Relationship Between the First Base of the Donor Splice Site of Waxy Gene Intron 1 and Amylose Content in Yunnan Indigenous Rice Varieties

There exists a single nucleotide polymorphism, G or T, at the first base of the donor splice site of waxy gene intron 1 in rice. In order to study the relationship between the first base of the donor splice site of waxy gene intron 1 and amylose content in rice, the one-step PCR method was used to determine whether it is G or T in 220 Yunnan indigenous rice varieties from 14 districts, 55 towns/counties of Yunnan Province, and 101 varieties of which were validated by the PCR-Acc I method. According to the G/T polymorphism, 164 rice varieties showed GG-genotype, while the other 56 fell into TT- genotype, accounting for 74.5% and 25.5% of all the test varieties, respectively. When all the rice varieties were divided into indica and japonica subspecies, it was found that 80.5% of indica rice and 67.0% of japonica rice belonged to GG-genotype. The rice varieties with GG-genotype had significantly higher amylose content （18.95% on average） than those with TT- genotype （all below 16%）, but 33 rice varieties with GG-genotype still had low amylose content ranging from 3.91% to 15.93%, and most of them came from the Dai minority area in the Southwest of Yunnan Province. However, there was no significant difference in the mean amylose content of the same GG or TT genotypes between indica and japonica rice, suggesting that different genetic backgrounds, indica or japonica, had no effect on amylose content. The coefficient of correlation between the genotype and amylose content was 0.733 （P〈0.01）.

A novel splice site mutation of CRYBA3/A 1 gene associated with congenital cataract in a Chinese family

AIM： To identify the disease-causing mutation responsible for the presence of congenital cataract in a Chinese family. METHODS： The study recruited a four-generation Chinese pedigree affected by autosomal dominant congenital cataract （ADCC）. Family history and the history of cataract extraction were recorded. Blood samples were collected from individuals for DNA extraction. Direct sequencing of congenital cataract-associated genes was performed. Single-strand conformational polymorphism and bioinformatic analysis were conducted to further study the mutation. RESULTS： Direct sequencing revealed a novel splice site mutation of c.30-2 A〉G in the CRYBA3/A1 gene. The mutation co-segregated within all affected individuals in the family and was not found in unaffected members or 100 unrelated normal controls. These results were further confirmed by single-strand conformational polymorphism and bioinformatic analysis using the Human Splicing Finder and MaxEnt online software and Annovar computer software. CONCLUSION： c,30-2 A〉G mutation of CRYBA3/A1 gene is a novel mutation and broadens the genetic spectrum of ADCC, KEYWORDS： splice site mutation; congenital cataract; CRYBA3/A1 gene

A BRCA1 Splice Site Variant Responsible for Familial Ovarian Cancer in a Han-Chinese Family

Objective Ovarian cancer(OC)is one of the most common and most lethal gynecological malignancies.OC has an age-dependent incidence and occurs more commonly in females older than 50 years old.Most OC patients are diagnosed at an advanced stage and have a poor prognosis.Germline mutations in the BRCA1 DNA repair associated gene(BRCA1)and the BRCA2 DNA repair associated gene(BRCA2)account for 20%–25%of epithelial ovarian cancer(EOC).BRCA1 germline mutations are more common in Chinese EOC patients.Methods This study reported a three-generation Han-Chinese family containing four EOC patients and a rectal adenocarcinoma patient.Whole-exome sequencing was performed on two EOC patients and an unaffected individual.Variant validation was also performed in all available members by Sanger sequencing.Results A heterozygous splice site variant,c.4358-2A>G in the BRCA1 gene,was identified.Bioinformatic analysis showed that the variant may change the splicing machinery.Conclusion The BRCA1 splice site variant,c.4358-2A>G was identified as the likely genetic cause for EOC,and may also be associated with the increased risk of rectal adenocarcinoma in the family.The findings were beneficial for genetic counseling,helpful for cancer prevention in other family members,and may facilitate therapy decision-making in the future to reduce cancer lethality.

A nucleotide substitution at the 5′ splice site of intron 1 of rice HEADING DATE 1(HD1) gene homolog in foxtail millet, broadly found in landraces from Europe and Asia

We investigated genetic variation of a rice HEADING DATE 1(HD1) homolog in foxtail millet.First, we searched for a rice HD1 homolog in a foxtail millet genome sequence and designed primers to amplify the entire coding sequence of the gene. We compared full HD1 gene sequences of 11 accessions(including Yugu 1, a Chinese cultivar used for genome sequencing) from various regions in Europe and Asia, found a nucleotide substitution at a putative splice site of intron 1, and designated the accessions with the nucleotide substitution as carrying a splicing variant. We verified by RT-PCR that this single nucleotide substitution causes aberrant splicing of intron 1. We investigated the geographical distribution of the splicing variant in 480 accessions of foxtail millet from various regions of Europe and Asia and part of Africa by d CAPS and found that the splicing variant is broadly distributed in Europe and Asia. Differences of heading times between accessions with wild type allele of the HD1 gene and those with the splicing variant allele were unclear. We also investigated variation in 13 accessions of ssp. viridis, the wild ancestor, and the results suggested that the wild type is predominant in the wild ancestor.

Analysis and prediction of exon, intron, intergenic region and splice sites for A. thaliana and C. elegans genomes

Although a great deal of research has been undertaken in the area of the annotation of gene structure, predictive techniques are still not fully developed. In this paper, based on the characteristics of base composition of sequences and conservative of nucleotides at exon/intron splicing site, a least increment of diversity al-gorithm (LIDA) is developed for studying and predicting three kinds of coding exons, introns and intergenic regions. At first, by selecting the 64 trinucleotides composition and 120 position parameters of the four bases as informational parameters, coding exon, intron and intergenic sequence are predicted. The results show that overall predicted accuracies are 91.1% and 88.4%, respectively for A. thaliana and C. ele-gans genome. Subsequently, based on the po-sition frequencies of four kinds of bases in regions near intron/coding exon boundary, initia-tion and termination site of translation, 12 position parameters are selected as diversity source. And three kinds of the coding exons are predicted by use of the LIDA. The predicted successful rates are higher than 80%. These results can be used in sequence annotation.

The Research on Identification of Gene Splice Sites by Support Vector Machine

The recognition of splicing sites is a very important step in the eukaryotic DNA se-quence analysis. Many scholars are working hard to improve the accuracy of identifi-cation. Our team carried out research on this issue based on support vector machine, which is one famous algorithm in data mining. The training and testing data is from the HS3D dataset, and excellent accuracy rate is achieved by nucleic acid sequence orthogonal coding and RBF core function, and the cross validation experiment hints that base pattern information is mainly located within 20 nucleotides upstream and downstream splice sites.

Comprehensive genetic diagnosis of patients with Duchenne/Becker muscular dystrophy(DMD/BMD) and pathogenicity analysis of splice site variants in the DMD gene

Duchenne muscular dystrophy(DMD)and Becker muscular dystrophy(BMD)are caused by mutations in the DMD gene.The aim of this study is to identify pathogenic DMD variants in probands and reduce the risk of recurrence of the disease in affected families.Variations in 100 unrelated DMD/BMD patients were detected by multiplex ligation-dependent probe amplification(MLPA)and next-generation sequencing(NGS).Pathogenic variants in DMD were successfully identified in all cases,and 11 of them were novel.The most common mutations were intragenic deletions(69%),with two hotspots located in the 5'end(exons 2–19)and the central of the DMD gene(exons 45–55),while point mutations were observed in 22%patients.Further,c.1149+1G>A and c.1150?2A>G were confirmed by hybrid minigene splicing assay(HMSA).This two splice site mutations would lead to two aberrant DMD isoforms which give rise to severely truncated protein.Therefore,the clinical use of MLPA,NGS,and HMSA is an effective strategy to identify variants.Importantly,eight embryos were terminated pregnancies according to prenatal diagnosis and a healthy boy was successfully delivered by preimplantation genetic diagnosis(PGD).Early and accurate genetic diagnosis is essential for prenatal diagnosis/PGD to reduce the risk of recurrence of DMD in affected families.

Effects of splice sites on the intron retention in histamine H_3 receptors from rats and mice

In the alternative splicing, intron retention, of histamine H3 receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats and are undetectable in mice using the regular PCR protocol. The retained introns have common 5＇ splice site and different 3＇ splice sites. The detailed mechanism for the special alternative splicing remains largely unclear. In this study, we developed a minigene splicing system to recapitulate natural alternative splicing of the receptors and investigated the effects of 5＇ and 3＇ splice sites on intron retention in HeLa cells. Mutating weak 5＇ and 3＇ splice sites of the alternatively spliced introns toward the canonical consensus sequences promoted the splicing of the corresponding introns in rat and mouse minigenes. The effect of splice site strength was context-dependent and much more sigiaificant for the 3＇ splice site of the longer alternative intron than for the 3＇ splice site of the shorter alternative intron and the common 5＇ splice sites; it was also more significant in the rat minigene than in the mouse minigene. Mutating the 3＇ splice site of the longer alternative intron resulted in almost complete splicing of the intron and made the corresponding isoform to become the nearly exclusive transcript in the rat minigene.

Comparative Analysis of Splice Site Regions by Information Content

We have applied concepts from information theory for a comparative analysis of donor （gt） and acceptor （ag） splice site regions in the genes of five different organisms by calculating their mutual information content （relative entropy） over a selected block of nucleotides. A similar pattern that the information content decreases as the block size increases was observed for both regions in all the organisms studied. This result suggests that the information required for splicing might be contained in the consensus of -6-8 nt at both regions. We assume from our study that even though the nucleotides are showing some degrees of conservation in the flanking regions of the splice sites, certain level of variability is still tolerated, which leads the splicing process to occur normally even if the extent of base pairing is not fully satisfied. We also suggest that this variability can be compensated by recognizing different splice sites with different spliceosomal factors.

Identification of a novel splice site mutation in the DNAAF4 gene of a Chinese patient with primary ciliary dyskinesia

Primary ciliary dyskinesia(PCD)is a rare hereditary orphan condition that results in variable phenotypes,including infertility.About 50 gene variants are reported in the scientific literature to cause PCD,and among them,dynein axonemal assembly factor 4(DNAAF4)has been recently reported.DNAAF4 has been implicated in the preassembly of a multiunit dynein protein essential for the normal function of locomotory cilia as well as flagella.In the current study,a single patient belonging to a Chinese family was recruited,having been diagnosed with PCD and asthenoteratozoospermia.The affected individual was a 32-year-old male from a nonconsanguineous family.He also had abnormal spine structure and spinal cord bends at angles diagnosed with scoliosis.Medical reports,laboratory results,and imaging data were investigated.Whole-exome sequencing,Sanger sequencing,immunofluorescence analysis,hematoxylin-eosin staining,and in silico functional analysis,including protein modeling and docking studies,were used.The results identified DNAAF4 disease-related variants and confirmed their pathogenicity.Genetic analysis through whole-exome sequencing identified two pathogenic biallelic variants in the affected individual.The identified variants were a hemizygous splice site c.784-1G>A and heterozygous 20.1 Kb deletion at the DNAAF4 locus,resulting in a truncated and functionless DNAAF4 protein.Immunofluorescence analysis indicated that the inner dynein arm was not present in the sperm flagellum,and sperm morphological analysis revealed small sperm with twisted and curved flagella or lacking flagella.The current study found novel biallelic variants causing PCD and asthenoteratozoospermia,extending the range of DNAAF4 pathogenic variants in PCD and associated with the etiology of asthenoteratozoospermia.These findings will improve our understanding of the etiology of PCD.

A new method for splice site prediction based on the sequence patterns of splicing signals and regulatory elements

It is of significance for splice site prediction to develop novel algorithms that combine the sequence patterns of regulatory elements such as enhancers and silencers with the patterns of splicing signals.In this paper,a statistical model of splicing signals was built based on the entropy density profile(EDP) method,weight array method(WAM) and κ test;moreover,the model of splicing regulatory elements was developed by an unsupervised self-learning method to detect motifs associated with regulatory elements.With two models incorporated,a multi-level support vector machine(SVM) system was de-vised to perform ab initio prediction for splice sites originating from DNA sequence in eukaryotic ge-nome.Results of large scale tests on human genomic splice sites show that the new method achieves a comparative high performance in splice site prediction.The method is demonstrated to be with at least the same level of performance and usually better performance than the existing SpliceScan method based on modeling regulatory elements,and shown to have higher accuracies than the traditional methods with modeling splicing signals such as the GeneSplicer.In particular,the method has evident advantage over splice site prediction for the genes with lower GC content.

A novel splice mutation of HERG in a Chinese family with long QT syndrome

Congenital long QT syndrome (LQTS) is a genetically heterogeneous disease in which six ion-channel genes have been identified. The phenotype-genotype relationships of the HERG (human ether-a-go-go-related gene) mutations are not fully understood. The objective of this study is to identify the underlying genetic basis of a Chinese family with LQTS and to characterize the clinical manifestations properties of the mutation. Single strand conformation polymorphism (SSCP) analyses were conducted on DNA fragments amplified by polymerase chain reaction from five LQT-related genes. Aberrant conformers were analyzed by DNA sequencing. A novel splice mutation in C-terminus of HERG was identified in this Chinese LQTS family,leading to the deletion of 11-bp at the acceptor splice site of Exon9 [Exon9 IVS del (-12→-2)]. The mutation might affect,through deficient splicing, the putative cyclic nucleotide binding domain (CNBD) of the HERG K+ channel. This mutation resulted in a mildly affected phenotype. Only the proband had a history of syncopes, while the other three individuals with long QT interval had no symptoms. Two other mutation carriers displayed normal phenotype. No sudden death occurred in the family. The 4 affected individuals and the two silent mutation carriers were all heterozygous for the mutation. It is the first splice mutation of HERG reported in Chinese LQTS families. Clinical data suggest that the CNBD mutation may be less malignant than mutations occurring in the pore region and be partially dominant over wild-type function.

三维多站点云拼接新技术在文物保护中的应用

三维激光扫描技术是一项新型的立体测绘技术,具有高效率、高分辨率、高精度、高穿透性等优点,在变形监测、灾害监测、三维重建、文物保护、数字城市等方面应用前景广泛。本文对三维激光扫描中的三维点云拼接技术的难点进行了方法性的研究,提出了新的点云拼接方法,通过实例应用天宝Surphaser对扫描的数据进行拼接处理,比较各种拼接技术精度的高低,得出相应结论,该方法可以很好地应用于文物保护等领域,具有十分重要的参考意义。

一成骨不全家系的COL1A1基因突变检测

成骨不全(Osteogenesisimperfecta,OI)是一种由于Ⅰ型胶原形成障碍,导致骨脆性增强为主要症状的 常染色体显性遗传性疾病。临床上主要表现为骨质脆弱、蓝巩膜、耳聋和中等程度的关节畸形等症状。成骨不全 基因分别定位于17q21.31 q22和7q22.1,其致病基因分别为COL1A1和COL1A2。对一常染色体显性遗传的 成骨不全家系进行连锁分析,在COL1A1遗传位点发现紧密连锁(LOD=9.31;θ=.00)。突变检测发现在 COL1A1基因第26内含子5′端剪接位点处存在一由GT转换为AT的致病突变,该突变引起的异常剪接是导致成 骨不全的致病原因之一。

基于支持向量机(SVM)的剪接位点识别

剪接位点的识别作为基因识别中的一个重要环节， 一直受到研究人员的关注。考虑到剪接位点附近存在的序列保守性，已有一些基于统计特性的方法被用于剪接位点的识别中，但效果仍有待进一步改进。支持向量机（Ｓｕｐｐｏｒｔ Ｖｅｃｔｏｒ Ｍａｃｈｉｎｅｓ） 作为一种新的基于统计学习理论的学习机，近几年有了很大的发展，已被应用在模式识别的许多问题中。文中将其用于剪接位点的识别中，并针对满足ＧＴ－ ＡＧ 规则的序列样本中虚假剪接位点的样本数远大于真实位点这一特性， 提出了一种基于ＳＶＭ 的平衡取小法以获得更好的识别效果。实验结果表明，应用支持向量机进行剪接位点的识别能更好地提取位点附近保守序列的统计特征，对测试集具有更好的推广能力，并且使用上更加简单。这一结果为剪接位点的识别提供了一种新的方法，同时也为生物大分子研究中结构和位点的识别问题的解决提供了新的线索。

用神经网络法预测mRNA的剪接位点

用神经网络预测了mRNA的剪接位点,比较了在各种不同的情况下,神经网络的学习与预测的情况,讨论了能反映真实剪接位点预测情况的有效预测成功率,指出它可达64%,而且总的预测成功率可达98%.预测的相关系数为0.66.

核小体定位与RNA剪接

根据核小体定位序列和缺失序列的碱基分布特征,应用多样性增量二次判别方法(IDQD)构建模型对这两类序列进行了区分,受试者操作特性曲线下的面积达到了0.958.应用这一模型研究了核小体在人类基因组剪接位点(GT/AG)邻近序列中的分布方式,发现外显子所对应的DNA序列通常倾向参与核小体的形成,并且由它所转录的RNA统计上具有较强的刚性,而剪接位点及其邻近的内含子对应的DNA序列则避免参与核小体的形成,所转录的RNA统计上具有较强的柔性.进一步还发现,DNA序列的核小体定位/缺失和RNA的刚性/柔性具有统计相关性,为从机制上解释为何前体RNA剪接事件与DNA序列中的核小体定位信息有关提供了依据.

基于支持向量机的人类5'非翻译区剪接位点识别

基因非编码区域剪接位点的识别是基因识别中一个非常具有挑战性的问题,尤其是5'非翻译区中剪接位点的识别。与一般剪接位点不同,5'非翻译区剪接位点的两侧不存在由编码到非编码的状态转移,所以通常的剪接位点识别算法在非翻译区的性能不太理想。文章采用了基于支持向量机的方法对5'非翻译区中的剪接位点进行识别。为了提高识别精度,采用了基于矩阵相似性度量的核函数参数选取方法,它能够简单快速地确定合适的核函数参数,进而提高核函数的识别性能。通过实验验证,经过参数选择后的支持向量机能够较好地识别5'非翻译区剪接位点。

拟南芥和线虫基因序列及剪切位点的理论预测

将拟南芥(A.thaliana)和线虫(C.elegans)基因组按外显子、内含子及基因间序列区分为3类。分别选取64、40、20 种三联体的概率作为信号参数构建离散源,根据离散增量预测序列所属类型。结果表明:拟南芥各条染色体标准集总预测成功率达到82.19%,检验集为87.95%;线虫各条染色体标准集总预测成功率达到79.67%,检验集达到81.93%。另外,将两种基因序列中的外显子分别划分成3类, 用外显子剪切位点、翻译起始和结束位点附近的三联体的3个位点作为3条子链,以各条子链的12个参数构建离散源,用离散增量对3种序列类型进行预测,预测成功率都达80%以上。

两个遗传性对称性色素异常症家系的DSRAD基因剪切突变

目的研究两个遗传性对称性色素异常症(DSH)家系中的DSRAD基因突变情况。方法收集了2个遗传性对称性色素异常症家系的外周血标本,用聚合酶链反应(PCR)扩增DSRAD基因的全部外显子并测序,检测2个家系中的患者及正常人和100例无关正常人的DSRAD基因。结果家系1中所有患者的DSRAD基因第6号内含子与第7号外显子交界处检测到一新的c.2271-3A>G剪切突变。家系2中所有患者的DSRAD基因第12号外显子与第12号内含子交界处检测到一新的c.3202+5G>A剪切突变。2家系中的正常人及100例无关正常人未发现突变。结论 2个DSH家系患者均有DSRAD基因剪切部位突变,可能由此引起非正常的基因剪切,导致编码蛋白的结构和功能改变,致皮肤色素异常。