Low Gene Copy Number Research Articles

Association between C4, C4A, and C4B copy number variations and susceptibility to autoimmune diseases: a meta-analysis

Although several studies have investigated the association between C4, C4A, and C4B gene copy number variations (CNVs) and susceptibility to autoimmune diseases, the results remain inconsistency for those diseases. Thus, in this study, a comprehensive meta-analysis was conducted to assess the role of C4, C4A, and C4B CNVs in autoimmune diseases in different ethnic groups. A total of 16 case-control studies described in 12 articles (8663 cases and 11099 controls) were included in this study. The pooled analyses showed that a low C4 gene copy number (GCN) (<4) was treated as a significant risk factor (odds ratio [OR] = 1.46, 95% confidence interval [CI] = 1.19–1.78) for autoimmune diseases compared with a higher GCN (>4). The pooled statistical results revealed that low C4 (<4) and low C4A (<2) GCNs could be risk factors for systemic lupus erythematosus (SLE) in Caucasian populations. Additionally, the correlation between C4B CNVs and all type of autoimmune diseases could not be confirmed by the current meta-analysis (OR = 1.07, 95% CI = 0.93–1.24). These data suggest that deficiency or absence of C4 and C4A CNVs may cause susceptibility to SLE.

Connective Tissue Growth Factor Transgenic Mouse Develops Cardiac Hypertrophy, Lean Body Mass and Alopecia.

Connective Tissue Growth Factor (CTGF/CCN2) is one of the six members of cysteine-rich, heparin-binding proteins, secreted as modular protein and recognised to play a major function in cell processes such as adhesion, migration, proliferation and differentiation as well as chondrogenesis, skeletogenesis, angiogenesis and wound healing. The capacity of CTGF to interact with different growth factors lends an important role during early and late development, especially in the anterior region of the embryo. CTGF Knockout (KO) mice have several craniofacial defects and bone miss shaped due to an impairment of the vascular system development during chondrogenesis. The aim of the study was to establish an association between multiple modular functions of CTGF and the phenotype and cardiovascular functions in transgenic mouse. Bicistronic cassette was constructed using pIRES expressing vector (Clontech, Palo Alto, CA). The construct harbours mouse cDNA in tandem with LacZ cDNA as a reporter gene under the control of Cytomegalovirus (CMV) promoter. The plasmid was linearised with NotI restriction enzyme, and 50 ng of linearised plasmid was injected into mouse pronucleus for the chimaera production. Immunohistochemical methods were used to assess the colocalisation renin and CTGF as well as morphology and rheology of the cardiovascular system. The chimeric mice were backcrossed against the wild-type C57BL/6 to generate hemizygous (F1) mouse. Most of the offsprings died as a result of respiratory distress and those that survived have low CTGF gene copy number, approximately 40 molecules per mouse genome. The copy number assessment on the dead pups showed 5×103 molecules per mouse genome explaining the threshold of the gene in terms of toxicity. Interestingly, the result of this cross showed 85% of the progenies to be positive deviating from Mendelian first law. All F2 progenies died excluding the possibility of establishing the CTGF transgenic mouse line, situation that compelled us to work at the level of hemizygosity. The histological characterisation of left ventricle shows cardiac hypertrophy together with decrease in body mass and alopecia, this compared to the wild type. The immunohistochemical staining of aorta root showed hyperplasia with increased expression and colocalisation of renin and CTGF demonstrating that CTGF may be involved in vascular tone control. Genetic engineering is a noble avenue to investigate the function of new or existing genes. Our data have shown that CTGF transgenic mouse has cardiac and aorta root hypertrophy and abnormal renin accumulation in aorta root as compared to the wild-type animals. The transgenic animals developed alopecia and lean body mass adding two new functions on pre-existing CTGF multiple functions.

Assessment of epidermal growth factor receptor mutation/copy number and K-ras mutation in esophageal cancer.

The molecular status of epidermal growth factor receptor (EGFR) in esophageal cancer has not been well elucidated. The purpose of the study was to investigate the prevalence of EGFR and K-ras mutation, and EGFR gene copy number status as well as its association with clinicopathologic characteristics, and also to identify the prognostic value of EGFR gene copy number in esophageal cancer. EGFR mutation in exon 19/exon 21 and K-ras mutation in codon 12/codon 13 were detected by real-time PCR method, while EGFR gene copy number status was analyzed by fluorescent in situ hybridization (FISH). EGFR gene amplification and high polysomy were defined as high EGFR gene copy number status (FISH-positive), and all else were defined as low EGFR gene copy number status (FISH-negative). The relationship between EGFR gene copy number status and clinicpathologic characteristics was analyzed. Kaplan-Meier method and Cox proportional hazards regression model were employed to evaluate the effects of EGFR gene copy number status on the patients' survival. A total of 57 esophageal squamous cell carcinoma (ESCC) patients and 9 esophageal adenocarcinoma (EADC) patients were enrolled in the study. EGFR mutation was identified in one patient who was diagnosed as ESCC with stage IIIC disease. K-ras mutation was identified in one patient who was diagnosed as EADC. In all, 34 of 66 (51.5%) samples were detected as FISH-positive, which includes 30 ESCC and 4 EADC tumor samples. The correlation analysis showed that FISH-positive was significantly associated with the tumor stage (P=0.019) and lymph node metastasis (P=0.005) in esophageal cancer patients, and FISH-positive was also significantly associated with the tumor stage (P=0.007) and lymph node metastasis (P=0.008) in ESCC patients. Cox regression analysis showed that high EGFR gene copy number was not a significant predictor of a poor outcome for esophageal cancer patients (P=0.251) or for ESCC patients (P=0.092), but esophageal cancer patients or ESCC patients with low EGFR gene copy number may have longer survival than those with high EGFR gene copy number according to the survival curve trends. The results indicated that EGFR or K-ras mutation was rare in esophageal cancer, but high EGFR gene copy number is frequent, and correlated with advanced pathologic stage and more number of the metastatic regional lymph nodes, especially in ESCC. In addition, high EGFR gene copy number is likely to have a deleterious effect on prognosis of esophageal cancer patients or ESCC patients, although no statistical significance was reached in the study.

Low α-defensin gene copy number increases the risk for IgA nephropathy and renal dysfunction

Although a major source of genetic variation, copy number variations (CNVs) and their involvement in disease development have not been well studied. Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. We performed association analysis of the DEFA1A3 CNV locus in two independent IgAN cohorts of southern Chinese Han (total of 1189 cases and 1187 controls). We discovered three independent copy number associations within the locus: DEFA1A3 [P = 3.99 × 10(-9); odds ratio (OR), 0.88], DEFA3 (P = 6.55 × 10(-5); OR, 0.82), and a noncoding deletion variant (211bp) (P = 3.50 × 10(-16); OR, 0.75) (OR per copy, fixed-effects meta-analysis). While showing strong association with an increased risk for IgAN (P = 9.56 × 10(-20)), low total copy numbers of the three variants also showed significant association with renal dysfunction in patients with IgAN (P = 0.03; hazards ratio, 3.69; after controlling for the effects of known prognostic factors) and also with increased serum IgA1 (P = 0.02) and galactose-deficient IgA1 (P = 0.03). For replication, we confirmed the associations of DEFA1A3 (P = 4.42 × 10(-4); OR, 0.82) and DEFA3 copy numbers (P = 4.30 × 10(-3); OR, 0.74) with IgAN in a Caucasian cohort (531 cases and 198 controls) and found the 211bp variant to be much rarer in Caucasians. We also observed an association of the 211bp copy number with membranous nephropathy (P = 1.11 × 10(-7); OR, 0.74; in 493 Chinese cases and 500 matched controls), but not with diabetic kidney disease (in 806 Chinese cases and 786 matched controls). By explaining 4.96% of disease risk and influencing renal dysfunction in patients with IgAN, the DEFA1A3 CNV locus may be a potential therapeutic target for developing treatments for this disease.

Enhanced expression of human prostaglandin H synthase-2 in the yeast Pichia pastoris and removal of the C-terminal tag with bovine carboxypeptidase A

Vertebrate prostaglandin H synthases (PGHSs) are membrane-bound disulphide-containing hemoglycoproteins. Therefore, eukaryotic expression systems are required for the production of recombinant PGHSs. Recently we announced the expression of human PGHS-2 (hPGHS-2) in the yeast Pichia pastoris. Here we report improved production of hPGHS-2 in P. pastoris and a convenient method for the purification and de-tagging of the protein. An affinity tag comprised of a proline, a glycine and eight histidines was introduced into the C-terminal end of hPGHS-2. The tagged hPGHS-2 was expressed intracellularly in P. pastoris under the control of a constitutive or methanol-inducible promoter. Compared to constitutive expression, methanol-induced expression yielded approximately four times more protein. The analysis of high and low gene copy number recombinants revealed a positive correlation between the gene copy number and the expression level of hPGHS-2. The recombinant hPGHS-2 was purified using immobilised metal ion affinity chromatography. A novel elution method, treatment of the affinity resin with bovine carboxypeptidase A, was employed. The yield of pure de-tagged hPGHS-2 from 1l of yeast culture was approximately 3mg. The protein purification process with simultaneous removal of the C-terminal polyhistidine tag could be easily applied for the affinity purification of other proteins.

Low AMY1 Gene Copy Number Is Associated with Increased Body Mass Index in Prepubertal Boys.

BackgroundGenome-wide association studies have identified more than 60 single nucleotide polymorphisms associated with Body Mass Index (BMI). Additional genetic variants, such as copy number variations (CNV), have also been investigated in relation to BMI. Recently, the highly polymorphic CNV in the salivary amylase (AMY1) gene, encoding an enzyme implicated in the first step of starch digestion, has been associated with obesity in adults and children. We assessed the potential association between AMY1 copy number and a wide range of BMI in a population of Italian school-children.Methods744 children (354 boys, 390 girls, mean age (±SD): 8.4±1.4years) underwent anthropometric assessments (height, weight) and collection of saliva samples for DNA extraction. AMY1 copies were evaluated by quantitative PCR.ResultsA significant increase of BMI z-score by decreasing AMY1 copy number was observed in boys (β: -0.117, p = 0.033), but not in girls. Similarly, waist circumference (β: -0.155, p = 0.003, adjusted for age) was negatively influenced by AMY1 copy number in boys. Boys with 8 or more AMY1 copy numbers presented a significant lower BMI z-score (p = 0.04) and waist circumference (p = 0.01) when compared to boys with less than 8 copy numbers.ConclusionsIn this pediatric-only, population-based study, a lower AMY1 copy number emerged to be associated with increased BMI in boys. These data confirm previous findings from adult studies and support a potential role of a higher copy number of the salivary AMY1 gene in protecting from excess weight gain.

Effects of Complement C4 Gene Copy-Number Variations, Gene Size Dichotomy and C4A-Deficiency on Genetic Risk and Clinical Presentation of East-Asian and European Systemic Lupus Erythematosus (SLE)

Abstract Human complement C4 is complex with multiple layers of diversity. This study aims to elucidate the copy-number variations (CNVs) of C4A and C4B in disease risk of SLE, and compare the basis of race-specific C4A-deficiency in East-Asians (EA) and Europeans. Our study-populations included (a) 999 EA-SLE patients and 1,347 healthy subjects; and (b) 232 European SLE patients and 500 healthy subjects. Variations in gene copy-numbers (GCNs) for total C4, C4A, C4B, long and short genes were determined and validated by independent genotyping technologies. Genomic regions with C4B96 were investigated to determine the basis of the most basic C4B protein that is concurrent with C4A-deficiency. In EA, strong protective effects for high GCNs of total C4 and C4A against SLE were notable; low and medium GCNs of total C4 and C4A, and the absence of short genes were risk factors for SLE. Homozygous C4A-deficiency was infrequent but had an odds-ratio (OR) of 12.4 (p=0.0015) in SLE. Low serum complement levels were strongly associated with low GCNs of total C4 (OR=3.27, p=7.0×10−7) and C4B (OR=2.55, p=2.5×10−5). Patients with low complement had high frequencies of anti-dsDNA (OR=4.96, p=9.7×10−17), hemolytic anemia (OR=3.89, p=3.6×10−10) and renal disease (OR=2.18, p=8.5×10−6). The monomodular-short haplotype with C4A-deficiency and in linkage-disequilibrium with HLA-DRB1*0301 prevalent in Europeans was scarce in EA. Instead, most EA-subjects with C4A-deficiency shared a recombinant haplotype with bimodular-LS encoding C4B1 and C4B96, which was linked to HLA-DRB1*1501. DNA-sequencing revealed the E920K polymorphism for C4B96. In conclusion, C4 CNVs and C4A-deficiency are important in the risk and manifestations of East-Asian and European SLE.

Early Components of the Complement Classical Activation Pathway in Human Systemic Autoimmune Diseases.

The complement system consists of effector proteins, regulators, and receptors that participate in host defense against pathogens. Activation of the complement system, via the classical pathway (CP), has long been recognized in immune complex-mediated tissue injury, most notably systemic lupus erythematosus (SLE). Paradoxically, a complete deficiency of an early component of the CP, as evidenced by homozygous genetic deficiencies reported in human, are strongly associated with the risk of developing SLE or a lupus-like disease. Similarly, isotype deficiency attributable to a gene copy-number (GCN) variation and/or the presence of autoantibodies directed against a CP component or a regulatory protein that result in an acquired deficiency are relatively common in SLE patients. Applying accurate assay methodologies with rigorous data validations, low GCNs of total C4, and heterozygous and homozygous deficiencies of C4A have been shown as medium to large effect size risk factors, while high copy numbers of total C4 or C4A as prevalent protective factors, of European and East-Asian SLE. Here, we summarize the current knowledge related to genetic deficiency and insufficiency, and acquired protein deficiencies for C1q, C1r, C1s, C4A/C4B, and C2 in disease pathogenesis and prognosis of SLE, and, briefly, for other systemic autoimmune diseases. As the complement system is increasingly found to be associated with autoimmune diseases and immune-mediated diseases, it has become an attractive therapeutic target. We highlight the recent developments and offer a balanced perspective concerning future investigations and therapeutic applications with a focus on early components of the CP in human systemic autoimmune diseases.

The pig X and Y Chromosomes: structure, sequence, and evolution.

We have generated an improved assembly and gene annotation of the pig X Chromosome, and a first draft assembly of the pig Y Chromosome, by sequencing BAC and fosmid clones from Duroc animals and incorporating information from optical mapping and fiber-FISH. The X Chromosome carries 1033 annotated genes, 690 of which are protein coding. Gene order closely matches that found in primates (including humans) and carnivores (including cats and dogs), which is inferred to be ancestral. Nevertheless, several protein-coding genes present on the human X Chromosome were absent from the pig, and 38 pig-specific X-chromosomal genes were annotated, 22 of which were olfactory receptors. The pig Y-specific Chromosome sequence generated here comprises 30 megabases (Mb). A 15-Mb subset of this sequence was assembled, revealing two clusters of male-specific low copy number genes, separated by an ampliconic region including the HSFY gene family, which together make up most of the short arm. Both clusters contain palindromes with high sequence identity, presumably maintained by gene conversion. Many of the ancestral X-related genes previously reported in at least one mammalian Y Chromosome are represented either as active genes or partial sequences. This sequencing project has allowed us to identify genes—both single copy and amplified—on the pig Y Chromosome, to compare the pig X and Y Chromosomes for homologous sequences, and thereby to reveal mechanisms underlying pig X and Y Chromosome evolution.

The correlation between human chemokine type 1 chemokine ligand 3 and chemokine ligand 4 gene and the immune reconstitution of acquired immunodeficiency syndrome patients after antiretroviral therapy

Objective To investigate the correlation between the human chemokine type 1 chemokine ligand 3 (CCL3L1) and chemokine ligand 4 (CCL4L) gene expression and the immune reconstitution of acquired immunodeficiency syndrome (AIDS) patients after antiretroviral therapy. Methods The gene copy numbers of CCL3L1 and CCL4L were detected by real time polymerase chain reaction in 217 AIDS patients before antiretroviral therapy. And the correlation between CCL3L1 and CCL4L gene copy numbers and the level of CD4+ and CD8+ T lymphocytes were analyzed. The changes of CD4+ and CD8+ T lymphocytes were defined as mean change value per month after 48 months treatment. The change rates of CD4+ and CD8+ T lymphocytes were defined as the logarithm of the ratio of the value after 48 month to that at baseline. Comparison between groups was conducted using analysis of variance. Results The median of gene copy numbers of CCL3L1 and CCL4L were 2 (range: 0-8) and 3 (range: 0-7), respectively. After antiviral treatment, there were significantly different changes of CD8+ T lymphocyte level (F=3.054, P<0.05) and change rate of CD4+ /CD8+ (F=3.520, P<0.05) among groups of high (gene copy 4-8), median (gene copy 2-3) and low (gene copy 0-1) CCL3L1 gene copy numbers. The changes of CD8+ T lymphocyte levels (P=0.023) and change rates (P=0.038) in high and low CCL3L1 gene copy groups were both significantly different. There were significant differents changes rate of ratio of CD4+ /CD8+ T lymphocyte among high and median (P=0.010), high and low CCL3L1 gene copy numbers (F=4.397, P<0.05). The significant difference of the change rates of CD4+ /CD8+ were found between the gene copy 3 group and gene copy 4-7 group CCL4L (P=0.005) and between the gene copy 4-7 group and gene copy 0-2 group of CCL4L (P=0.030). The change ratio of CD4+ /CD8+ T lymphocytes increased with the increase of copy numbers of CCL4L gene. Conclusions The gene expressions of CCL3L1 and CCL4L might be associated with the ability of immune reconstitution of AIDS patients after antiretroviral therapy. Key words: Acquired immunodeficiency syndrome; Chemokine CCL3; Chemokine CCL4; Gene copy numbers; Immune reconstitution

Optimization protein productivity of human interleukin-2 through codon usage, gene copy number and intracellular tRNA concentration in CHO cells

Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host’s tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.

Diversity and distribution of &amp;lt;i&amp;gt;amoA&amp;lt;/i&amp;gt;-type nitrifying and &amp;lt;i&amp;gt;nirS&amp;lt;/i&amp;gt;-type denitrifying microbial communities in the Yangtze River estuary

Abstract. Coupled nitrification–denitrification plays a critical role in the removal of excess nitrogen, which is chiefly caused by humans, to mitigate estuary and coastal eutrophication. Despite its obvious importance, limited information about the relationships between nitrifying and denitrifying microbial communities in estuaries, and their controlling factors have been documented. We investigated the nitrifying and denitrifying microbial communities in the estuary of turbid subtropical Yangtze River (YRE), the largest river in Asia, by analyzing the ammonia monooxygenase gene amoA, including archaeal and bacterial amoA, and the dissimilatory nitrite reductase gene nirS using clone libraries and quantitative PCR (qPCR). The diversity indices and rarefaction analysis revealed a quite low diversity for both β-proteobacterial and archaeal amoA genes, but qPCR data showed significantly higher amoA gene copy numbers for archaea than β-proteobacteria. Compared with the amoA gene, a significantly higher level of diversity but lower gene copy numbers were found for the nirS gene. Nitrification and denitrification rates based on 15N incubation experiments supported gene abundance data as denitrification rates were below detection limit, suggesting lower denitrification than nitrification potential. In general, the abundances of the amoA and nirS genes were significantly higher in the bottom samples than the surface ones, and in the high-turbidity river mouth, were significantly higher in the particle-associated (&gt; 3 μm) than the free-living (0.2 ~ 3 μm) communities. Notably, positive correlations between the amoA and nirS gene abundances suggested potential gene-based coupling between nitrification and denitrification, especially for the particle-associated assemblages. Statistical analysis of correlations between the community structure, gene abundances and environmental variables further revealed that dissolved oxygen and total suspended material might be the key factors controlling community spatial structure and regulating nitrification and denitrification potentials in the YRE ecosystem.

CCL3L1 copy number, HIV load, and immune reconstitution in sub-Saharan Africans

BackgroundThe role of copy number variation of the CCL3L1 gene, encoding MIP1α, in contributing to the host variation in susceptibility and response to HIV infection is controversial. Here we analyse a sub-Saharan African cohort from Tanzania and Ethiopia, two countries with a high prevalence of HIV-1 and a high co-morbidity of HIV with tuberculosis.MethodsWe use a form of quantitative PCR called the paralogue ratio test to determine CCL3L1 gene copy number in 1134 individuals and validate our copy number typing using array comparative genomic hybridisation and fiber-FISH.ResultsWe find no significant association of CCL3L1 gene copy number with HIV load in antiretroviral-naïve patients prior to initiation of combination highly active anti-retroviral therapy. However, we find a significant association of low CCL3L1 gene copy number with improved immune reconstitution following initiation of highly active anti-retroviral therapy (p = 0.012), replicating a previous study.ConclusionsOur work supports a role for CCL3L1 copy number in immune reconstitution following antiretroviral therapy in HIV, and suggests that the MIP1α -CCR5 axis might be targeted to aid immune reconstitution.

Pediatric Immunological Disorders Biorepository (PIDBioRep) at University of São Paulo Medical School, Brazil.

Pediatric Immunological Disorders Biorepository (PIDBioRep) at University of São Paulo Medical School, Brazil.

Low C4 gene copy numbers are associated with superior graft survival in patients transplanted with a deceased donor kidney

Complement C4 is a central component of the classical and the lectin pathways of the complement system. The C4 protein exists as two isotypes C4A and C4B encoded by the C4A and C4B genes, both of which are found with varying copy numbers. Deposition of C4 has been implicated in kidney graft rejection, but a relationship between graft survival and serum C4 concentration as well as C4 genetic variation has not been established. We evaluated this using a prospective study design of 676 kidney transplant patients and 211 healthy individuals as controls. Increasing C4 gene copy numbers significantly correlated with the C4 serum concentration in both patients and controls. Patients with less than four total copies of C4 genes transplanted with a deceased donor kidney experienced a superior 5-year graft survival (hazard ratio 0.46, 95% confidence interval: 0.25-0.84). No significant association was observed in patients transplanted with a living donor. Thus, low C4 copy numbers are associated with increased kidney graft survival in patients receiving a kidney from a deceased donor. Hence, the degree of ischemia may influence the clinical impact of complement.

Low C4 gene copy numbers are associated with superior graft survival in patients transplanted with a deceased donor kidney

Complement C4 is a central component of the classical and the lectin pathways of the complement system. The C4 protein exists as two isotypes C4A and C4B encoded by the C4A and C4B genes, both of which are found with varying copy numbers. Deposition of C4 has been implicated in kidney graft rejection, but a relationship between graft survival and serum C4 concentration as well as C4 genetic variation has not been established. We evaluated this using a prospective study design of 676 kidney transplant patients and 211 healthy individuals as controls. Increasing C4 gene copy numbers significantly correlated with the C4 serum concentration in both patients and controls. Patients with less than four total copies of C4 genes transplanted with a deceased donor kidney experienced a superior 5-year graft survival (hazard ratio 0.46, 95% confidence interval: 0.25–0.84). No significant association was observed in patients transplanted with a living donor. Thus, low C4 copy numbers are associated with increased kidney graft survival in patients receiving a kidney from a deceased donor. Hence, the degree of ischemia may influence the clinical impact of complement.

THU0187 Complement C4 gene polymorphisms, serum protein levels and protein functional activity in relationship with clinical manifestations in patients with systemic lupus erythematosus

BackgroundThe complement component C4 and its associated polymorphisms have a strong association with systemic lupus erythematosus (SLE). A low gene copy number (GCN) of C4 and its two isotypes (C4A...

Abstract LB-145: Results of a phase I study of AZD4547, an inhibitor of fibroblast growth factor receptor (FGFR), in patients with advanced solid tumors .

Abstract Background: AZD4547 is an orally bioavailable, selective inhibitor of FGFR 1, 2, and 3, with activity in a wide range of cell lines and xenografts dependent upon FGFR signalling, including patient-derived explant models with FGFR gene amplification. Methods: A 3-part study of AZD4547 was initiated in patients with advanced solid tumors (NCT00979134): Part A to determine the maximum tolerated dose (MTD) and/or continuous tolerable dose (RD); Part B to characterize the pharmacokinetic and safety profile (Parts A and B unselected for FGFR amplification); Part C1 to assess safety and clinical activity of AZD4547 (80 mg bid continuous dosing) in patients with advanced solid tumors prospectively selected for amplification of FGFR 1 and 2. FGFR gene amplification status was determined using fluorescent in situ hybridization (FISH) analysis of archival or fresh tumor tissue. Pharmacodynamic biomarkers including phosphate and FGF23 were assessed in plasma samples. Results: At data cut-off (15 January 2013), 43 patients had been treated (dose range 20-200 mg bid) in the dose-escalation phase (Part A) of this study, and the RD was determined as 80 mg bid continuous dosing. Dose-limiting toxicities included increased liver enzymes, stomatitis, renal failure, hyperphosphataemia, and mucositis. In the dose-expansion phase of the study (Part B), a total of 6 patients were treated to confirm the tolerability of the RD. In Part C1, 21 patients with FGFR 1 or 2 amplified tumors received AZD4547 80 mg bid; these patients had diverse tumor types and a range of gene copy number gain (mostly low gene copy number &amp;lt;6). One squamous NSCLC patient in the C1 arm, with high copy number FGFR 1 amplification and who had received 2 previous lines of anti-cancer therapy, experienced a partial response lasting 12 weeks (patient discontinued study treatment due to RECIST disease progression at 24 weeks). An additional 4 patients in Part C1 had long periods of disease stabilization (≥24 weeks; advanced breast cancer [n=1], squamous NSCLC [n=1] and transitional cell carcinoma [n=2]). Pharmacokinetic data were generally both predictable and linear. The most common adverse events (AEs) reported were hyperphosphataemia, dry skin and mucous membranes, and retinal pigmented epithelial detachment (RPED). AEs were generally mild to moderate and reversible. Parts C2 (squamous NSCLC) and C3 (gastric cancer) are ongoing. Conclusions: AZD4547 80 mg bid continuous dosing was generally tolerated. Encouraging evidence of anti-tumor activity was seen in some patients, most notably a partial response in a patient with squamous NSCLC who had a high level FGFR gene amplification. Pharmacodynamic plasma biomarker data will be presented. Studies remain ongoing in patients with tumors selected for high levels of FGFR amplification. Citation Format: Fabrice Andre, Malcolm Ranson, Emma Dean, Andrea Varga, Ruud van der Noll, Paul K. Stockman, Dana Ghiorghiu, Elaine Kilgour, Paul D. Smith, Merran Macpherson, Peter Lawrence, Andrew Hastie, Jan HM Schellens. Results of a phase I study of AZD4547, an inhibitor of fibroblast growth factor receptor (FGFR), in patients with advanced solid tumors . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-145. doi:10.1158/1538-7445.AM2013-LB-145

Varying Tolerance to Glyphosate in a Population of Palmer Amaranth with Low EPSPS Gene Copy Number

A Palmer amaranth population (seeds collected in the year 2000; Washington Co., MS) suspected to be susceptible to glyphosate was examined as a population and as individual plants and found to exhibit varying tolerance or resistance to glyphosate. Whole plant spraying of glyphosate (0.84 kg·ha?1) to the population revealed that approximately 40% of this population were resistant to glyphosate and an LD50 of 0.75 kg·ha?1 was determined. Spray application of glyphosate indicated that some plants displayed varying degrees of resistance 14 days after treatment. Initial tests using leaf disc bioassays on 10 individual plants selected randomly from the population, allowed characterization of glyphosate resistance using both visual ratings of injury and quantitative measurement via chlorophyll content analysis. After initial bioassays and spray application, five plants with a range of tolerance to glyphosate were selected for cloning so that further studies could be accomplished on these individuals. Q-PCR analysis of these clones showed that resistance was not due to elevated EPSPS gene copy number. Shikimate levels were lower in the resistant and higher in the susceptible clones which correlated with varying degrees of resistance demonstrated in bioassays and spray application of glyphosate of these clones. Results demonstrate that individuals in a population can vary widely with respect to herbicide resistance and suggest that uptake, translocation, sequestration, metabolism or altered target site may contribute to the resistance in some individuals of this population.

Cancer cells preferentially lose small chromosomes

Genetic and genomic aberrations are the primary cause of cancer. Chromosome missegregation leads to aneuploidy and provides cancer cells with a mechanism to lose tumor suppressor loci and gain extra copies of oncogenes. Using cytogenetic and array-based comparative genomic hybridization data, we analyzed numerical chromosome aneuploidy in 43,205 human tumors and found that 68% of solid tumors are aneuploid. In solid tumors, almost all chromosomes are more frequently lost than gained with chromosomes 7, 12 and 20 being the only exceptions with more frequent gains. Strikingly, small chromosomes are lost more readily than large ones, but no such inverse size correlation is observed with chromosome gains. Because of increasing levels of proteotoxic stress, chromosome gains have been shown to slow cell proliferation in a manner proportional to the number of extra gene copies gained. However, we find that the extra chromosome in trisomic tumors does not preferentially have a low gene copy number, suggesting that a proteotoxicity-mediated proliferation barrier is not sustained during tumor progression. Paradoxically, despite a bias toward chromosome loss, gains of chromosomes are a poor prognostic marker in ovarian adenocarcinomas. In addition, we find that solid and non-solid cancers have markedly distinct whole-chromosome aneuploidy signatures, which may underlie their fundamentally different etiologies. Finally, preferential chromosome loss is observed in both early and late stages of astrocytoma. Our results open up new avenues of enquiry into the role and nature of whole-chromosome aneuploidy in human tumors and will redirect modeling and genetic targeting efforts in patients.