Analysis of the mechanism of high incidence of thanatophoric dysplasia type I

致死性侏儒症（thanatophoric dysplasia,TD）是重型短肢畸形病中相对常见的致死性遗传性骨病,分为TD-I和TD-II 2型,它们都是由于FGFR3基因发生致死突变所致。TD-I型存在多种致病性突变,其中以c.742C〉T/p.R248C突变最为常见。本病为常染色体显性遗传（autosomal dominant,AD）,杂合突变即可致死,但为何表型、基因型正常的父母会生出带有p.R248C突变的患胎并且是纯合突变的死胎？如果是新生突变,又为何会接连数胎生出同样的患胎？p.R248C高发突变的机制是什么？本文重点围绕这些问题,从＂FGFR3（Fibroblast growth factor receptor 3）基因和FGFR3受体（Fibroblast growth factor receptor 3）的结构和功能,TD-I型p.R248C高发突变的发生机制,正常父母生出纯合致死突变的可能机制＂几方面进行剖析,指出：（1）FGFR3基因及其受体蛋白结构和功能的特殊性是高发突变发生的物质基础;（2）位于IgⅡ和IgⅢ结构域连接区的氨基酸是极性很强的亲水氨基酸,很容易与带电离子结合而影响α螺旋结构,故易受外来理化因素的攻击、诱变而发生改变;（3）正常父母生出纯合致死突变,推测有两种可能：一种是夫妇一方的生殖腺已带有该高发突变,当胎儿从亲代遗传了一次突变后,只需在另一位点上再发生一次新生突变即可产生;另一种是夫妇双方的生殖腺都是该突变的嵌合体,当两者结合在一起,就有可能产生纯合突变。此外,还对未来发展趋势进行了展望。本文旨在探讨c.742C〉T/p.R248C高发突变和纯合突变发生的机制,进而为其今后的诊断和防治工作提供理论依据。Thanatophoric dysplasia（TD）,divided into TD type I and TD type II,is a genetic bone disease caused by the fatal mutation of FGFR3 gene.It is relatively common lethal hereditary osteopathy among severe short limb malformations.TD-I type has many kinds of pathogenic mutations,of which c.742CT/p.R248 C mutation is the most common.This disease is an autosomal dominant（AD） genetic disease which means heterozygous mutations can be lethal.However,why some parents with normal genotypes and phenotypes can give birth to a stillbirth who carried the homozygous mutation p.R248C？ If it is a novel mutation,why the successive fetuses with the same mutation were born？ What is the high incidence mechanism of p.R248 C mutation？ This paper focus mainly around these problems,from＂The structure and function of FGFR3 gene and FGFR3 receptor;The pathogenetic mechanism of high incidence of p.R248 C in TD type I;The possible mechanisms of normal parents producing homozygous lethal mutation＂,several aspects are analyzed and pointed out：（1）FGFR3 gene and the structure and function of its receptor protein is the material base of high frequent mutation;（2） Some amino acids,located in the connected region of Ig Ⅱ and Ig Ⅲ domain,are strong polar hydrophilic amino acids,which tend to bind charged ions to influence the alpha helical structure,therefore,it is easy to be affected by external physical and chemical factors;（3） Normal parents give birth to fetus with homozygous lethal mutation,there are 2 possibilities：one is the reproductive gonad of one of the parents has carried the hot mutation,and the fetus inherited the hot mutation,under these circumstances,as long as a same mutation happen in its allele,which will lead to the generation of homozygous mutation;another is the reproductive glands of couples are the mutant chimeras,when the two are combined together,it is possible to generate homozygous mutation.In addition,the future development trend is prospected.The purpose of this paper is to explore th闽粤合作科研基金（71010025

Research advance in genetic diagnosis of monogenic inherited diseases

单基因遗传病(简称单基因病)种类、分型繁多,常规诊断难以确诊,而基因诊断技术在遗传病特别是单基因病的确诊、分型等方面都发挥着不可或缺的作用。近一、二十年来,基因诊断技术进展迅猛,各种检测方法层出不穷。本文重点围绕基因诊断技术的最新进展进行综述,以期对临床诊断和预防工作提供一些有益的启示。Monogenic diseases are caused by inheritance of single mutated gene, but the type of the diseases are diversified depending on the type and locus of genetic mutation inherited. While conventional laboratory methods may contribute to an initial identification, a definitive diagnosis and classification of these inherited disorders often need molecular/genetic testing. In the past twenty years, diagnostic techniques for the genetic disorders have rapidly advanced, leading to the invention of many laboratory tests for the detection of genetic disorders. In this article, we review the recent advance in the diagnostic modalities for the genetic disorders, in hope of setting light on improvement of the clinical diagnosis and prevention.国家自然科学基金(30772069);; 闽粤横向科研基金(71010025

Maroteaux-Lamy综合征的ARSB基因分析及新突变的致病性鉴定

【目的】对7家拟诊为Maroteaux_Lamy综合征(MPSⅥ)的患儿及其父母进行ARSB基因的突变检测和新突变的致病性鉴定,以揭示其分子发病机制,为将来的产前/植入前基因诊断等创造前提条件。【方法】在临床初诊及GAG尿检和MPS酶检的基础上,抽取患儿及其父母EDTA抗凝血,进行ARSB基因的PCR扩增和Sanger测序。对所发现的新突变,在经HGMD、1000G和ExAC等数据库核实排查后,首先用SWISS-MODEL软件分析、比对突变蛋白和正常蛋白的空间构象,然后用Clustal X软件分析跨物种氨基酸的保守性,用PROVEAN、SIFT、PolyPhen-2软件预测新突变的致病性,最后用ACMG标准对新突变的致病性进行综合分析鉴定。【结果】1)7个家系先证者的基因检测结果分别为:No1:c.574T> C,p.C192R纯合错义突变;No2:c.160G> A/p.D54N(来自其母)和c.1197C> G/p.F399L(来自其父)的复合杂合子;No3:仅检出c.1072G> A/p.V358M和IVS5 as(-27)A>C变异,但酶检和临床表型符合Ⅵ型;No4:为c.281C> T,p.S94L(新突变,来自其母)和IVS5 as(-27)A> C(来自其父)的复合杂合子;No5:为c.1197 C> G,p.F399L纯合错义突变;No6:为c.1197 C> G,p.F399L(来自其母)和c.1379 C> T,p.S460F(新突变,来自其父)的复合杂合子;No7:为c.499 G> A,p.G167R(来自其父)和c.1325C>T,p.T442M(来自其母)的复合杂合子。2)新突变鉴定结果:对正常ARSB酶蛋白和p.S94L突变酶蛋白的空间构象的预测比对结果显示,两者有明显区别;跨物种保守性分析结果显示,p.94突变点所在氨基酸(S)在物种进化过程中具有高度保守性;PROVEAN、SIFT和PolyPhen-2软件对p.S94L预测结果分别为:Deleterious、Damaging和Probably damaging。用上述方法对p.S460F的预测结果及ACMG的综合分析结果也显示该突变可能是致病性的。【结论】1)家系4的p.S94L和家系6的p.S460F新突变可能都是新的致病性突变,有可能都是引起患儿发病的内在原因之一。2)家系1,2,5,6,7可以确诊为MPS Ⅵ型,其基因型和表现型具有显著的相关性,家系3虽经酶检确诊,临床症状和尿检结果也都与MPS Ⅵ型符合,但却未能在DNA水平查到明确的突变类型,其表现型与基因型的相关性还有待进一步证实。国家自然科学基金(30772069);;闽粤合作科研基金(71010025和71020010

Fast detection of thanatophoric dysplasia type I p.R248C mutation hot spots and rapid prenatal diagnosis of three TD type I high-risk fetuses

目的针对致死性侏儒症I型（thanatophoric dysplasia typeⅠ,TD-Ⅰ）FGFR3基因的突变热点＂p.R248C＂,建立快速特异的酶切鉴定法（restriction endonuelease testing,RE）和扩增受阻突变系统（amplification refractory mutation system,ARMS）/RE法,并应用于后续3例疑似TD-I高危胎儿的快速产前诊断,以及时防止患胎出生,同时为今后的胚胎植入前遗传学诊断（preimplantation genetic diagnosis,PGD）打下良好基础。方法首先,针对突变热点p.R248C突变前后的序列特点,选择合适的内切酶＂Afe I＂,建立酶切鉴定法。其次,设计双错配碱基特异引物结合Apa LI酶切,建立ARMS/RE双重特异鉴定法。对阴性和阳性结果均再用普通引物扩、测的结果进行验证。结果用Afe I酶切FGFR3基因exons（6-7）普通引物的PCR产物（535 bp）,正常对照及非p.R248C突变的TD病例均能被切成255 bp和280 bp 2个片段,而胎1~胎3均切出255 bp、280 bp和535 bp 3个片段。用特异引物E7（p.R248C）扩增,正常对照和非p.R248C突变的TD病例均扩增阴性,无法进一步做酶切鉴定;而p.R248C突变均扩增阳性,当再用Apa LI酶切PCR产物（365 bp）时,胎1~胎3均切出22 bp和343 bp 2个片段。通过引物扩、测结果显示：胎1~胎3均是p.R248C杂合突变。结论该法快速特异、准确可靠,可用于p.R248C突变热点的快速检测及含该突变高危胎儿的快速产前诊断。该法还可用于含p.R248C突变TD-I型家系的PGD。胎1~胎3都是TD-I患胎,建议尽早终止妊娠。Objective To build up the specific rapid methods of RE and ARMS/RE for mutation hotspot＂p.R248C＂in the FGFR3 gene of thanatophoric dysplasia type I（TD-I）,then use the method for rapid prenatal diagnosis of 3 follow-up high-risk fetuses of TD-I to stop the birth of suffering fetuses,at the same time,lay a good foundation of preimplantation genetic diagnosis（PGD） for the future.Methods First,according to the characteristics of the sequence of the mutation hot spot p.248 C before and after mutation,the appropriate enzyme＂Afe I＂was selected to establish the identification method of enzyme digestion.After that,specific primers of double mismatch bases were devised,combining the method of digestion of Apa LI（ARMS/RE） to achieve the double identification.In the end,the sequences obtained by common primers were used to verify the negative and positive results through DNA sequencing.Results For the normal control and the patient without p.R248 C mutation of TD-I,PCR products（535 bp） of exons（6-7） of FGFR3 gene,amplified by common primers,could be digested into 2 fragments（255 bp and 280 bp） by Afe I.However,for fetuses 1~3,the PCR products could be digested into 3 fragments（255 bp,280 bp and 535 bp）.The normal control and the patient without p.R248 C mutation of TD-I were negative using the specific primers E7（p.R248C）,which could not be further identified by enzyme digestion.For the fetuses with p.R248 C mutation of TD-I,PCR products（365 bp） of exon 7 of FGFR3 gene,amplified by specific primers,could be digested by Apa LI and all the digested products were 22 bp and 343 bp.Sequencing results of common primers＇ products indicated：fetuses 1~3 were p.R248 C heterozygous mutation.Conclusions The method is fast,accurate and reliable,which can be available for the fast detection of mutation hot spot p.R248 C and the rapid prenatal diagnosis of high-risk fetus with p.R248 C mutation.This method can also be applied to PGD of high-risk fetus of TD-I with the mutation＂p.R248C＂.Fo闽粤合作科研基金（71010025

Construction of Hybrid Plasmid for shuttling uses between Escherichia coli and Cyanobacterium Synechococcus sp.T3

本文利用PUb1质粒中含有Ori.和蓝细菌plectonemaboryanum小质粒的Bgll酶切大片段和 p35S-CAT质粒中含有CaMV35Spromoter-Cm'-rbsS-3'终止末端”的Begll酶切片段，连接后转 化HB101，从高于对照50倍Cm抗性的LB平板上筛选出转化子，用强碱法检测，获得了新的 杂合质粒PEb。。经HPa酶切分析证明PEb3含有P.boryanum小质粒。将其转化蓝细菌Syne- clwcoccussp.T3获得抗ppmCm的转化藻株，并从中检测到转化的质粒，表明已初步构建成了 一种新的菌藻共用质粒载体。A new hybrid plasmid pEb3, originated from plasmids of E.coli and a cyanobacterium sp. Plectonema boryanum, containing a fragment of "CaMV 35S promoter-CAT gene-rbcS-3'end" from the plasmid p35S-CAT' has been reconstructed. Resistance to Cm in E.coli HB101 was proved to be risen from 1ppm to more than 50 ppm on LB plate medium after pEb3 transformation. Presence of the 1.55 Kb plasmid of P.bo...学位：理学硕士院系专业：生命科学学院生物学系_生物化学与分子生物学学号：YL00017

热带珍贵树种青梅苗木分级研究

为评价青梅苗木质量,应用相关分析确定苗高和地径作为青梅苗木分级指标,采用逐步聚类和平均值(M)±标准差(SD)法探究青梅苗木分级标准,比较分析2种方法各级别苗木的造林效果,从而确定适宜的分级方法和分级标准。结果表明,采用地径(D)、苗高(H)作为2年生青梅苗木分级指标是可行的;从各级别苗木比率以及造林效果分析,青梅苗木适宜采用平均值±标准差法进行苗木分级,其分级标准为Ⅰ级苗H≥45.3cm,D≥4.7mm;Ⅱ级苗21.8cm≤H<45.3cm,3.3mm≤D<4.7mm;Ⅲ级苗H<21.8cm,D<3.3mm

果实成熟度对青皮种子萌发及幼苗生长的影响

青皮果实成熟后易脱落,且常受台风、暴雨影响而采种困难。为了保障大规模果实采收和苗木培育,研究了其果实成熟度对种子萌发与幼苗生长的影响。结果表明:随着果实成熟度的增加,千果重、种子发芽率、发芽指数、发芽势以及幼苗成活率、苗高和地径均极显著增加(p0.05),因此青皮果皮颜色由青色向褐色过渡、被少量毛时即可采种