软骨发育不全植入前遗传学诊断的方法学研究

目的：针对软骨发育不全（ACH）高危家系的 FGFR3基因的突变类型（c.1138G 〉 A, p.G380R）,建立多种快速特异、行之有效的植入前遗传学诊断（PGD）方法,为实施该 ACH 家系的 PGD创造必要的前提条件。方法在确诊该 ACH 患者特定突变类型的基础上,首先用外周血建立各种PGD方法,包括： A.直接测序法; B. ARMS法; C.酶切鉴定法;D. ARMS／RE法。然后对单个卵裂球的全基因组扩增（WGA）产物进行相应方法学的研究。最后,对各种方法进行优化优选。结果 A.直接测序法：正常对照c.1138为G 纯合子,而患者为G／A 杂合峰; B. ARMS 法：正常对照扩增阴性,而患者有445 bp 的特异扩增条带。 C.酶切鉴定法：正常对照酶切后仍为513 bp,而患者酶切后产生205 bp、308 bp、513 bp三条带; D. ARMS／RE法：正常对照扩增阴性,无法做酶切鉴定。而患者有445 bp扩增产物,经SfcI酶切后可产生27 bp和418 bp两个片段。结论本研究已成功建立针对c.1138 G 〉 A 杂合错义突变的直接测序法、ARMS 法、酶切鉴定法和ARMS／RE 法。所建立的4种方法快速、特异,但各有利弊,同时使用可相互弥补,结合STR 连锁分析可把误诊风险降到最低程度。在正常情况下,可在24 h 内完成对ACH高危胚胎的PGD。     

突变特异性扩增系统和变性高效液相色谱分析法结合DNA测序法快速产前诊断黏多糖贮积症Ⅱ型高危胎儿

目的为了快速、准确地对黏多糖贮积症Ⅱ型(MPSⅡ)高危胎儿做出产前基因诊断,从而及时而高效地防止病胎的出生。方法在阐明先证者病因及其父母基因型的基础上,采用已建立的突变特异性扩增系统(ARMS)特异引物直接鉴定法、变性高效液相色谱(DHPLC)快速筛检法,结合DNA序列分析法,对已孕20周的高危胎儿的艾杜糖-2-硫酸酯酶(IDS)基因相应突变位点进行快速检测和鉴定。由于MPSⅡ为XR病,故又用SRY引物对胎儿进行性别鉴定,以避免可能出现的母血污染而导致的误诊。结果所怀胎儿未获得母源性的c.1344delA突变,是1例基因型完全正常的女胎。后经随访和复检,证实所生小孩的基因型、表现型都与产前诊断结果完全一致。结论 ARMS特异引物直接鉴定法、DHPLC快速筛检法结合DNA序列分析法是快速、准确产前诊断MPSⅡ高危胎儿的有效方法。其中,所建立的ARMS和DHPLC方法,既快速特异,又简便经济,可在一天之内送报结果 ,特别适用于已明确病因的高危胎儿的早期诊断以及正常组和患者试验组的快速鉴别。这一产前基因诊断工作的成功实施为今后继续开展本病以及其他相关遗传病的遗传咨询、基因诊断和产前诊断积累了宝贵的经验,也为今后开展...     

致死性侏儒症p.R248C突变热点的快速检测和三例TD-Ⅰ型高危胎儿的快速产前诊断

目的 针对致死性侏儒症Ⅰ型(thanatophoric dysplasia type Ⅰ,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“AfeI”,建立酶切鉴定法.其次,没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AfeI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.     

肾上腺皮质肿瘤与MEN1基因的相关性研究

目的 从分子水平上揭爪散发性肾上腺皮质肿瘤的发病与MEN1基因突变的相互关系,为今后开展症状前诊断和产前基因诊断创造前提条件.方法 在对先证者进行临床诊断的基础上,用微量快提法制备模板DNA,用自行设计的9对引物对先证者MEN1基因的所有编码区以及外显子与内含子的交界部位进行PCR扩增和DNA序列分析.结果 先证者MEN1基因第4内含子存在隐蔽性拼接位点ⅣS4as（-9）G〉A的杂合突变,而正常对照无此突变.结论 所用检测方法快速简便、微量经济,可用于肾上腺皮质肿瘤的快速确诊.所发现的MEN1基因的ⅣS4 as（-9）G〉A隐蔽性拼接位点突变为国内首报的病理性突变.     

软骨发育不全患者的FGFR3基因诊断

目的建立软骨发育不全(ACH)患者的FGFR3基因检测方法。方法用PCR扩增和测序技术对临床上疑似ACH的14例患者的FGFR3基因突变进行测序。结果 FGFR3基因的外显子10测序结果显示,14例疑似ACH患者中10例检出FGFR3基因1138位G→A突变。对4例未检出突变的样本进行FGFR3基因的全部外显子测序,有2例样本检出FGFR3基因882位T→C杂合突变,最后确定其为已知的单核苷酸多态性(SNP)位点(rs2234909)。对4例样本中的1例进行了后续检查,证实该受检者不是ACH患者。结论 FGFR3基因的突变检测有助于疑似ACH患者的明确诊断,对遗传咨询具有重要意义。     

一例成骨不全Ⅱ型高危胎儿的产前基因诊断

目的对广东一疑似致死性侏儒症或成骨不全Ⅱ型的高危胎儿实施产前基因诊断,以阐明胎儿骨发育异常的真实病因,及时预防患胎出生。方法对经超声检查初诊为致死性侏儒症或成骨不全、已孕25周的高危胎儿,在抽取脐血制备DNA模板后,采用PCR-DNA直接测序法,分别对胎儿的FGFR3基因和COL1A1基因进行突变检测,然后对所发现的突变进行分析和鉴定。结果 FGFR3基因未发现病理性突变,而COL1A1基因发现一典型的杂合错义突变(c.3065G>T,p.G1022V),经查HGMD数据库证实为成骨不全Ⅱ型的致病性突变。结论 (1)此高危胎儿为成骨不全Ⅱ型患胎,应及时终止妊娠(胎儿已经引产,经复查证实与产前基因诊断结果完全一致)。(2)在超声初诊基础上,采用产前基因诊断可快速、有效对高危胎儿做出确诊,为出生缺陷的预防提供技术保障。     

婴儿型多囊肾家系PKHD1基因的突变分析

目的对1个婴儿型多囊肾家系的致病基因PKHD1进行突变分析。方法先证者母亲怀孕两次,胎儿均在围产期死亡,且两次胎儿彩超结果相似,符合多囊肾改变。提取先证者父母及其他成员外周血基因组DNA,PCR扩增PKHD1基因编码区并进行序列测定;找到突变后,对相应PCR产物进行酶切鉴定或变性高效液相色谱分析,以证实突变。结果先证者家系成员序列分析表明,其父和祖母PKHD1基因第59外显子存在杂合突变（c．9901G〉T）,其母和外祖母PKHD1基因第53外显子存在杂合的缺失突变（c．8317delG）。两个突变均导致mRNA提前出现终止密码子。结论发现两个新的PKHD1基因突变;若胎儿同时遗传了这两个突变,将导致婴儿型多囊肾的发生。     

1个糖原累积症家系的分子诊断与产前诊断

目的对1个糖原累积症(glycogen storage disease,GSD)家系进行基因突变分析,并对该家系中的一高危胎儿进行产前分子诊断。方法采集该家系先证者及其父母外周血,采用二代测序方法查找先证者致病基因及突变位点,Sanger测序进行突变验证。确定先证者及其父母基因型后采集羊水标本,采用PCR扩增及直接测序方法进行产前分子诊断。结果该家系先证者为G6PC基因c.648GT纯合突变。双亲均为G6PC基因c.648GT杂合突变。胎儿携带与父母相同的c.648GT杂合突变。结论建立了对GSD进行分子诊断和产前分子诊断的方法,并成功应用于1个GSD家系。     

一例肥厚型心肌病家系的产前诊断和遗传分析

摘要：目的：对1例肥厚型心肌病（HCM）家系的孕妇进行产前诊断和遗传分析，探讨HCM致病基因突变与临床表型的联系。 方法：搜集该先证者及其家系成员临床资料，提取先证者外周血DNA、羊水细胞DNA、经培养的羊水细胞DNA，二代测序（NGS）筛查先证者的致病基因位点，PCR扩增产物直接测序验证HCM致病候选基因 MYH7 和 MYBPC3 的可疑致病突变序列，并检测羊水细胞潜在的致病突变。用遗传学数据资料和Mutation taster、PolyPhen-2、ANTHEPROT等生物信息学软件预测突变的致病性。 结果：测序结果显示先证者存在 MYH7 c.1988G＞A（p.Arg663His）和 MYBPC3 c.151G＞A （p.Ala51Thr）杂合突变，其父亲表型正常且未检出异常突变，[JP2]其表型正常的母亲仅携带 MYBPC3 c.151G＞A杂合突变。胎儿仅存在 MYH7 c.1988G＞A[JP]杂合突变，而 MYBPC3 无异常变异。突变效应预测和蛋白质结构功能分析显示这2种错义突变分别影响蛋白质的疏水性和抗原性，且遗传学数据资料显示 MYH7 c.1988G＞A为明确致病性突变。 结论：先证者 MYH7 c.1988G＞A为新发致病性突变或由于其父母为生殖嵌合所致， MYBPC3 c.151G＞A突变可能促进HCM的发生。胎儿虽然仅携带 MYH7 c.1988G＞A，但其表型可能仍为HCM。     

一罕见成骨不全IV型的基因诊断

目的对一疑似成骨不全IV型或其他类型的患儿实施基因诊断,以揭示患儿发病及频繁骨折的内在原因,为今后实施对症治疗和产前基因诊断创造必要的前提条件。方法对经症状、体征观察和x线检查初诊为成骨不全Iv型或其他类型的患儿,在抽取外周血制备DNA模板后,采用PCR、DNA直接测序法,对患儿的COLlAl基因进行突变检测,然后对所发现的突变进行分析和鉴定。结果在COLlAl基因的编码区内发现一典型的错义突变（c．823G〉C1／p．G275R）,经查HGMD数据库证实为成骨不全Iv型的致病性突变。结论先证者为一罕见的成骨不全IV型患儿,所发现的p．G275R突变为中国人群首次报道的病理性突变。     

Accurate diagnosis of a homozygous G1138A mutation in the fibroblast growth factor receptor 3 gene responsible for achondroplasia

Achondroplasia is the most common genetic form of dwarfism inherited as an autosomal dominant disorder. Individuals affected with achondroplasia have impaired ability to form bone from cartilage (endochondral bone formation). Homozygous achondroplasia is a neonatal lethal condition. The vast majority of patients with achondroplasia have a G-to-A transition at position 1138 of the fibroblast growth factor receptor 3 (FGFR3) cDNA sequence, resulting in the Gly-to-Arg substitution at position 380 of the FGFR3 protein. This mutation has been diagnosed by SfcI digestion of amplified genomic DNA. However, it has also been demonstrated that the SfcI digestion protocol does not consistently distinguish between DNA samples heterozygous and homozygous for the G1138A substitution. This study was designed to improve the molecular diagnosis based on the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques for the FGFR3 G1138A mutation. The newly designed forward primer contains one mismatch (G at position 1136) from the FGFR3 cDNA sequence (A at position 1136), thereby creating a PstI site (CTGCAG at positions 1134 to 1139) in the amplified DNA from alleles containing the G1138A mutation. The PCR-RFLP technique based on the PstI digestion of amplified genomic DNA with a novel forward primer shows 100% accuracy in diagnosis of the G1138A mutation in heterozygous and homozygous individuals.     

An improved tetra-primer PCR approach for the detection of the FGFR3 G380R mutation responsible for achondroplasia

Achondroplasia is the most common form of dwarfism and has an incidence of approximately 1/7500. In more than 98% of cases, the disease is associated with a G to A or G to C substitution at nucleotide position 1138 (p.G380R) of the fibroblast growth factor receptor 3 (FGFR3) gene. We have developed a sensitive single tube tetra-primer PCR assay to detect both the c.1138G>A and c.1138G>C mutations and can successfully distinguish DNA samples that are homozygous and heterozygous for the c.1138G>A mutation. Titration studies showed that the assay could reliably detect one copy of the mutant allele in a mix of 100 wild-type alleles. The assay has been tested in 50 healthy controls, 3 known patients with achondroplasia, and 5 amniotic fluids suspected of having achondroplasia and for whom we had previously determined the genotypes for the c.1138G>A mutation by PCR-RFLP. We have observed complete concordance between methods. Our tetra-primer PCR assay is sensitive, low-cost, and easy to use method for FGFR3 p.G380R genotyping, which could be used even in "low-tech" laboratories.     

Genotyping of the G1138A mutation of the FGFR3 gene in patients with achondroplasia using high-resolution melting analysis

OBJECTIVES: The fibroblast growth factor receptor 3 gene (FGFR3) plays a critical role in cartilage growth-plate differentiation and bony development. It has been shown that 97% of patients with achondroplasia have a G to A transition mutation at position 1138 (c.1138 G>A) of codon 380 of the FGFR3 gene. DESIGN AND METHODS: Exon 8 of the FGFR3 gene was analyzed in 40 patients with achondroplasia, as well as in 50 control individuals for the presence of the c.1138G>A variant using melting curve analysis with a high-resolution melting instrument (HR-1). RESULTS: The high-resolution melting curve analysis successfully genotyped the c.1138G>A mutation in exon 8 of the FGFR3 gene in all 40 patients with achondroplasia without the need of further assays. The technique had a sensitivity and specificity of 100%. CONCLUSION: High-resolution melting analysis is a simple, rapid, and sensitive one tube assay for genotyping the FGFR3 gene. The technique is a low cost high-throughput FGFR3 screening assay.     

采用ARMS分析口腔脱落细胞ApoE基因型

以口腔脱落细胞ＤＮＡ为模板,采用扩增不应突变系统（ＡＲＭＳ）,设计了等位基因４个特异性寡核苷酸引物和一个公共引物,分析了７４例个体ＡｐｏＥ３个常见共显性等位基因∈２、∈３和∈４。这一系统扩增出１８１ｂｐ和３１９ｂｐ两种ＡｐｏＥ基因顺序,当携某一等位基因的模板ＤＮＡ与相应的等位基因特异性寡核着酸引物及公共引物反应时,扩增出相应的基因顺序。省略了限制性核酸内切酶的消化或等位基因特异性寡核苷酸探针的杂交。方法简单、可靠、非放射性。     

山东地区甲状腺异位的先天性甲状腺功能减退症患儿TSHR基因突变研究

目的：对山东地区由甲状腺异位导致的先天性甲状腺功能减退症（CH）患儿 TSHR 基因第10外显子进行突变筛查,阐明山东地区CH患者TSHR基因突变类型和特点。方法以89例甲状腺异位导致CH的患儿为研究对象,采集外周血提取DNA,PCR扩增第10外显子,采用直接测序的方法对TSHR基因进行突变筛查。结果在89例研究对象第10外显子测序结果中,发现1个错义突变 c.1269G＞A （p.V424I）,1个SNP位点（rs1991517,c.2181G＞C,变异频率18.5%）。对小鼠、大鼠、猫、猪、牛、斑马鱼和人的TSHR蛋白进行同源性比较,结果表明第424密码子编码的氨基酸位于TSHR的保守区,该位点的缬氨酸被异亮氨酸取代（p.V424I）。结论山东地区甲状腺异位导致CH的患者中,TSHR基因第10外显子突变率较低。     

Rapid detection of common CARD15 variants in patients with inflammatory bowel disease.

BACKGROUND: Three mutations (R702W, G908R, and 1007fs) within the CARD15 gene have been identified as independent risk factors for the development of Crohn's disease (CD). Virtually all studies investigating the occurrence of these mutations in patients with CD have used separate PCR-based methods to screen patient DNA, here we describe a novel multiplex amplification refractory mutation system (ARMS) assay that allows the simultaneous detection of R702W, G908R, and 1007fs, and a fourth CARD15 variant, P268S, at a fraction of the cost of the pre-existing genotyping assays. METHODS: Allele-specific primer sets were designed for each CARD15 variant, optimized separately for annealing temperature and MgCl2 and then multiplexed. The mutant- and wild-type-specific primers were split across two tubes so that each multiplex reaction was internally controlled for amplification failure. An additional primer pair specific to beta2-microglobulin was included as an independent control for DNA quality. The specificity of each primer set was tested using positive controls that had been validated by sequencing, and the robustness of the final ARMS assay was assessed by genotyping 111 Caucasian patients with inflammatory bowel disease (IBD). RESULTS: The specificity of each primer set was confirmed using a sequence validated positive control for each of the four CARD15 variants. Of the 111 DNA samples screened with our ARMS assay, a clear CARD15 genotype was obtained for 109 patients. DISCUSSION AND CONCLUSIONS: Given the potential predictive value of R702W, G980R, and 1007fs, a robust genotyping method for these variants would be of considerable value both in diagnostic and research settings. Our ARMS assay only takes 3-4 hours to perform once DNA has been extracted and requires only 1U of Taq DNA polymerase, making it a rapid, reliable, and cost-effective alternative to current CARD15 genotyping methods.