Investigation on the Interface between Exons and Introns in the Genomic DNA of Arabidopsis thaliana in the Phase Space

In this study,three weight vectors L1,L2 and L3 were set.After calculating the probability of three bases in the exons or introns in the genomic DNA of Arabidopsis thaliana,64-dimensional vector P was obtained.Dot products of P vector and three weight vectors were the feature coordinates for the exons and introns in 3-dimensional phase space.The expression for the interface between the exons and the introns in the genomic DNA of Arabidopsis thaliana in 3-dimensional phase space was established,which could be used to distinguish the exons and the introns in the genomic DNA of Arabidopsis thaliana with an accuracy higher than85%in 3-dimensional phase space.

Clinical outcomes of EGFR-TKI treatment and genetic heterogeneity in lung adenocarcinoma patients with EGFR mutations on exons 19 and 21

Background:Epidermal growth factor receptor(EGFR) mutations,including a known exon 19 deletion(19 del) and exon 21 L858 R point mutation(L858R mutation),are strong predictors of the response to EGFR tyrosine kinase inhibitor(EGFR-TKI) treatment in lung adenocarcinoma.However,whether patients carrying EGFR 19 del and L858 R mutations exhibit different responsiveness to EGFR-TKls and what are the potential mechanism for this difference remain controversial.This study aimed to investigate the clinical outcomes of EGFR-TKI treatment in patients with EGFR 19 del and L858 R mutations and explore the genetic heterogeneity of tumors with the two mutation subtypes.Methods:Of 1127 patients with advanced lung adenocarcinoma harboring EGFR 19 del or L858 R mutations,532 received EGFR-TKI treatment and were included in this study.EGFR 19 del and L858 R mutations were detected by using denaturing high-performance liquid chromatography(DHPLC).T790 M mutation,which is a common resistant mutation on exon 20 of EGFR,was detected by amplification refractory mutation system(ARMS).Next-generation sequencing(NGS) was used to explore the genetic heterogeneity of tumors with EGFR 19 del and L858 R mutations.Results:Of the 532 patients,319(60.0%) had EGFR 19 del,and 213(40.0%) had L858 R mutations.The patients with EGFR 19 del presented a significantly higher overall response rate(ORR) for EGFR-TKI treatment(55.2%vs.43.7%,P = 0.017) and had a longer progression-free survival(PFS) after first-line EGFR-TKI treatment(14.4 vs.11.4 months,P = 0.034) compared with those with L858 R mutations.However,no statistically significant difference in overall survival(OS) was observed between the two groups of patients.T790 M mutation status was analyzed in 88 patients before EGFR-TKI treatment and 134 after EGFR-TKI treatment,and there was no significant difference in the co-existence of T790 M mutation with EGFR 19 del and L858 R mutations before EGFR-TKI treatment(5.6%vs.8.8%,P = 0.554)or after treatment(24.4%vs.35.4%,P = 0.176).In addition,24 patients with EGFR 19 del and 19 with L858 R mutations were analyzed by NGS,and no significant difference in the presence of multiple somatic mutations was observed between the two genotypes.Conclusions:Patients with EGFR 19 del exhibit longer PFS and higher ORR compared with those with L858 R mutations.Whether the heterogeneity of tumors with EGFR 19 del and L858 R mutations contribute to a therapeutic response difference needs further investigation.

Deletion and Mutation of WWOX Exons 6-8 in Human Non-small Cell Lung Cancer

Summary: To examine the deletion and point mutation of WWOX (WW domain containing oxidoreductase) exons 6-8 in human non-small cell lung cancer and their possible relationship with pathological stages, tumor tissues and the corresponding normal tissues were obtained from 44 Chinese patients who had undergone surgery for non-small cell lung cancer. RNA was extracted from each sample and deletion and mutation of WWOX exons 6-8 were analyzed by RT-PCR and DNA sequencing. Our results showed that 28 of 44 (63.6 %) lung cancer samples showed loss of WWOX exons 6-8 transcript and the deletion was detected in only 3 of 44 (6.8 %) corresponding adjacent normal tissues (P<0.05). The transcript sequencing analyses of the 16 lung cancer samples without transcript loss of WWOX exons 6-8 revealed no difference from the sequence of GenBank. Moreover, the deletion of WWOX exons 6-8 was significantly higher in the smokers when compared with the non-smokers. It is also higher in the men and squamous carcinomas than in women and adenocarcinomas (P<0.05). The deletion, however, was not found to be associated with pathological stages of the tumors. Our study documented a high incidence of deletion of WWOX exons 6-8 in non-small cell lung cancer in Chinese patients and suggested that the frequent loss of WWOX exons 6-8 might play an important role in the tumorigenesis of non-small cell lung cancer in Chinese. WWOX exons 6-8 may serves as a candidate molecular target of smoking carcinogenesis, and point mutation is not a predominant way of alteration of WWOX exons 6-8.

SCN1A基因Exons7-21C>T多态位点与全面性癫痫伴热性惊厥附加症相关性研究

目的研究SCN1A基因Exon7-21C>T多态位点在全面性癫痫伴热性惊厥附加症患者中的分布。方法中国汉族人中收集155例全面性癫痫伴热性惊厥附加症患者和135例正常人标本,采用聚合酶链反应-变性高效液相色谱法检测SCN1A基因的第7编码外显子及与mRNA剪接有关的内含子进行筛查,对发现异常洗脱峰者进行测序并应用SPSS11.5分析结果。结果 135例正常人中,SCN1A Exon7-21C>TCC、CT、TT基因型频率分别为:0.474、0.444、0.082,C、T等位基因频率分别为:0.696、0.304。155例全面性癫痫伴热性惊厥附加症患者中,SCN1AExon7-21C>TCC、CT、TT基因型频率分别为:0.490、0.439、0.071,C、T等位基因频率分别为:0.710、0.290。实验组的基因型频率和等位基因频率与正常对照组比较,差异无统计学意义(P>0.05)。结论 SCN1A基因单核苷酸多态位点Exon7-21C>T与全面性癫痫伴热性惊厥附加症无相关性。

CRISPR/Cas9-mediated NlInR2 mutants:Analyses of residual mRNA and truncated proteins

CRISPR/Cas9 technology is a powerful genome manipulation tool in insects.However,little is known about whether mRNA and protein of a target gene are completely cleared in homozygous mutants.This study generated homozygous mutants of the insulin receptor gene 2(NlInR2)in the brown planthopper(Nilaparvata lugens)using CRISPR/Cas9 genome editing.Both frameshift mutants,E5_D17 and E6_I7,differentiated towards long wings,but there were differences in wing morphology,with E5_D17 showing wing deformities.Subsequent investigations revealed the presence of residual expression of NlInR2 mRNA in both mutants,as well as the occurrence of spliceosomes featuring exon skipping splicing in E5_D17.Additionally,the E5_D17 exhibited the detection of N-terminally truncated NlInR2 protein.RNA interference experiments indicated that the knockdown of NlInR2 expression in the E5_D17 mutant line increased the proportion of wing deformities from 11.1 to 65.6%,suggesting that the residual NlInR2 mRNA of the E5_D17 mutant might have retained some genetic functions.Our results imply that systematic characterization of residual protein expression or function in CRISPR/Cas9-generated mutant lines is necessary for phenotypic interpretation.

Physiological and druggable skipping of immunoglobulin variable exons in plasma cells

The error-prone V(D)J recombination process generates considerable amounts of nonproductive immunoglobulin(Ig)pre-mRNAs.We recently demonstrated that aberrant Ig chains lacking variable(V)domains can be produced after nonsense-associated altered splicing(NAS)events.Remarkably,the expression of these truncated Ig polypeptides heightens endoplasmic reticulum stress and shortens plasma cell(PC)lifespan.Many questions remain regarding the molecular mechanisms underlying this new truncated Ig exclusion(TIE-)checkpoint and its restriction to the ultimate stage of B-cell differentiation.To address these issues,we evaluated the extent of NAS of Ig pre-mRNAs using an Ig heavy chain(IgH)knock-in model that allows for uncoupling of V exon skipping from TIE-induced apoptosis.We found high levels of V exon skipping in PCs compared with B cells,and this skipping was correlated with a biallelic boost in IgH transcription during PC differentiation.Chromatin analysis further revealed that the skipped V exon turned into a pseudo-intron.Finally,we showed that hypertranscription of Ig genes facilitated V exon skipping upon passive administration of splice-switching antisense oligonucleotides(ASOs).Thus,V exon skipping is coupled to transcription and increases as PC differentiation proceeds,likely explaining the late occurrence of the TIE-checkpoint and opening new avenues for ASO-mediated strategies in PC disorders.

Computational analysis and prediction for exons of PAC579 genomic sequence

To isolate the novel genes related to human hepatocellular carcinoma (HCC), we sequenced P1-derived artificial chromosome PAC579 (D17S926 locus) mapped in the minimum LOH (loss of heterozygosity) deletion region of chromosome 17p13.3 in HCC, Four novel genes mapped in this genomic sequence area were isolated and cloned by wet-lab experiments, and the exons of these genes were located. 0-60 kb of this genomic sequence including the genes of interest was scanned with five different computational exon prediction programs as well as four splice site recognition programs. After analyzing and comparing the computationally predicted results with the wet-lab experiment results, some potential exons were predicted in the genomic sequence by using these programs.

Identification of 1q21.1 microduplication in a family:A case report

BACKGROUND Copy number variation(CNV)has become widely recognized in recent years due to the extensive use of gene screening in developmental disorders and epilepsy research.1q21.1 microduplication syndrome is a rare CNV disease that can manifest as multiple congenital developmental disorders,autism spectrum disorders,congenital malformations,and congenital heart defects with genetic heterogeneity.CASE SUMMARY We reported a pediatric patient with 1q21.1 microduplication syndrome,and carried out a literature review to determine the correlation between 1q21.1microduplication and its phenotypes.We summarized the patient’s medical history and clinical symptoms,and extracted genomic DNA from the patient,her parents,elder brother,and sister.The patient was an 8-mo-old girl who was hospitalized for recurrent convulsions over a 2-mo period.Whole exon sequencing and whole genome low-depth sequencing(CNV-seq)were then performed.Whole exon sequencing detected a 1.58-Mb duplication in the CHR1:145883867-147465312 region,which was located in the 1q21.1 region.Family analysis showed that the pathogenetic duplication fragment,which was also detected in her elder brother’s DNA originated from the mother.CONCLUSION Whole exon sequencing combined with quantitative polymerase chain reaction can provide an accurate molecular diagnosis in children with 1q21.1 microduplication syndrome,which is of great significance for genetic counseling and early intervention.

Townes–Brocks syndrome with adult renal impairment in a Chinese family:A case report

BACKGROUND Townes–Brocks syndrome(TBS)is a rare autosomal dominant syndrome that is characterized by a triad of imperforate anus,dysplastic ears,and thumb malformations.Heterozygous variants of SALL1 are responsible for this syndrome.Renal structural abnormalities and functional impairments are often reported in TBS patients.CASE SUMMARY We report a case of TBS in a Chinese family.The index patients showed obvious renal atrophy and renal failure.TBS was suggested after a physical examination and pedigree analysis.Whole exome sequencing revealed a heterozygous variant of SALL1.The variant(NM_001127892 c.1289_c.1290 insC)led to a read-frame shift of the encoded protein,which was confirmed by Sanger sequencing.The variant cosegregated with the phenotype among affected members.CONCLUSION A novel variant in SALL1 gene may be the molecular pathogenic basis of this disorder.

12个水牛群体催乳素基因第4外显子遗传特征分析

催乳素对水牛乳腺发育、泌乳和乳蛋白基因的表达有明显的调控作用。该研究采用直接测序并结合PCR-SSCP技术,分析沼泽型水牛和河流型水牛12个群体385个个体的催乳素基因(PRL)第4外显子(exon4)的遗传特征。结果表明:水牛PRLexon4由180个核苷酸组成,在不同物种中具有高度保守性。序列分析发现水牛中该外显子的第109位碱基处有C>T替换,为沉默突变,与泌乳性状之间无显著相关性。在9个沼泽型水牛群体中均检测到该突变位点,其中PBA基因在7个沼泽型水牛群体中为优势基因,PBB基因在德宏和富钟群体中为优势基因,沼泽型水牛群体中PBA基因频率在0.400～0.917之间,群体平均值为0.629±0.049,具有地域分布特征。在3个河流型水牛群体中,第109位核苷酸处均为C,未检测到多态。提示河流型水牛与沼泽型水牛已有较大的遗传分化。

西农萨能羊乳腺脂肪酸合酶基因exon 9-15的克隆与序列分析

【目的】山羊FAS基因exon 9-15编码的乙酰/丙二酸单酰基转移酶(acetyl-CoA and malonyl-CoA transacylases,AT/MT)区域对山羊乳短、中链脂肪酸的合成起重要调控作用。本研究针对西农萨能羊乳腺FAS基因exon 9-15进行克隆和序列分析。【方法】以处于泌乳期28d的西农萨能羊乳腺组织mRNA反转录的cDNA为模板,通过RT-PCR方法首次扩增出西农萨能羊乳腺FAS基因exon 9-15的cDNA序列全长及exon 8的3′端和exon 16的5′端部分cDNA序列(GenBank收录号为DQ915966),并对其进行了同源性分析和功能预测。【结果】克隆片段全长1449bp,其中包括exon 9-15共1388bp、exon 8的3′端9bp和exon 16的5′端52bp,编码483个氨基酸,包含编码的AT/MT区域951bp(454～1404nt);西农萨能羊乳腺FAS基因exon 9-15与牛(NM_001012669),人(NM_004104),大鼠(NM_017332)和鸡(NM_205155)核苷酸序列相似度分别为95%、85.7%、82.7%、73.2%,氨基酸相似度分别为92.9%、77.7%、82.3%、64.7%;exon 10较其它物种缺失1个氨基酸。【结论】克隆获得西农萨能羊FAS基因片段全长1449bp,外显子9-15大小分别为463、185、190、95、135、204和116bp;核苷酸及氨基酸序列与牛相应序列的同源性均高于其它物种;AT/MT区域的活性位点在各物种间均为高度保守的丝氨酸,但西农萨能羊exon 10较其它物种缺失1个氨基酸,这可能对AT/MT区域的空间构象及其生理功能产生重要影响。本研究为西农萨能羊FAS基因cDNA全长克隆以及基因功能研究奠定了重要基础。

中国大陆野猪SLA DRB基因exon2的序列变异分析

本文基于135头野猪样本中检测到的15种SLA DRB基因exon 2序列的遗传变异分析,探讨DRB基因在不同地理区域中自然选择的变化特征。研究发现DRB基因exon 2抗原结合位点的非同义替换率为同义替换率的2.95倍,表明该位点受到平衡选择的强烈作用。在系统发生分析中,在基于核苷酸序列构建的NJ树中,野猪DRB位点的15种等位基因全部聚为一枝,没有发现跨种多态现象;而在基于氨基酸序列构建的系统发生树中,野猪SLA-DRB*nnu6和人DRB基因exon 2片段聚在一起,这种氨基酸残基的高度相似可能是由于自然选择压力下产生的趋同。野猪DRB基因exon 2的序列多态性分析表明,东北野猪基因多态性低于四川野猪和华南野猪,这可能与不同气候条件和地理环境下的寄生虫等因素对SLA等位基因变异的影响存在差异有关。

GHR基因exon 9和exon 10多态性与西农萨能奶山羊产奶性能的相关分析

针对GHR基因exon 9和exon 10设计了2对引物,采用PCR-SSCP技术检测该基因这2个位点在西农萨能羊群上的单核苷酸多态性,同时研究其对产奶性能的影响。结果表明:exon 9具有多态,exon 10不存在多态性。Exon 9位点在西农萨能山羊中检测到NN型和NT型,纯化测序发现有1个SNP位点,NN型与NT型相比在该片段第241 bp处有1个G→T的突变;NN和NT基因型之间产奶量的最小二乘均值差异显著(P<0.05):NN基因型在各胎次产奶量和平均产奶量等指标上均好于NT基因型,其中在第3胎的产奶量和第4胎产奶量2项指标上都达到显著水平(P<0.05)。研究结果提示:GHR基因exon 9对奶山羊产奶量有显著影响,因此认为GHR基因exon 9位点可作为奶山羊高产奶量的标记辅助选择的有效分子标记。

a-1,3-N-乙酰半乳糖胺转移酶基因EXON 7突变形成A311血型基因亚型

本研究对1例产生抗A抗体的血清学正反定型不符的A血型标本进行血型血清学分析及基因分析,以了解其分子基础。采用试管法进行血型血清学的检测,采用PCR-SSP对A基因进行扩增分析,采用Sanger测序法对A血型糖基转移酶基因进行测序分析。结果表明:血清学正反定型不符,初定为A亚型;PCR-SSP扩增检测结果有O、A血型基因的存在;对ABO基因第6外显子测序发现存在c.261delG,显示有O基因;对A、O基因第7外显子进行特异性扩增测序分析显示,在A单倍型基因第7外显子出现c.467 C>T、c.626 G>A的突变,在O单倍型基因第7外显子出现c.646T>A、681G>A、771C>T、829G>A等突变。结论:该献血者在A基因第7外显子的c.626G>A是新的等位基因突变,c.467 C>T是SNP;新突变的GenBank序列号是KC690281,被BGMUT命名为1个新的A等位基因亚型A311。

内蒙古地区3个马品种ELA-DRA＊exon2的PCR-SSCP分析

为了检测内蒙古地区3个马品种ELA_DRA*exon2的多态性,通过PCR_SSCP技术内蒙古地区3个马品种(三河马、锡尼河马和巴尔虎马)各50匹的ELA_DRA*exon2进行检测,用12%非变性聚丙烯酰胺凝胶电泳将已变性的PCR扩增产物分离,并用银染法显色。结果表明,150匹马中共出现6种基因型:3种纯合子分别记为AA,BB和CC型,均为3条带;3种杂合子分别记为AB,BC和AC型,均为4条带。其中A等位基因频率最高,B次之,而C只在三河马中出现。因此说三河马ELA_DRA*exon2的多态性比锡尼河马和巴尔虎马丰富。

水牛催乳素基因第3外显子群体遗传特征分析

催乳素基因(PRL)第3外显子(exon 3)对奶牛产奶量、乳脂产量和乳蛋白产量有显著影响,但在水牛中该基因的遗传特征及其与产奶性状是否有遗传关联目前还不清楚。本研究采用PCR-SSCP并结合PCR产物直接测序技术,分析了沼泽型水牛和河流型水牛共12个群体275个个体PRL exon 3的遗传变异,结果表明:水牛PRL exon 3由108个核苷酸组成,编码36个氨基酸,在水牛群体中未检测到单核苷酸多态性位点(SNPs),PRL exon 3与水牛产奶量之间没有直接相关性。通过序列比对发现在不同物种中PRL exon 3具有高度保守性。但在PRL第2内含子(intron 2)的2 265 nt和2 335 nt分别检测到G>A突变和C>T突变,其中2 335 nt位点突变仅在2头槟榔江水牛中检测到。

利用PCR-RFLP分析eNOS基因exon7BanⅡ酶切位点的遗传多态性

目的 :探讨内皮型一氧化氮合酶基因多态性与妊高征的相关性。方法 :应用PCR -RFLP技术检测了 12例正常女性的内皮型一氧化氮合酶基因第 7外显子。结果 :发现 12例正常女性中有 4例的基因型是eNOS/GG ,有 8例的基因型是eNOS/GT ,未检测到eNOS/TT型的个体。等位基因A1(eNOS/G)的频率为 66 7% ,等位基因A2 (eNOS/T)的频率为 33 3%。结论 :广东女性人群存在内皮型一氧化氮合酶第 7外显子BanⅡ酶切位点遗传多态性。

常见JAK2基因突变的筛检及在BCR-ABL阴性骨髓增殖病中的临床意义

目的建立多重等位基因特异性聚合酶链反应检测JAK2基因5种常见突变的方法,并评价在3种常见的骨髓增殖性疾病中的临床意义。方法对149例BCR-ABL融合基因为阴性的骨髓增殖性疾病(MPD)患者,包括73例真性红细胞增多症(PV)、51例原发性血小板增多症(ET)和25例原发性骨髓纤维化(IMF)患者;并结合临床及实验室其他检查资料,对其在该类疾病诊断和鉴别诊断中的价值进行评估。20名急性白血病患者和15名健康志愿者作为对照组进行研究。所有病例的骨髓或外周血标本抽提DNA后用建立的多重PCR方法进行平行检测。结果①149例MPD患者中共检出118例患者存在JAK2基因突变,总突变率为79.2%。其中PV患者阳性68例,JAK2V617F突变65例、JAK2exon12突变3例,阳性率93.2%(68/73);ET患者检测出阳性35例,其中34例为V617F突变,JAK2exon12突变1例,阳性率为68.6(35/51);IMF患者阳性15例,均为JAK2V617F突变,阳性率为60%(15/25)。②JAK2突变阳性的MPD患者和突变阴性MPD的临床资料相比较,两组在患者性别,发病年份方面的差异无统计学意义。研究发现两组PV患者的白细胞[(19.5士7.6)×109/L vs(7.1土1.1)×109/L,P=0.0005]、血小板计数[(498士172)×109/L vs(206士114)×109/L,P=0.0004]、ET患者的血红蛋白[(152士18)g/L vs(112士11)g/L,P<O.OO01]、白细胞计数[(16.2士5.2)×109/L vs(9.3士3.4)×109/L,P<0.0001]以及IMF患者的白细胞[(15.1士3.8)×109/L vs(9.3土1.6)×109/L,P=0.0001]差异均有统计学意义。结论建立的多重PCR方法可以同时检测5种JAK2基因突变,可在临床上推广使用。与JAK2突变阴性的MPD患者相比较,突变阳性的患者有更明显的骨髓增殖紊乱特征。