PWS region located on 15q11.2. It is usually caused by either maternal uniparental disomy of chromosome 15 (UPD15) or 15q11.2 recurrent deletion(s). Here, we report a healthy carrier of a balanced X;15 translocation and her two daughters, both with the karyotype 45,X,der(X)t(X;15)(p22;q11.2),-15. Both daughters display symptoms consistent with haploinsufficiency of the SHOX gene and PWS. We explored the architecture of the derivative chromosomes and investigated effects on gene expression in patient-derived neural cells. First, a multiplex ligation-dependent probe amplification methylation assay was used to determine the methylation status of the PWS-region revealing maternal UPD15 in daughter 2, explaining her clinical symptoms. Next, short read whole genome sequencing and 10X genomics linked read sequencing was used to pinpoint the exact breakpoints of the translocation. Finally, we performed transcriptome sequencing on neuroepithelial stem cells from the mother and from daughter 1 and observed biallelic expression of genes in the PWS region (including SNRPN) in daughter 1. In summary, our multi-omics analysis highlights two different PWS mechanisms in one family and provide an example of how structural variation can affect imprinting through long-range interactions.

A Randomized Sham-Controlled Mixed Methods Pilot Study of the Feasibility of Acupuncture for Chemotherapy-Induced Neuropathy: Lessons Learned From Patient Experiences in Integrative Cancer Care.

OBJECTIVE: Since there is a lack of effective pharmacological therapies for chemotherapy-induced neuropathy and many patients ask for integrative cancer therapies such as acupuncture, the objective of this pilot study was to describe patients' experiences, and to study the feasibility and short-term effects of genuine acupuncture for chemotherapy-induced neuropathic pain and unpleasant sensations compared to sham acupuncture. METHODS: The pilot study used mixed methods, collecting quantitative and qualitative data. Patients (n = 12) with chemotherapy-induced neuropathy after colorectal cancer were blindly randomized to genuine acupuncture or telescopic sham acupuncture. Individual interviews were conducted, and were analyzed using qualitative content analysis. The patients registered pain and unpleasant sensations (100 mm Visual Analog Scales) before and after n = 120 sessions, n = 60 genuine and n = 60 sham acupuncture sessions. RESULTS: Five categories of patient experiences were described. The neuropathy negatively affected life. Physical activity was perceived to be important for health, but neuropathy was a barrier. The neuropathy required symptom-managing strategies. Acupuncture was pleasant and valuable, but some patients presented doubts regarding its effect mechanisms. After the genuine acupuncture sessions, pain (mean -2.0 steps relief during each session) and unpleasant sensations (-2.4) in the face was reduced more than after sham acupuncture (+0.1 steps worse pain, P = .018, +0.1 steps worse unpleasant sensations, P = .036). After genuine acupuncture, unpleasant sensations in the hands were reduced less (-0.23) compared to after sham acupuncture (-5.5, P = .002). Pain or unpleasant sensations in the feet did not change. CONCLUSIONS: Patients experienced that the neuropathy negatively changed their life and that acupuncture was pleasant and valuable. Patients receiving genuine acupuncture had short-term effects regarding pain and unpleasant sensations in the face compared to patients receiving sham acupuncture, while hands and feet did not improve. The patients were successfully blinded and complied with the acupuncture. We welcome future full-scaled randomized sham-controlled acupuncture studies.

COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status.

BACKGROUND: Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. METHOD: Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. RESULTS: Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1ß, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4) showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3) were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. CONCLUSIONS: Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue.

The clinical phenotype of Lynch syndrome due to germ-line PMS2 mutations.

BACKGROUND & AIMS: Although the clinical phenotype of Lynch syndrome (also known as hereditary nonpolyposis colorectal cancer) has been well described, little is known about disease in PMS2 mutation carriers. Now that mutation detection methods can discern mutations in PMS2 from mutations in its pseudogenes, more mutation carriers have been identified. Information about the clinical significance of PMS2 mutations is crucial for appropriate counseling. Here, we report the clinical characteristics of a large series of PMS2 mutation carriers. METHODS: We performed PMS2 mutation analysis using long-range polymerase chain reaction and multiplex ligation-dependent probe amplification for 99 probands diagnosed with Lynch syndrome-associated tumors showing isolated loss of PMS2 by immunohistochemistry. Penetrance was calculated using a modified segregation analysis adjusting for ascertainment. RESULTS: Germ-line PMS2 mutations were detected in 62% of probands (n = 55 monoallelic; 6 biallelic). Among families with monoallelic PMS2 mutations, 65.5% met revised Bethesda guidelines. Compared with the general population, in mutation carriers, the incidence of colorectal cancer was 5.2-fold higher, and the incidence of endometrial cancer was 7.5-fold higher. In North America, this translates to a cumulative cancer risk to age 70 years of 15%-20% for colorectal cancer, 15% for endometrial cancer, and 25%-32% for any Lynch syndrome-associated cancer. No elevated risk for non-Lynch syndrome-associated cancers was observed. CONCLUSIONS: PMS2 mutations contribute significantly to Lynch syndrome, but the penetrance for monoallelic mutation carriers appears to be lower than that for the other mismatch repair genes. Modified counseling and cancer surveillance guidelines for PMS2 mutation carriers are proposed.

A new susceptibility locus for hypospadias on chromosome 7q32.2-q36.1.

Hypospadias is a common malformation (1/300 boys) where the urethra opens on the ventral side of the penis. It is considered a complex disorder with both genetic and environmental factors involved in the pathogenesis. To identify the chromosomal loci involved in the pathogenesis of hypospadias, we performed a genome-wide linkage analysis in a three-generational family showing autosomal dominant inheritance of hypospadias. Fifteen individuals, whereof seven affected, were genotyped within a total of 426 microsatellite markers and the genotyping results were analyzed using parametric and non-parametric linkage analyses. The genome-wide linkage analysis and subsequent fine mapping gave a maximum linkage in both parametric (LOD score 2.71) and non-parametric (NPL score 5.01) single-point analyses for marker D7S640. A susceptibility haplotype shared by all affected boys was identified with the centromeric and telomeric boundaries defined by markers D7S2519 and D7S2442, respectively. This suggests a novel hypospadias locus at chromosome 7q32.2-q36.1 that encompasses 18.2 Mb (25 cM) and harbors hundreds of genes. Mutation analysis of two genes within the region, the AKR1D1 (aldo-keto reductase family 1, member D1) gene involved in the androgen pathway and the PTN gene coding for pleiotrophin, an embryonic differentiation and growth factor, was performed but without putative findings.

Preoperative treatment with a non-steroidal anti-inflammatory drug (NSAID) increases tumor tissue infiltration of seemingly activated immune cells in colorectal cancer.

This study evaluates HLA gene expression and tumor infiltration by B-cells, macrophages, dendritic cells, T-helper and cytotoxic T-lymphocytes in response to short-term preoperative treatment with cyclooxygenase inhibitors. Patients with colorectal carcinoma were randomized to receive oral NSAID (indomethacin or celebrex) for three days preoperatively; controls received esomeprazol. Peroperative tumor biopsies and normal colon tissue were analyzed by microarray, quantitative PCR and immunohistochemistry. Efficacy of short-term systemic NSAID treatment was confirmed by measurement of PGE2 production in blood monocytes from healthy volunteers. NSAID treatment upregulated genes at the MHC locus on chromosome 6p21 in tumor tissue, but not in normal colon tissue, from the same patient. 23 of the 100 most upregulated genes belonged to MHC class II. HLA-DM, -DO (peptide loading), HLA-DP, -DQ, -DR (antigen presentation), granzyme B, H, perforin and FCGR3A (CD16) (cytotoxicity) displayed increased expression, as did CD8, a marker of cytotoxic T-lymphocytes, while HLA-A and -C expression were not increased by NSAID treatment. MHC II protein (HLA-DP, -DQ, -DR) levels and infiltration by CD4+ T-helper cells of tumor stroma increased upon NSAID treatment, while CD8+ cytotoxic T-lymphocytes increased in both tumor stroma and epithelium. Molecules associated with immunosuppressive T regulatory cells (FOXP3, IL-10) were significantly decreased in indomethacin-exposed tumors. Standard oral administration of NSAID three days preoperatively was enough to increase tumor infiltration by seemingly activated immune cells. These findings agree with previous information that high prostanoid activities in colorectal cancer increase the risk for reduced disease-specific survival following tumor resection.

Understanding rationales for acupuncture treated individuals' beliefs in acupuncture effects, to be able to maximize therapeutic results: A qualitative analysis.

OBJECTIVE: To investigate how individuals expressed rationales for their beliefs regarding efficacy of acupuncture. METHODS: Qualitative data from participants of two different randomized sham-controlled trials, of relaxing (non-cancer volunteers of the general population) or antiemetic (patients with cancer undergoing radiotherapy) effects of acupuncture was analyzed. Participants (n = 441) received genuine (n = 120 and n = 100) or sham (n = 121 and n = 100) (telescopic blunt sham-needle) relaxing or antiemetic acupuncture. The participants (n = 428; 97% response rate) expressed their belief regarding the efficacy of acupuncture, and n = 264 delivered qualitative rationales for their belief, analyzed using qualitative content analysis. RESULTS: Of the 428 participants, 35 (8%) believed entirely that the acupuncture was effective, 209 (49%) believed much, 136 (32%) believed moderately, 39 (9%) believed a little, and 9 (2%) did not believe that the acupuncture was effective. Five categories and seven subcategories represented the meaning units of the central message of the rationales for the treatment belief. Participants with positive beliefs (believed entirely/much, n = 244) presented rationales related to: "Experienced positive effects", "Knowledge regarding effect-mechanisms of acupuncture", and "General trustworthiness of acupuncture". Participants with more negative beliefs (believed a little or not, n = 48) presented rationales related to: "Lack of feasibility of the acupuncture", "Varying effects", and "The effect is individual, not available for everybody". CONCLUSION: In order to strengthen acupuncture treated patients' beliefs in the efficacy of acupuncture during clinical practice or research, acupuncture therapists may consider emphasizing these aspects in the therapeutic situation.

FGFR2, FGF8, FGF10 and BMP7 as candidate genes for hypospadias.

Hypospadias is a common malformation, which results from failure of urethral tube closure, and whose molecular mechanisms are still largely unknown. The normal genital development is orchestrated by the urethral plate epithelium (UPE), at the genital tubercle (GT), which has polarizing activity, controlling a network of epithelial-mesenchymal interactions, which, when disturbed, may lead to hypospadias. Homeobox proteins (HOXs), fibroblast growth factors (FGFs) and bone morphogenic proteins (BMPs) are essential in this process. Hypospadias in the Hoxa13 -/- mice occurs as a result of the combined loss of Fgf8 and Bmp7 expression in the UPE. In both Fgf10 and Fgfr2 deficient mutant hypospadic male mice, cell proliferation is arrested prematurely and the maturation of the urethral epithelium is disrupted. Fgf8, Fgf10, and their receptor Fgfr2 are downstream targets of androgens (AR) during external genital development, an important fact given the pivotal role of AR in male sex differentiation. Therefore, we examined FGFR2, FGF10, FGF8, and BMP7 as candidate genes for hypospadias. DNA from 60 boys with familial, isolated, hypospadias was screened for mutations in FGFR2, FGF10, FGF8, and BMP7 genes, using DHPLC and DNA sequence analysis. The sequence variations c.590C>G and c.582-62G>A in FGF8, and, c.550+27C>T, c.727+180T>G, c.830T>C (p.Me186Thr), and c.2454C>T in FGFR2 were found uniquely in patients with hypospadias, as compared with 96 controls. No genetic variant in the other genes was detected. These results indicate that mutations are rare in FGF8 and FGFR2 in hypospadias, but gene variants may influence the risk.

The added value of PMS2 immunostaining in the diagnosis of hereditary nonpolyposis colorectal cancer.

Identification and characterization of the genetic background in patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is important since control programmes can in a cost-effective manner prevent cancer development in high-risk individuals. HNPCC is caused by germline mismatch repair (MMR) gene mutations and the genetic analysis of HNPCC therefore includes assessment of microsatellite instability (MSI) and immunohistochemical MMR protein expression in the tumor tissue. MSI is found in >95% of the HNPCC-associated tumors and immunostaining using antibodies against the MMR proteins MLH1, MSH2, and MSH6 has been found to correctly pinpoint the affected gene in about 90% of the cases. The PMS2 antibody was the most recently developed and we have in a clinical material assessed the added value of PMS2 immunostaining in 213 patients with suspected hereditary colorectal cancer. All 119 MSS tumors showed retained expression for all four antibodies and PMS2 did thus not identify any underlying MMR defect in these cases. However, PMS2 immunostaining contributed to the characterization of the MMR defect in a subset of the MSI tumors. Concomitant loss of MLH1 and PMS2, which functionally interact in the MutLalpha complex, was found in 98% of the tumors from patients with germline MLH1 mutations. Among the 12 MSI-high tumors with retained expression of MLH1, MSH2 and MSH6, 8 tumors showed loss of PMS2 staining, and mutations in MLH1 were identified in 2 and mutations in PMS2 in 3 of these individuals. In summary, isolated loss of PMS2 was found in 8% of the MSI-high tumors in our series, including 8/12 previously unexplained MSI-high tumors, in which mutations either in MLH1 or in PMS2 were identified in five cases.

The Expression Pattern of the Pre-B Cell Receptor Components Correlates with Cellular Stage and Clinical Outcome in Acute Lymphoblastic Leukemia.

Precursor-B cell receptor (pre-BCR) signaling represents a crucial checkpoint at the pre-B cell stage. Aberrant pre-BCR signaling is considered as a key factor for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) development. BCP-ALL are believed to be arrested at the pre-BCR checkpoint independent of pre-BCR expression. However, the cellular stage at which BCP-ALL are arrested and whether this relates to expression of the pre-BCR components (IGHM, IGLL1 and VPREB1) is still unclear. Here, we show differential protein expression and copy number variation (CNV) patterns of the pre-BCR components in pediatric BCP-ALL. Moreover, analyzing six BCP-ALL data sets (n = 733), we demonstrate that TCF3-PBX1 ALL express high levels of IGHM, IGLL1 and VPREB1, and are arrested at the pre-B stage. By contrast, ETV6-RUNX1 ALL express low levels of IGHM or VPREB1, and are arrested at the pro-B stage. Irrespective of subtype, ALL with high levels of IGHM, IGLL1 and VPREB1 are arrested at the pre-B stage and correlate with good prognosis in high-risk pediatric BCP-ALL (n = 207). Our findings suggest that BCP-ALL are arrested at different cellular stages, which relates to the expression pattern of the pre-BCR components that could serve as prognostic markers for high-risk pediatric BCP-ALL patients.

The valine allele of the V89L polymorphism in the 5-alpha-reductase gene confers a reduced risk for hypospadias.

CONTEXT: Hypospadias is one of the most common malformations in man, with an incidence of 1:300 in newborn boys. No gene has been identified that causes isolated hypospadias, but the androgenic influence is important during male genital development. OBJECTIVE: A key enzyme for the androgenic function is steroid 5-alpha-reductase (SRD5A2). The V89L polymorphism in the SRD5A2 gene has been studied and found to be of functional importance. The leucine version of the enzyme is 30% less efficient than the valine variant. DESIGN, SETTING, PATIENTS, AND RESULTS: We have genotyped 158 hypospadias cases and 96 unaffected controls for this polymorphism and found a significant negative association for the V89 allele in hypospadias (odds ratio, 0.24; 95% confidence interval, 0.14-0.41 for homozygous individuals). This indicates that a fully functional 5-alpha-reductase enzyme (homozygous for V89) protects the male urethral development. This association is shown regardless of heredity, ethnicity, and severity of phenotype. We have also sequenced a selected material of 37 sporadic cases of more severe hypospadias for mutations in the androgen receptor AR, SRD5A2, and 17beta-hydroxysteroid dehydrogenase HSD17B3 genes and found only two previously described mutations, one in the AR and one in the SRD5A2 gene. CONCLUSION: This finding is in accordance with the assumption that functional polymorphisms may play an important role in complex disorders such as hypospadias when several genes as well as environmental factors contribute to the etiology.

Microsatellite instability analysis and/or immunostaining for the diagnosis of hereditary nonpolyposis colorectal cancer?

Hereditary nonpolyposis colorectal cancer (HNPCC) represents 2-4% of colorectal cancers and is caused by a constitutional defect in a mismatch repair (MMR) gene, most commonly affecting the genes MLH1, MSH2, and MSH6. The MMR defect results in an increased cancer risk with the greatest lifetime risks for colorectal cancer and endometrial cancer. The HNPCC-associated tumor phenotype is generally characterized by microsatellite instability (MSI) and immunohistochemical loss of expression of the affected MMR protein. We have evaluated the information obtained from MSI analysis and immunostaining for MLH1, MSH2, and MSH6 in a series of 128 tumors from patients suspected of having HNPCC. A MSI-high pattern was present in 59 of 128 (46%) tumors. Loss of immunohistochemical expression for at least one of these MMR proteins was found in 54 of 59 (92%) evaluable MSI tumors. This loss affected MLH1 in 28, MSH2 in 22, and MSH6 in 21 tumors (with MSH6 as the only loss in 4 tumors). Five (8%) MSI-high tumors showed normal MMR protein expression. All 69 microsatellite stable or MSI-low tumors showed normal immunostaining for all three proteins. In 28 patients, all with MSI-H tumors, germ-line mutations of MLH1, MSH2, or MSH6 had been identified, and a corresponding immunohistochemical loss of MMR protein expression was identified in all these cases. In summary, immunostaining for the MMR proteins MLH1, MSH2, and MSH6 had a sensitivity of 92% and a specificity of 100% for detecting MMR-deficient tumors. MMR protein immunostaining facilitates mutation analysis in suspected HNPCC patients, since it pinpoints the mutated gene, but until the genetic background to the MSI tumors with retained MMR protein expression has been clarified, we suggest that MSI and MMR protein immunostaining should optimally be combined in clinical HNPCC analysis.

New ZMPSTE24 (FACE1) mutations in patients affected with restrictive dermopathy or related progeroid syndromes and mutation update.

Restrictive dermopathy (RD) is a rare and extremely severe congenital genodermatosis, characterized by a tight rigid skin with erosions at flexure sites, multiple joint contractures, low bone density and pulmonary insufficiency generally leading to death in the perinatal period. RD is caused in most patients by compound heterozygous or homozygous ZMPSTE24 null mutations. This gene encodes a metalloprotease specifically involved in lamin A post-translational processing. Here, we report a total of 16 families for whom diagnosis and molecular defects were clearly established. Among them, we report seven new ZMPSTE24 mutations, identified in classical RD or Mandibulo-acral dysplasia (MAD) affected patients. We also report nine families with one or two affected children carrying the common, homozygous thymine insertion in exon 9 and demonstrate the lack of a founder effect. In addition, we describe several new ZMPSTE24 variants identified in unaffected controls or in patients affected with non-classical progeroid syndromes. In addition, this mutation update includes a comprehensive search of the literature on previously described ZMPSTE24 mutations and associated phenotypes. Our comprehensive analysis of the molecular pathology supported the general rule: complete loss-of-function of ZMPSTE24 leads to RD, whereas other less severe phenotypes are associated with at least one haploinsufficient allele.

Uncoupling of natural IgE production and CD23 surface expression levels.

CD23, the low affinity receptor for immunoglobulin E (IgE), has been proposed to play a critical role in the regulation of IgE production, based on altered IgE levels in CD23-deficient mice and transgenic mouse models, as well as in mouse strains with mutations in the CD23 gene, e.g. 129 substrains. Here, we have investigated a mouse line termed LxT1 that expresses reduced CD23 surface levels on B cells, and its influence on natural IgE production. Extensive phenotypic analysis showed that CD23 surface expression was reduced in LxT1 compared to the control, without affecting B cell development in general. This CD23(low) surface level in LxT1 mice is not as a result of reduced CD23 mRNA expression levels or intracellular accumulation, but linked to a recessive locus, a 129-derived region spanning 28 Mb on chromosome 8, which includes the CD23 gene. Sequence analysis confirmed five mutations within the CD23 coding region in LxT1 mice, the same as those present in New Zealand Black (NZB) and 129 mice. However, this CD23(low) phenotype was not observed in all 129 substrains despite carrying these same CD23 mutations in the coding region. Moreover, serum IgE levels in LxT1 mice are as low as those in the C57BL/6 (B6) strain, and much lower than those in 129 substrains. These data indicate that the CD23 surface level and serum IgE level are uncoupled and that neither is directly regulated by the mutations within the CD23 coding region. This study suggests that caution should be taken when interpreting the immunological data derived from mice with different genetic background, especially if the gene of interest is thought to influence CD23 surface expression or serum IgE level.

DNA repair genes are selectively mutated in diffuse large B cell lymphomas.

DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis.

Receptor and enzyme expression for prostanoid metabolism in colorectal cancer related to tumor tissue PGE2.

Prostaglandins support progression of colorectal cancer by several mechanisms. This conclusion is based on epidemiological and drug intervention long-term studies or retrieved from animal and cell culture experiments. The aim of the present study was to map receptor and enzyme expression for prostanoid metabolism in the presence of high or low PGE2 content within colon cancer tissue at primary tumor operation and after short-term preoperative provision of non-steroidal anti-inflammatory drug (NSAID). Twenty-three unselected patients with colon cancer were randomly selected to receive indomethacin (NSAID) or sham treatment for 3 days before surgery. Normal colon and tumor tissue were collected at operation for RNA extraction. Tissue PGE2 levels were measured by radioimmunoassay. Gene expression was quantified by microarray and real-time PCR. COX-1 expression increased proportionally to COX-2 expression in colon cancer tissue from untreated patients. Indomethacin reduced PGE2 content in normal and tumor tissue with subsequently decreased IP, HPGD and PPARgamma receptor expression in both tumor and normal colon tissue, while subtype EP1-4 receptors were not significantly influenced by indomethacin treatment. MPGES-1 expression was not related to overall PGE2 content in tumor and colon tissue, but decreased significantly in normal tissue during indomethacin exposure. Reduction of tumor tissue PGE2 was related to significant alteration in expression of several hundred genes indicating decreased cell cycling and increased apoptosis during indomethacin treatment, probably related to upregulation of acute phase reactants in tumor tissue. Increased prostanoid activity in colon cancer tissue is related to cross-talk between tumor and stroma cells.

Genes with relevance for early to late progression of colon carcinoma based on combined genomic and transcriptomic information from the same patients.

BACKGROUND: Genetic and epigenetic alterations in colorectal cancer are numerous. However, it is difficult to judge whether such changes are primary or secondary to the appearance and progression of tumors. Therefore, the aim of the present study was to identify altered DNA regions with significant covariation to transcription alterations along colon cancer progression. METHODS: Tumor and normal colon tissue were obtained at primary operations from 24 patients selected by chance. DNA, RNA and microRNAs were extracted from the same biopsy material in all individuals and analyzed by oligo-nucleotide array-based comparative genomic hybridization (CGH), mRNA- and microRNA oligo-arrays. Statistical analyses were performed to assess statistical interactions (correlations, co-variations) between DNA copy number changes and significant alterations in gene and microRNA expression using appropriate parametric and non-parametric statistics. RESULTS: Main DNA alterations were located on chromosome 7, 8, 13 and 20. Tumor DNA copy number gain increased with tumor progression, significantly related to increased gene expression. Copy number loss was not observed in Dukes A tumors. There was no significant relationship between expressed genes and tumor progression across Dukes A-D tumors; and no relationship between tumor stage and the number of microRNAs with significantly altered expression. Interaction analyses identified overall 41 genes, which discriminated early Dukes A plus B tumors from late Dukes C plus D tumor; 28 of these genes remained with correlations between genomic and transcriptomic alterations in Dukes C plus D tumors and 17 in Dukes D. One microRNA (microR-663) showed interactions with DNA alterations in all Dukes A-D tumors. CONCLUSIONS: Our modeling confirms that colon cancer progression is related to genomic instability and altered gene expression. However, early invasive tumor growth seemed rather related to transcriptomic alterations, where changes in microRNA may be an early phenomenon, and less to DNA copy number changes.

Stickler syndrome caused by COL2A1 mutations: genotype-phenotype correlation in a series of 100 patients.

Stickler syndrome is an autosomal dominant connective tissue disorder caused by mutations in different collagen genes. The aim of our study was to define more precisely the phenotype and genotype of Stickler syndrome type 1 by investigating a large series of patients with a heterozygous mutation in COL2A1. In 188 probands with the clinical diagnosis of Stickler syndrome, the COL2A1 gene was analyzed by either a mutation scanning technique or bidirectional fluorescent DNA sequencing. The effect of splice site alterations was investigated by analyzing mRNA. Multiplex ligation-dependent amplification analysis was used for the detection of intragenic deletions. We identified 77 different COL2A1 mutations in 100 affected individuals. Analysis of the splice site mutations showed unusual RNA isoforms, most of which contained a premature stop codon. Vitreous anomalies and retinal detachments were found more frequently in patients with a COL2A1 mutation compared with the mutation-negative group (P<0.01). Overall, 20 of 23 sporadic patients with a COL2A1 mutation had either a cleft palate or retinal detachment with vitreous anomalies. The presence of vitreous anomalies, retinal tears or detachments, cleft palate and a positive family history were shown to be good indicators for a COL2A1 defect. In conclusion, we confirm that Stickler syndrome type 1 is predominantly caused by loss-of-function mutations in the COL2A1 gene as >90% of the mutations were predicted to result in nonsense-mediated decay. On the basis of binary regression analysis, we developed a scoring system that may be useful when evaluating patients with Stickler syndrome.

Dystrophia Helsinglandica: a new type of hereditary corneal recurrent erosions with late subepithelial fibrosis.

PURPOSE: To describe the phenotype of an autosomal-dominant corneal dystrophy with an early onset of recurrent corneal erosions and development of subepithelial fibrosis in the cornea, and also to exclude genetic linkage to known corneal dystrophies with autosomal-dominant inheritance and clinical resemblance. METHODS: We describe the medical history and clinical findings in individuals from a seven-generation family with recurrent corneal erosions. A total of 43 individuals were evaluated by ophthalmological examination. Genomic DNA was prepared from peripheral blood and polymorphic microsatellite markers were analysed to study haplotypes surrounding genes causing corneal dystrophies with similar phenotypes. RESULTS: Erosive symptoms usually lasted for between 1 and 10 days. By the age of 7 almost all of the affected individuals suffered from recurrent corneal erosions. The attacks generally declined in frequency and intensity from the late 20s, but all examined individuals had developed subepithelial fibrosis by the age of 37. The fibrosis generally started in the mid periphery and was followed in some family members by central fibrosis and the development of gelatinous superficial elevations. Only a marginal reduction of visual acuity was seen in a few individuals. The affected individuals did not share haplotypes for genetic microsatellite markers surrounding genes that are known to cause autosomal-dominant corneal dystrophies. CONCLUSION: We describe a new type of autosomal-dominant corneal disorder with recurrent corneal erosions and subepithelial fibrosis not significantly affecting visual acuity.