Genome-wide investigation to assess copy number variants in the Italian local chicken population

Background Copy number variants(CNV)hold significant functional and evolutionary importance.Numerous ongoing CNV studies aim to elucidate the etiology of human diseases and gain insights into the population structure of livestock.High-density chips have enabled the detection of CNV with increased resolution,leading to the identification of even small CNV.This study aimed to identify CNV in local Italian chicken breeds and investigate their distribution across the genome.Results Copy number variants were mainly distributed across the first six chromosomes and primarily associated with loss type CNV.The majority of CNV in the investigated breeds were of types 0 and 1,and the minimum length of CNV was significantly larger than that reported in previous studies.Interestingly,a high proportion of the length of chromosome 16 was covered by copy number variation regions(CNVR),with the major histocompatibility complex being the likely cause.Among the genes identified within CNVR,only those present in at least five animals across breeds(n=95)were discussed to reduce the focus on redundant CNV.Some of these genes have been associated to functional traits in chickens.Notably,several CNVR on different chromosomes harbor genes related to muscle development,tissue-specific biological processes,heat stress resistance,and immune response.Quantitative trait loci(QTL)were also analyzed to investigate potential overlapping with the identified CNVR:54 out of the 95 gene-containing regions overlapped with 428 QTL associated to body weight and size,carcass characteristics,egg production,egg components,fat deposition,and feed intake.Conclusions The genomic phenomena reported in this study that can cause changes in the distribution of CNV within the genome over time and the comparison of these differences in CNVR of the local chicken breeds could help in preserving these genetic resources.

Relationship Between Gene-Phenotype and Clinical Manifestations of Chromosomal Copy Number Variations Indicated by Non-Invasive Prenatal Testing

Objective:To analyze the clinical value of non-invasive prenatal testing(NIPT)in detecting chromosomal copy number variations(CNVs)and to explore the relationship between gene expression and clinical manifestations of chromosomal copy number variations.Methods:3551 naturally conceived singleton pregnant women who underwent NIPT were included in this study.The NIPT revealed abnormalities other than sex chromosome abnormalities and trisomy 13,18,and 21.Pregnant women with chromosome copy number variations underwent genetic counseling and prenatal ultrasound examination.Interventional prenatal diagnosis and chromosome microarray analysis(CMA)were performed.The clinical phenotypes and pregnancy outcomes of different prenatal diagnoses were analyzed.Additionally,a follow-up was conducted by telephone to track fetal development after birth,at six months,and one year post-birth.Results:A total of 53 cases among 3551 cases showed chromosomal copy number variation.Interventional prenatal diagnosis was performed in 36 cases:27 cases were negative and 8 were consistent with the NIPT test results.This indicates that NIPT’s positive predictive value(PPV)in CNVs is 22.22%.Conclusion:NIPT has certain clinical significance in screening chromosome copy number variations and is expected to become a routine screening for chromosomal microdeletions and microduplications.However,further interventional prenatal diagnosis is still needed to identify fetal CNVs.

Copy number variation of B1 controls awn length in wheat

Wheat awns contribute to photosynthesis and grain production.In this study,an F2population and F2:3families from a cross between the awned line 7D12 and the Chinese awnless variety Shiyou 20(SY20)were used to identify loci associated with awn length.Bulked-segregant RNA sequencing and linkage mapping identified a single dominant locus in a 0.3 cM interval on chromosome 5AL.Five genes were in the interval,including the recently cloned awn inhibitor B1.Although a single copy of the B1 gene was detected in 7D12,SY20 carried five copies of the gene.Increased copy number of B1 in SY20enhanced gene expression.Based on sequence variation among the promoter regions of five B1 gene copies in SY20,two dominant markers were developed and found to cosegregate with B1 in a population of 931 wheat accessions.All 77 awnless accessions harbored sequence variations in the B1 promoter regions similar to those of SY20 and thus carried multiple copies of the gene,whereas 15 randomly selected awned wheats carried only one copy.These results suggest that an increase in copy number of the B1 gene is associated with inhibition of awn length.

De novel heterozygous copy number deletion on 7q31.31-7q31.32 involving TSPAN12 gene with familial exudative vitreoretinopathy in a Chinese family

AIM:To investigate the genetic and clinical characteristics of patients with a large heterozygous copy number deletion on 7q31.31-7q31.32.METHODS:A family with familial exudative vitreoretinopathy(FEVR)phenotype was included in the study.Whole-exome sequencing(WES)was initially used to locate copy number variations(CNVs)on 7q31.31-31.32,but failed to detect the precise breakpoint.The long-read sequencing,Oxford Nanopore sequencing Technology(ONT)was used to get the accurate breakpoint which is verified by quantitative real-time polymerase chain reaction(QPCR)and Sanger Sequencing.RESULTS:The proband,along with her father and younger brother,were found to have a heterozygous 4.5 Mb CNV deletion located on 7q31.31-31.32,which included the FEVRrelated gene TSPAN12.The specific deletion was confirmed as del(7)(q31.31q31.32)chr7:g.119451239_123956818del.The proband exhibited a phase 2A FEVR phenotype,characterized by a falciform retinal fold,macular dragging,and peripheral neovascularization with leaking of fluorescence.These symptoms led to a significant decrease in visual acuity in both eyes.On the other hand,the affected father and younger brother showed a milder phenotype.CONCLUSION:The heterozygous CNV deletion located on 7q31.31-7q31.32 is associated with the FEVR phenotype.The use of long-read sequencing techniques is essential for accurate molecular diagnosis of genetic disorders.

Benchmark Dose Assessment for Coke Oven Emissions-Induced Mitochondrial DNA Copy Number Damage Effects

Objective The study aimed to estimate the benchmark dose(BMD)of coke oven emissions(COEs)exposure based on mitochondrial damage with the mitochondrial DNA copy number(mtDNAcn)as a biomarker.Methods A total of 782 subjects were recruited,including 238 controls and 544 exposed workers.The mtDNAcn of peripheral leukocytes was detected through the real-time fluorescence-based quantitative polymerase chain reaction.Three BMD approaches were used to calculate the BMD of COEs exposure based on the mitochondrial damage and its 95%confidence lower limit(BMDL).Results The mtDNAcn of the exposure group was lower than that of the control group(0.60±0.29 vs.1.03±0.31;P<0.001).A dose-response relationship was shown between the mtDNAcn damage and COEs.Using the Benchmark Dose Software,the occupational exposure limits(OELs)for COEs exposure in males was 0.00190 mg/m^(3).The OELs for COEs exposure using the BBMD were 0.00170 mg/m^(3)for the total population,0.00158 mg/m^(3)for males,and 0.00174 mg/m^(3)for females.In possible risk obtained from animal studies(PROAST),the OELs of the total population,males,and females were 0.00184,0.00178,and 0.00192 mg/m^(3),respectively.Conclusion Based on our conservative estimate,the BMDL of mitochondrial damage caused by COEs is0.002 mg/m^(3).This value will provide a benchmark for determining possible OELs.

Copy number variation sequencing for diagnosis of cytomegalovirus infection based low-depth whole-genome sequencing technology in fetus:Three cases and literature review

Objective:To summarize the application value of copy number variant sequencing(CNV-seq)in the detection of fetal chromosome and cytomegalovirus load.Methods:The study analyzed the clinical basic data,relevant laboratory tests,treatment process,and outcomes of three patients with positive cytomegalovirus load detected by CNV-seq for fetal chromosomes and cytomegalovirus load,and literature review was done simutaneoubly.Results:In all three cases,the amniotic fluid cytomegalovirus load was less than 105 Copies/ml,and there were no significant neurological abnormalities observed during pregnancy or postpartum follow-up.There is no literature review on the application of CNV-seq technology in the detection of cytomegalovirus infection,only literature reports on genome analysis of CMV-DNA in confirmed patients were available.Conclusion:CNV-seq can be used to detect cytomegalovirus load,which may have a certain degree of predictive value for fetal outcome.CNV-seq can simultaneously detect fetal chromosomes and pathogenic microorganisms,which is of great significance for the prevention and control of birth defects.

Isolation of Rice EPSP Synthase cDNA and Its Sequence Analysis and Copy Number Determination

In order to isolate the total cDNA of rice (Oryza sativa L.) epsps gene, RT-PCR was carried out with template of rice first-strand cDNA and primers designed according to rice EPSP synthase genomic sequence obtained in previous study. A 1 585-bp cDNA fragment was amplified and cloned. The 1 585-bp cDNA contains an open reading frame (ORF) comprising of 1 533 nucleotides (nt) which encodes a 511 residue polypepetides, including 67 amino acids chloroplast transit peptide and 444 amino acids EPSP synthase mature peptide. A comparison between the EPSP synthase of different sources indicates that the mature peptide shows more than 51% identity except for the fungi EPSP synthase and the transit peptide shows considerably less sequence conservation. The copy number of rice epsps gene is estimated to be one copy per haploid rice genome using southern blot. RT-PCR indicated that rice epsps gene is expressed in rice leaves, endosperms and roots and has the highest expression level in leaves.

Determining the Copy Number of Exogenous Gene in Transgenic Plant by SYBR Green Real-time Quantitative PCR

[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.

Target genes discovery through copy number alteration analysis in human hepatocellular carcinoma

High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma(HCC)cohorts confirmed previously identified frequently mutated somatic genes,such as TP53,CTNNB1 and AXIN1,and identified several novel genes with moderate mutation frequencies,including ARID1A,ARID2,MLL,MLL2,MLL3,MLL4,IRF2,ATM,CDKN2A,FGF19,PIK3CA,RPS6KA3,JAK1,KEAP1,NFE2L2,C16orf62,LEPR,RAC2,and IL6ST.Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling,Wnt/β-catenin signaling,JAK/STAT signaling,and oxidative stress play critical roles in HCC tumorigenesis.Nevertheless,because there are few druggable genes used in HCC therapy,the identification of new therapeutic targets through integrated genomic approaches remains an important task.Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain,copy number alteration(CNA)analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons,homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression.Moreover,integration of CNAs with other high-throughput genomic data,such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models,provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients.

Prognostic value of MET, cyclin D1 and MET gene copy number in non-small cell lung cancer

The aim of this study was to analyze the correlation of the expression of MET and cyclin D1 and MET gene copy number in non-small cell lung cancer （NSCLC） tissues and patient clinicopathologic characteristics and sur- vival. Sixty-one NSCLC tissue specimens were included in the study. The expression of MET and cyclin D1 was evaluated by immunohistochemistry and MET gene copy number was assessed by quantitative real-time polymer- ase chain reaction （Q-PCR）. Positive expression of MET and cyclin D1 protein and increased MET gene copy number occurred in 59.0%, 59.0% and 18.0% of 61 NSCLC tissues, respectively. MET-positivity correlated with poor differentiation （P = 0.009）. Increased MET gene copy number was significantly associated with lymph node metastasis （P = 0.004） and advanced tumor stage （P = 0.048）, while the expression of cyclin D1 was not associ- ated with any clinicopathologic parameters. There was a significant correlation between the expression of MET and MET gene copy number （P = 0.002）. Additionally, the expression of cyclin D1 had a significant association with the expression of MET as well as MET gene copy number （P = 0.002 and P = 0.017, respectively）. MET- positivity and increased MET gene copy number were significantly associated with poor overall survival （P = 0.003 and P 〈 0.001, respectively） in univariate analysis. Multivariate Cox proportional hazard analysis confirmed that the expression of MET and MET gene copy number were prognostic indicators of NSCLC （P = 0.003 and P = 0.001, respectively）. The overexpression of MET and the increased MET gene copy number might be adverse prognostic factors for NSCLC patients. The activation of the MET/cyclin D1 signaling pathway may contribute to carcino- genesis and the development of NSCLC, and may represent a target for therapy.

EGFR gene copy number as a predictive biomarker for resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer treatment:a meta-analysis

Objective：The epidermal growth factor receptor （EGFR） inhibitors monoclonal antibodies （MoAbs） have already shown the therapeutic effectiveness in patients with metastatic colorectal cancer （mCRC）.But many patients resist to the treatment.The aim of this meta-analysis was to assess EGFR gene copy number （GCN） as a candidate predictive biomarker for resistance to anti-EGFR MoAbs in mCRC treatment.Methods：Systematic computerized searches of the PubMed,EMBase and Cochrane Library were performed.The primary endpoint was objective response rate （ORR）.The second endpoints included progression-free survival （PFS）,and overall survival （OS）.The pooled odd ratio （OR） and pooled sensitivity,specificity,and summary receiver operator characteristic （SROC） for ORR were estimated.The pooled hazard ratios （HR） for PFS and OS were also calculated.Results：Fourteen studies with 1,021 patients were included.Increased EGFR GCN was associated with increased ORR （OR=6.905; 95％ CI：4.489-10.620）.It was also found in wild-type KRAS mCRC patients,with the pooled OR of 8.133 （95 ％ CI：4.316-15.326）.GCN has medium value for predicting ORR,with the pooled sensitivity of 0.79 （95％ CI：0.73-0.84）,the pooled specificity of 0.59 （95％ CI：0.55-0.62）.In wildtype KRAS mCRC patients,the sensitivity and the specificity were 0.80 （95％ CI：0.70-0.87） and 0.60 （95％CI：0.53-0.66）,respectively.Increased EGFR GCN was associated with increased PFS （HR=0.557; 95％ CI：0.382-0.732） and OS （HR=0.579; 95％ CI：0.422-0.737）.Conclusions：This meta-analysis suggests that EGFR GCN represents a predictive biomarker for tumor response in mCRC patients treated with MoAbs regardless of KRAS mutation.mCRC patients with increased EGFR GCN are more likely to have a better response,PFS,and OS when treated with cetuximab or panitumumab.

Scan of the endogenous retrovirus sequences across the swine genome and survey of their copy number variation and sequence diversity among various Chinese and Western pig breeds

In pig-to-human xenotransplantation,the transmission risk of porcine endogenous retroviruses(PERVs)is of great concern.However,the distribution of PERVs in pig genomes,their genetic variation among Eurasian pigs,and their evolutionary history remain unclear.We scanned PERVs in the current pig reference genome(assembly Build 11.1),and identified 36 long complete or near-complete PERVs(lc PERVs)and 23 short incomplete PERVs(si PERVs).Besides three known PERVs(PERV-A,-B,and-C),four novel types(PERV-JX1,-JX2,-JX3,and-JX4)were detected in this study.According to evolutionary analyses,the newly discovered PERVs were more ancient,and PERV-Bs probably experienced a bottleneck~0.5 million years ago(Ma).By analyzing63 high-quality porcine whole-genome resequencing data,we found that the PERV copy numbers in Chinese pigs were lower(32.0±4.0)than in Western pigs(49.1±6.5).Additionally,the PERV sequence diversity was lower in Chinese pigs than in Western pigs.Regarding the lc PERV copy numbers,PERV-A and-JX2 in Western pigs were higher than in Chinese pigs.Notably,Bama Xiang(BMX)pigs had the lowest PERV copy number(27.8±5.1),and a BMX individual had no PERV-C and the lowest PERV copy number(23),suggesting that BMX pigs were more suitable for screening and/or modification as xenograft donors.Furthermore,we identified 451 PERV transposon insertion polymorphisms(TIPs),of which 86 were shared by all 10 Chinese and Western pig breeds.Our findings provide systematic insights into the genomic distribution,variation,evolution,and possible biological function of PERVs.

Copy Number Aberrations of Multiple Genes Identified as Prognostic Markers for Extrahepatic Metastasis-free Survival of Patients with Hepatocellular Carcinoma

Extrahepatic metastasis confers unfavorable patient prognosis in patients with hepatocellular carcinoma(HCC),however,reliable markers allowing prediction of extrahepatic metastasis at the time of initial diagnosis are still lacking.This study was to identify gene-level copy number aberrations(CNAs)related to extrahepatic metastasis-free survival of HCC patients,and further examine the associations between CNAs and gene expression.Array comparative genomic hybridization(aCGH)and expression array were used to analyze gene CNAs and expression levels,respectively.The associations between CNAs of a panel of 20 genes and extrahepatic metastasis-free survival were analyzed in 66 patients with follow-up period of 1.6-90.5 months.The gene expression levels between HCCs with and without gene CNA were compared in 109 patients with HCC.We observed that gains at MDM4 and BCL2L1,and losses at APC and FBXW7 were independent prognostic markers for extrahepatic metastasis-free survival of HCC patients.Integration analysis of aCGH and expression data showed that MDM4 and BCL2L1 were significantly upregulated in HCCs with gene gain,while APC and FBXW7 were significantly downregulated in HCCs with gene loss.We concluded that gene gains at MDM4 and BCL2L1,and losses at APC and FBXW7,with concordant expression changes,were associated with extrahepatic metastasis-free survival of HCC patients and have potential to act as novel prognostic markers.

Low-depth whole genome sequencing reveals copy number variations associated with higher pathologic grading and more aggressive subtypes of lung non-mucinous adenocarcinoma

Objective:Histology grade,subtypes and TNM stage of lung adenocarcinomas are useful predictors of prognosis and survival.The aim of the study was to investigate the relationship between chromosomal instability,morphological subtypes and the grading system used in lung non-mucinous adenocarcinoma(LNMA).Methods:We developed a whole genome copy number variation(WGCNV)scoring system and applied next generation sequencing to evaluate CNVs present in 91 LNMA tumor samples.Results:Higher histological grades,aggressive subtypes and more advanced TNM staging were associated with an increased WGCNV score,particularly in CNV regions enriched for tumor suppressor genes and oncogenes.In addition,we demonstrate that 24-chromosome CNV profiling can be performed reliably from specific cell types(<100 cells)isolated by sample laser capture microdissection.Conclusions:Our findings suggest that the WGCNV scoring system we developed may have potential value as an adjunct test for predicting the prognosis of patients diagnosed with LNMA.

Prediction of early-stage hepatocellular carcinoma using Onco Scan chromosomal copy number aberration data

AIM To identify chromosomal copy number aberrations(CNAs) in early-stage hepatocellular carcinoma(HCC) and analyze whether they are correlated with patient prognosis.METHODS One hundred and twenty patients with early-stage HCC were enrolled in our study, with the collection of formalin fixed, paraffin-embedded(FFPE) specimens and clinicopathological data. Tumor areas were marked by certified pathologists on a hematoxylin and eosinstained slide, and cancer and adjacent non-cancerous tissues underwent extraction of DNA, which was analyzed with the Affymetrix Onco Scan platform to assess CNAs and loss of heterozygosity(LOH). Ten individuals with nonmalignant disease were used as the control group. Another cohort consisting of 40 patients with stage Ⅰ/Ⅱ HCC were enrolled to analyze gene expression and to correlate findings with the Onco Scan data.RESULTS Copy number amplifications occurred at chromosomes 1 q21.1-q44 and 8 q12.3-24.3 and deletions were found at 4 q13.1-q35.2, 8 p 23.2-21.1, 16 q23.3-24.3, and 17 p13.3-12, while LOH commonly occurred at 1 p32.3, 3 p21.31, 8 p23.2-21.1, 16 q22.1-24.3, and 17 p 13.3-11 in early-stage HCC. Using Cox regression analysis, we also found that a higher percentage of genome change(≥ 60%) was an independent factor for worse prognosis in early-stage HCC(P = 0.031). Among the 875 genes in the Onco Scan Gene Chip, six were independent predictors of worse disease-free survival, of which three were amplified(MYC, ELAC2, and SYK) and three were deleted(GAK, MECOM, and WRN). Further, patients with HCC who exhibited ≥ 3 CNAs involving these six genes have worse outcomes compared to those who had < 3 CNAs(P < 0.001). Similarly, Asian patients with stage I HCC from The Cancer Genome Atlas harboring CNAs with these genes were also predicted to have poorer outcomes.CONCLUSION Patients with early-stage HCC and increased genome change or CNAs involving MYC, ELAC2, SYK, GAK, MECOM, or WRN are at risk for poorer outcome after resection.

Association between TLR7 copy number variations and hepatitis B virus infection outcome in Chinese

AIM To explore whether copy number variations (CNVs) of toll-like receptor 7 (TLR7) are associated with susceptibility to chronic hepatitis B virus (HBV) infection. METHODS This study included 623 patients (495 males and 128 females) with chronic hepatitis B virus infection (CHB) and 300 patients (135 females and 165 males) with acute hepatitis B virus infection (AHB) as controls. All CHB patients were further categorized according to disease progression after HBV infection (CHB, liver cirrhosis, or hepatocellular carcinoma). Copy numbers of the TLR7 gene were measured using the AccuCopy method chi(2) tests were used to evaluate the association between TLR7 CNVs and infection type. P values, odds ratios, and 95% confidence intervals (CIs) were used to estimate the effects of risk. RESULTS Among male patients, there were significant differences between the AHB group and CHB group in the distribution of TLR7 CNVs. Low copy numberof TLR7 was significantly associated with chronic HBV infection (OR = 0.329, 95% CI: 0.229-0.473, P > 0.001). Difference in TLR7 copy number was also found between AHB and CHB female patients, with low copy number again associated with an increased risk of chronic HBV infection (OR = 0.292, 95% CI: 0.173- 0.492, P < 0.001). However, there were no significant differences in TLR7 copy number among the three types of chronic HBV infection (CHB, liver cirrhosis, or hepatocellular carcinoma). In addition, there was no association between TLR7 copy number and titer of the HBV e antigen. CONCLUSION Low TLR7 copy number is a risk factor for chronic HBV infection but is not associated with later stages of disease progression.

Relationship between epidermal growth factor receptor gene mutation and copy number in Chinese patients with non-small cell lung cancer

Epidermal growth factor receptor(EGFR) gene mutation and copy number are useful predictive markers that guide the selection of non-small cell lung cancer(NSCLC) patients for EGFR-targeting therapy.This study aimed to investigate the correlation between EGFR gene mutation and copy number and clinicopathologic characteristics of Chinese patients with NSCLC.NSCLC specimens collected from 205 patients between November 2009 and January 2011 were selected to detect EGFR gene mutations with real-time polymerase chain reaction(RT-PCR) and to detect EGFR gene copy number with fluorescence in situ hybridization(FISH).EGFR mutations primarily occurred in females,non-smokers,and patients with adenocarinomas(all P < 0.001).Tissues from 128(62%) patients were FISH-positive for EGFR,including 37(18%) with gene amplification and 91(44%) with high polysomy.EGFR gene mutation was correlated with FISH-positive status(R = 0.340,P < 0.001).Multivariate analysis showed that not smoking(OR = 5.910,95% CI = 2.363-14.779,P < 0.001) and having adenocarcinoma(OR = 0.122,95% CI = 0.026-0.581,P = 0.008) were favorable factors for EGFR gene mutation.These results show a high frequency of EGFR FISH positivity in NSCLC tissues from Chinese patients and a significant relevance between EGFR gene mutations and FISH-positive status.Among the FISH-positive samples,EGFR gene mutation occurred more frequently in samples with gene amplification compared to those with high polysomy,suggesting that EGFR mutation and gene amplification should be used as clinical decision parameters to predict response to EGFR-targeting therapy.

Copy number and zygosity determination of transgenic rapeseed by droplet digital PCR

Establishing an accurate and rapid method for copy number and zygosity determination can accelerate genetic engineering research process. In this study, droplet digital PCR (ddPCR), an emerging DNA absolute quantification technology, was used to identify single-copy homozygous transgenic lines from a batch of T0 transgenic rapeseed harboring 11 exogenous elements. Copy number of exogenous gene was evaluated in T0 generation based on calculated ratio between transgene and reference CruA gene, single-copy transformants were selected for selfing followed by subsequent zygosity analysis. Single-copy homozygous transgenic plants were successfully screened out in T1 generation by ddPCR.Segregation analysis with T2 seedlings verified that identification results of ddPCR were accurate and reliable. This study provides a novel rapid and accurate method for copy number and zygosity determination in transgenic rapeseed which overcomes disadvantages of traditional Southern analysis and recently developed real-time quantitative method.

AB015.The relationship between copy number variations and high myopia in Chinese:a case-control study

As a complex disease,myopia is the most common eye disease worldwide.Many myopia susceptibility genes or variants have been successfully identified in the past years by genome-wide genetic association studies(GWAS),which focus mainly on the single-nucleotide polymorphisms.Little attention has been paid to examine the role of copy number variations(CNVs)in refractive error and myopia.This study adopted a systematic strategy to investigate the role of CNVs in high myopia.In the discovery phase,a pilot GWAS suggests putative CNVs for follow-up.Multiplex ligation-dependent probe amplification was then used to quantify the copy number of 89 CNV segments in 737 case-control samples in the second phase and then 24 top-ranking CNVs in a second group of 1,029 case-control samples in the final validation phase.This validation phase identified 22 significant CNVs.Further work is needed to examine the role of these few CNVs in myopia development.

Identification of chloroquine resistance Pfcrt-K76T and determination of Pfmdr1-N86Y copy number by SYBR Green I qPCR

Objective:To identify prevalence of chloroquine resistance point mutation at(Pfcrt,K76T)and(Pfindr1.N86Y) copy number variation.Methods:SYBR Green I based real time PCR was used.One hundred and thirty-three samples were analyzed for(Pfcrt,K76T) and(Pfmdr1.N86Y) copy number from dried blood spot.Parasite DNA was extracted using high pure DNA preparation kit.The amplification of DNA was done by using AccuPower 2* GreenStar '' qPCR Master mix.For quantification purpose a new primer pair was designed for 178 base pair template from complete genome sequence of Plasmodium falciparum strain 3D7 at NCBI.Absolute quantification method was used to determine the Pfmdr1-N86 Y copy number variations.Standard curve was built from strain3D7 gDNA since it has single copy of Pfindr1 per haploid genome.The known positive controls with single and multi-copy number of Pfindr1 gene were included in each experiment.The copy number ratio of the samples to the standard calibrator was made to obtain the fold difference among the samples with respect to copy number variation.Results:Out of 133 samples 73(54.89%) were confirmed as mutant(Pfcrt,76T) and the remaining 60(45.11%) were genotyped as wild type(Pfcrt,K76).The(Pfindr1.N86Y) copy number variation was determined for 133 clinical samples.Out of these samples 61(45.86%)had single copy and the remaining 72(54.14%) had multi-copy numbers higher than 1.5 copies per genome.Thirty-four(25.56%) multi-copies were between 1.5 and 2.5 copies per genome while 38(28.57%) were more than 2.5 copies per genome.The minimum and maximum copies per genome were 0.474 and 4.741.respectively.Conclusions:The study showed high prevalence level and fixation of Pfcrt.76 T mutation after chloroquine withdrawal.The prevalence of Pfindr1 copy number variant suggested that the presence of modulating factor for emergence of Plasmodium falciparum strains with higher copy numbers.However,the prevalence level was not statistically significant.