PCR方法对单细胞SRY基因扩增效率的对比研究

目的探讨单细胞基因扩增时,巢式PCR和引物预扩增(PEP)-巢式PCR两种扩增方法对SRY基因脱扣的影响程度;并应用PEP-巢式PCR方法对单个卵裂球细胞进行SRY基因诊断。方法获取单个正常男女淋巴细胞,随机分为巢式PCR组和PEP-巢式PCR组。同时扩增SRY基因和ZP3基因位点。选用IVF-ET后冻存的4个胚胎,处理后获取单个卵裂球11个,用PEP-巢式PCR扩增,鉴定其性别。结果单个淋巴细胞经巢式PCR和PEP-巢式PCR方法扩增后,其基因扩增成功率分别为92.39%,98.91%;性别诊断正确率分别为86.00%,98.00%;SRY基因的脱扣率分别为16.67%,2.38%。两者有统计学检验均有显著性差异(P<0.05。对单个卵裂球应用PEP-巢式PCR方法进行SRY基因和ZP3基因扩增后,有3个胚胎的8个卵裂球被诊断为男性,而另外1个胚胎的3个卵裂球被诊断为女性。结论1.行单细胞基因扩增时,PCR扩增方法会影响等位基因脱扣的发生率。2.对性连锁遗传病进行植入前遗传学诊断时,采用PEP-巢式PCR方法扩增SRY基因和ZP3基因对单个细胞对进行性别鉴定时,可以有效地降低SRY基因脱扣的发生率,提高性别诊断的特异性和敏感性,能够用于单细胞的性别诊断,可用于性连锁遗传病的植入前遗传学诊断。     

X-连锁慢性肉芽肿病的植入前遗传学检测

目的 采用多重置换扩增结合单体型分析及致病基因检测进行X-连锁慢性肉芽肿病的植入前遗传学检测。方法 利用位于CYBB基因两侧的16个短串联重复序列位点对一个X-连锁慢性肉芽肿病的家系构建单体型进行分析。活检的胚胎细胞通过多重置换扩增技术进行全基因组扩增，利用扩增产物检测具有多态性的STR位点与CYBB基因，同时采用位点Amel进行性别诊断。结果 对一个家系共进行了2个周期的PGT，家系分析结果显示7个STR位点具有多态性。共对16个胚胎进行了诊断，均获得诊断结果，诊断效率为100%，其中8个为正常胚胎，1个为携带者，7个为异常胚胎。全基因组扩增成功率100%，后续PCR成功率为99.3%(143/144),ADO率为14.0%(6/43)。先后进行了三次胚胎移植，末次移植后获得单胎妊娠，最终足月顺产一个不携带致病基因的健康男婴。结论 基于多重置换扩增产物对短串联重复序列进行连锁分析结合致病基因检测可对X连锁慢性肉芽肿病进行准确且有效的植入前遗传学检测。     

单细胞巢式PCR诊断软骨发育不全

目的 探讨单细胞水平诊断软骨发育不全(achondroplasia，ACH)的可靠性，为开展ACH的胚胎植入前遗传学诊断(preimplantation genetic diagnosis，PGD)打下基础。方法 采用巢式PCR扩增单淋巴细胞及单卵裂球的成纤维细胞生长因子受体3基因的高发突变位点G380R区域，用限制酶Bfm I消化PCR产物，10％聚丙烯酰胺凝胶电泳检测。结果 单淋巴细胞的PCR扩增成功率为90．4％，等位基因脱扣发生率为8．2％，诊断准确率为91．8％；单卵裂球的扩增成功率为75．4％。结论 单细胞巢式PCR诊断ACH是比较稳定、可靠的。     

应用巢式PCR对单细胞进行性别诊断的初步研究

目的：应用巢式PCR技术对人类植入前胚胎进行性别诊断。方法：收集单个或两个淋巴细胞和卵裂球（50个／组），按不同的方法处理单细胞（纯水法、冻融法、碱法），而后行巢式PCR扩增牙釉质基因。结果：纯水法、冻融法、碱法处理后，扩增率分别为83％、94％、95％。后两种处理方法的扩增效率明显高于纯水法（P＜0．01），通过检测正常男性单淋巴细胞基因型发现3种方法等位基因脱失率分别为24％、12％、4％，差异有显著性（P＜0．05）。两个淋巴细胞或卵裂球的扩增率及等位基因脱失率与单细胞相比差异无显著性。结论：提高植入前遗传学诊断的准确性主要取决于如何克服单细胞PCR的缺点，采用碱法裂解单细胞及取两个卵裂球进行检测可提高用于性别诊断的单细胞PCR技术准确性和敏感性。     

胚胎植入前诊断α-地中海贫血一例

目的对1例α-地中海贫血患者进行植入前胚胎遗传学诊断。方法应用单细胞跨越断裂点荧光聚合酶链反应（PCR）检测技术对1例夫妇双方均为α-地贫杂合子进行了胚胎植入前诊断（PGD）。结果9个胚胎进行PGD,经PCR分析,共获得2个正常胚胎,其中一个胚胎体外发育停滞;2个杂合子胚胎,2个重型地贫胚胎,2个胚胎未检出;移植了3个胚胎,获得临床妊娠。孕17周时经脐带血穿刺,分别证实为完全正常胚胎和杂合子胚胎,现已出生两名健康男婴。结论应用PGD技术可对α-地贫进行胚胎植入前遗传筛查,达到优生的目的。     

引物延伸预扩增用于胚胎种植前诊断的实验研究

目的 用引物延伸预扩增（PEP）方法对种植前胚胎进行单基因病诊断的实验研究。方法 取IVF后剩余胚胎的一个卵裂球用PEP方法，扩增SRY、ZP3、RhCE、RhD和FVⅢ5个基因，而同一胚胎的另一个卵裂球双重套式PCR扩增SRY和ZP3基因。结果 PEP分析分别由6个胚胎获得的6个卵裂球，1-4号胚胎有19个基因呈阳性（19/20），1个基因阴性，5，6号胚胎除SRY均阴性，其余基因为阳性；而6个胚胎的卵裂球双重套式PCR扩增SRY和ZP3基因的结果与同一胚胎PEP的结果相同。结论 采用PEP方法进行种植前胚胎单基因病的诊断是可靠、准确的，且可用于无创性产前诊断。     

两种不同检测方案在α-地中海贫血植入前遗传学诊断中的应用分析

目的比较两种不同的检测方案在α-地中海贫血胚胎植入前遗传学诊断(preimplantation genetic diagnosis,PGD)中的应用。方法对2017年1月至2018年12月在本院进行α-地中海贫血PGD的囊胚检测结果进行回顾性分析,其中蛋白酶K裂解后荧光PCR直接检测致病位点方法(方案1)40个周期,全基因组扩增结合短串联重复序列单体型分析和致病位点检测方案(方案2)108个周期,将两组的诊断结果与随访结果进行比较。结果方案1检测成功率为95.15%(16/330),正常胚胎为21.97%(69/314),携带者胚胎为50.64%(159/314),重型地贫胚胎为27.39%(86/314);方案2检测成功率为93.02%(54/774),正常胚胎为19.17%(138/720),携带者胚胎为44.72%(322/720),重型地贫胚胎为25.83%(186/720),其他类型(包括疑似单体10个,疑似三体15个,疑似重组49个)胚胎为10.28%(74/720)。两种方案的检测成功率、正常胚胎率、携带者胚胎率、重型地贫胚胎率均无显著性差异(P0.05)。但方案1不能检出16号染色体拷贝数异常及HBA基因附近重组情况。结论全基因组扩增结合短串联重复序列单体型分析和致病位点检测方案较蛋白酶K裂解后荧光PCR直接检测致病位点方案更适合进行α-地中海贫血PGD。     

采用多重置换扩增进行脊肌萎缩症的植入前遗传学诊断研究

目的建立一种可靠、准确的植入前遗传学诊断方法,可广泛用于各种类型的脊肌萎缩症。方法采用多重置换扩增(MDA)对单细胞进行全基因组扩增,等位基因特异性扩增SMN1外显子7进行脊肌萎缩症(SMA)致病基因检测及采用与SMN基因紧密连锁的12个短串联重复序列(STR)进行SMA的单体型分析。结果共进行了80单个淋巴细胞及63个单卵裂球的MDA。MDA扩增效率在单淋巴细胞中为97.5%(78/80),在单卵裂球为96.8%(61/63)。在单淋巴细胞及单卵裂球MDA产物中,总的PCR扩增效率和等位基因脱扣率(ADO)为分别为96.2%(587/610)和8.7%(52/597),95.5%(722/756)和9.9%(54/547)。在单淋巴细胞MDA产物中,SMN1外显子7的PCR结果均与外周血结果相符,而单个卵裂球MDA产物中,有一个PCR扩增失败,扩增效率为98.4%(60/61)。结论该方法可用于常见的缺失型的SMA的PGD,而且可以应用于突变型的为SMA的PGD,为进一步的临床应用奠定了基础。     

PCR技术用于单个卵裂球基因诊断的实验研究

目的建立单卵裂球PCR技术,为开展单基因病的着床前遗传学诊断奠定基础。方法共对103个卵裂球进行了PCR扩增;其中30个扩增KG8位点,33个扩增D16S291位点,40个扩增D16S423位点。结果单卵裂球扩增KG8、D16S291和D16S423的成功率分别86.7%、87.9%和87.5%。结论建立的单卵裂球PCR技术是稳定可靠的,可以用于单基因病的着床前遗传学诊断。     

β-地贫合并α珠蛋白基因三联体导致中间型地贫的家系分析与产前诊断

目的 探讨分析β-地贫合并α珠蛋白基因三联体导致中间型地贫的家系分析和诊断流程，总结中间型地贫的诊断策略及产前诊断临床实践。方法 通过家系成员血液学表型、常规地贫基因检测和PCR电泳法及珠蛋白基因测序技术分析β-地贫合并α珠蛋白基因三联体的临床表现；利用羊水细胞DNA对该家系1例地贫高风险胎儿进行产前诊断。结果 先证者检出β-地贫CD41-42杂合突变合并αααanti4.2三联体，父亲检出αααanti4.2三联体，母亲检出β-地贫CD41-42杂合突变，胎儿结果未见异常。结论 临床表现为中重度贫血的β-地贫杂合突变且未见罕见型变异，应结合临床表现与基因型是否一致，考虑可能合并α珠蛋白基因三联体导致的中间型地贫。     

Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis

Sickle-cell and beta-thalassemia syndromes are priority genetic diseases for prevention programs involving population screening with the option of prenatal diagnosis for carrier couples. Preimplantation genetic diagnosis (PGD) represents a specialized alternative to prenatal diagnosis and is most appropriately used for couples with an unsuccessful reproductive history and/or undergoing assisted reproduction. However, clinical application of PGD has been hindered by difficulties in reliably transferring molecular diagnostic protocols to the single-cell level. We standardized and validated a protocol involving first-round multiplex PCR, amplifying the region of the beta-globin gene containing most of the common disease mutations world-wide and two unlinked microsatellite markers (GABRB3 and D13S314), followed by: 1) analysis of beta-globin genotypes with real-time PCR and 2) microsatellite sizing to exclude chance contamination. The protocol was standardized on 100 single lymphocytes from a beta-thalassemia heterozygote, including 15 artificially contaminated samples, the latter demonstrated through microsatellite analysis. PCR failure and allele drop-out (ADO) were observed in one (uncontaminated) sample each (1.2%). A pilot study in six clinical PGD cycles with five different beta-globin genotype interactions achieved results (in 5-6 hr) in 46 out of 50 single blastomeres (92%), all concordant with results from an established PGD method applied simultaneously; microsatellite analysis detected only parental alleles, excluding contamination. Beta-globin genotypes were also confirmed in two blastomeres through prenatal diagnosis (twin pregnancy), and in 11 out of 12 spare embryos, revealing one incident of ADO. Overall, the protocol proved to be sensitive, accurate, reliable, rapid, and applicable for many genotype interactions, with internal monitoring of contamination, thus fulfilling all requirements for clinical PGD application.     

Preimplantation genetic diagnosis for beta-thalassaemia using sequencing of single cell PCR products to detect mutations and polymorphic loci

In order to carry out preimplantation genetic diagnosis (PGD) for beta-thalassaemia, we have applied direct sequencing of single cell PCR products to detect mutations and polymorphic loci within the beta-globin gene. Conventional duplex PCR was used to amplify two regions of the beta-globin gene with an amplification efficiency of 79% for blastomeres. Sequencing data were obtained for 100% of amplified products, with 12% having confirmed allele drop-out (ADO). A double ADO event was observed at least twice, confirming the real risk of such an event during PGD. In one couple, the presence of a polymorphism linked to the female partner's mutation enabled us to eliminate the risk of misdiagnosis due to double ADO without having to amplify both mutations within the same PCR product. We present here the data from eight clinical PGD cycles for three couples resulting in a singleton pregnancy and a twin pregnancy with all babies confirmed to be free from beta-thalassaemia (major).     

Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis

Cystic fibrosis (CF) is the first monogenic disorder for which single cell preimplantation genetic diagnosis (PGD) has been successfully applied. The spectrum of mutations in CF is extremely heterogeneous, and hence, the development of mutation-specific PGD protocols is impracticable. The current study reports the development and evaluation of a general multiplex marker polymerase chain reaction (PCR) protocol for PGD of CF. Four closely linked highly polymorphic (CA)(n) repeat markers D7S523, D7S486, D7S480 and D7S490, flanking the cystic fibrosis transmembrane regulator (CFTR) gene, were used. In 99% of the single cells tested (100 leukocytes and 50 blastomeres), multiplex PCR results were obtained and the overall allelic drop out (ADO) rate varied from 2 to 5%. After validation for the presence of ADO and additional alleles, 95% of the multiplex PCR results were accepted to construct the marker genotypes. Depending on the genotype of the couple, and taking into account the embryos lost for transfer due to validation criteria (5%), ADO (0-2%) and single recombination (1.1-3%), in general >90% of the embryos could be reliably genotyped by PGD using a single blastomere. The risk of misdiagnosis equals the chance of a double recombination between informative flanking markers and is <0.05%. Therefore, this polymorphic and multi-allelic marker system is a reliable and generally applicable alternative for mutation-directed PGD protocols. Furthermore, it provides a test for the origin of the detected genotype and also gives an indication of the chromosomal ploidy status of the blastomere tested.     

Multiple displacement amplification improves PGD for fragile X syndrome

We report an improvement in the PGD test for fragile X syndrome (FXS). Recently, multiple displacement amplification (MDA) has been reported to yield large amounts of DNA from single cells. Taking into account this technique, we developed a new PGD test for FXS, enabling combined analysis of linked polymorphic markers with the study of the non-expanded CGG repeat. Single cell amplification efficiency was first assessed on single lymphocytes. Amplification rate of the different markers ranged from 85 to 95% with an allele drop-out (ADO) rate comprised between 7 and 34%. Using this test, eight PGD cycles were carried out for six couples, and 37 embryos were analysed after preliminary MDA. Amplification rate was increased by this technique from 41 to 66% so that embryos with no results were rarer (14 versus 45% without MDA). Reliability of the test was considerably improved by combining direct with indirect genetic analysis. Furthermore, in cases of fully expanded alleles too large to be amplified by PCR, this test gives an internal amplification control. Embryonic transfers were carried out in all but one PGD cycles. One biochemical and one clinical pregnancy resulted, and a healthy child was born. This single diagnosis procedure could be suitable to most patients carrying FXS.     

Strategies and clinical outcome of 250 cycles of Preimplantation Genetic Diagnosis for single gene disorders

BACKGROUND: We report on our experience with preimplantation genetic diagnosis (PGD) for single gene disorders (SGDs), from 1999 to 2004, describing strategies and overall clinical outcome of 250 cycles in 174 couples for 23 different genetic conditions. METHODS: PGD cycles included 15 for autosomal dominant, 148 for autosomal recessive and 19 for X-linked SGDs. In addition, 68 cycles of PGD for SGDs were performed in combination with HLA matching. The strategy in each case used an initial multiplex PCR, followed by minisequencing to identify the mutation(s) combined with multiplex PCR for closely linked informative markers to increase accuracy. Linkage analysis, using intragenic and/or extragenic polymorphic microsatellite markers, was performed in cases where the disease-causing mutation(s) was unknown or undetectable. RESULTS: In 250 PGD cycles, a total of 1961 cleavage stage embryos were biopsied. PCR was successful in 3409 out of 3149 (92.4%) biopsied blastomeres and a diagnosis was possible in 1849 (94.3%) embryos. Four hundred and twenty-seven embryos were transferred in 211 cycles, resulting in 71 pregnancies (33.6% per embryo transfer), including 15 biochemical pregnancies, six spontaneous miscarriages, two ectopic pregnancies, which were terminated, and nine pregnancies which are still ongoing. The remaining pregnancies were confirmed to be unaffected and went to term without complications, resulting in the birth of 35 healthy babies. CONCLUSIONS: Minisequencing for mutation detection combined with multiplex fluorescence PCR for linkage analysis is an efficient, accurate and widely applicable strategy for PGD of SGDs. Our experience provides a further demonstration that PGD is an effective clinical tool and a useful option for many couples with a high risk of transmitting a genetic disease.     

Multiplex sequence variation detection throughout the CFTR gene appropriate for preimplantation genetic diagnosis in populations with heterogeneity of cystic fibrosis mutations

Cystic fibrosis (CF) is one of the most important genetic diseases requiring prevention programmes. Preimplantation genetic diagnosis (PGD) represents an alternative to prenatal diagnosis, and is especially appropriate for couples with an unsuccessful reproductive history. For clinical application, protocols must be optimized to minimize PCR failure, allelic drop-out (ADO) and contamination, while simultaneously detecting a wide spectrum of CF genotypes. We have developed a flexible multiplex PCR protocol allowing analysis of sequence variations in any combination amongst seven CFTR gene exons (4, 10, 11, 13 in two parts, 14b, 17b and 21) by nested PCR and denaturing gradient gel electrophoresis analysis, along with analysis of a fluorescently labelled intragenic microsatellite (IVS8CA). The experiments were carried out on 390 single lymphocytes from three CF patients, one heterozygote and one non-CF individual. PCR efficiency of the exons ranged from 90 to 100%, and ADO from 0 to 3.8%. IVS8CA was co-amplified with a PCR efficiency of 92.4 and 10.8% ADO. The present method overcomes the need for separate assays for each CFTR gene mutation. Additionally, it facilitates analysis of any informative linked polymorphic sequence variation (within the seven exons) along with analysis of a microsatellite, which is useful (when informative) for minimizing misdiagnosis and/or indirect diagnosis. This method proved robust and flexible for diagnosing diverse CF genotype combinations in single cells.     

PGD for autosomal dominant polycystic kidney disease type 1

Autosomal dominant polycystic kidney disease (ADPKD) is primarily characterized by renal cysts and progression to renal failure. It is a genetically heterogeneous disease, with mutations in the PKD1 gene accounting for the majority of cases. Direct mutation detection for PKD1-linked ADPKD or type 1 is complicated by the large size and complex genomic structure of PKD1. This paper describes a microsatellite marker-based assay for PGD in couples at risk of transmitting ADPKD type 1. During PGD, genetic analysis is carried out on single blastomeres biopsied from preimplantation embryos obtained after IVF, and only embryos unaffected by the disease under investigation are selected for transfer. Single-cell genetic analysis relied on a fluorescent duplex-PCR of linked polymorphic markers followed by fragment length determination on an automated sequencer. The co-amplification of the intragenic KG8 and the extragenic D16S291 marker at the single-cell level was evaluated in pre-clinical tests on lymphoblasts and research blastomeres. The developed assay proved to be efficient (96.1% amplification) and accurate (1.4% allele drop-out and 4.3% contamination), and can be applied in all informative ADPKD type 1 couples. From five clinical cycles carried out for three couples, two pregnancies ensued, resulting in the birth of two healthy children.     

Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A

Charcot-Marie-Tooth (CMT) disease is the 'common' name for a range of hereditary peripheral neuropathies. CMT1 is the most common form and is transmitted in an autosomal dominant manner. CMT1A maps to chromosome 17p11.2 and is caused, in the majority of cases, by a 1.5 Mb DNA duplication, that includes the peripheral myelin protein 22 (PMP) gene. This paper reports on preimplantation genetic diagnosis (PGD) for CMT1A in five couples. The CMT1A duplication was detected by fluorescent PCR analysis using polymorphic (CA)n markers localized within the duplication. Single-cell PCR on blastomeres allowed genetic analysis of embryos obtained after ICSI. Only healthy unaffected embryos were transferred to the uterus. PCR experiments with single EBV-transformed lymphoblasts or with research blastomeres allowed the evaluation of amplification efficiencies, as well as contamination and allele drop-out (ADO) rates for each PCR protocol. Three simplex PCR protocols (using one primer pair) and two duplex PCR protocols (using two primer pairs) were developed for CMT1A. Additionally, a protocol using all three primer pairs in triplex was also established. Thirteen clinical ICSI-PGD cycles were performed for five couples (12 simplex PCR cycles and one duplex PCR cycle), resulting in seven embryo transfers. Three singleton pregnancies ensued in two couples and three healthy babies were delivered. This report describes different fluorescent PCR-based tests which allow efficient and accurate single-cell level detection of the CMT1A duplication. On the basis of the presence of the healthy allele of the affected parent-to-be (and/or absence of the affected one), healthy embryos can be selected for transfer. The assays are suitable for PGD for other couples who present with the same CMT1A duplication [depending on their informativity for the (CA)n markers available] as described here.     

Pregnancy following preimplantation genetic diagnosis for Crouzon syndrome

Crouzon syndrome is a dominantly inherited craniosynostosis syndrome which is caused by mutations in the fibroblast growth factor receptor 2 gene (FGFR2). However, a specific point mutation in the FGFR3 gene has also been shown to result in Crouzon syndrome associated with acanthosis nigricans. We report here the first method for preimplantation genetic diagnosis (PGD) of Crouzon syndrome based on multiplex PCR amplification followed by the direct detection of the causative mutation by single-stranded conformational polymorphism (SSCP) analysis. A highly polymorphic short tandem repeat (STR) locus was simultaneously analysed as a control against some forms of contamination. The mutation, carried by the female partner, was a de-novo substitution at codon 338 of the FGFR2 gene. The couple were found to be informative at the D21S11 STR locus. Two clinical PGD cycles were performed, resulting in the biopsy of 36 blastomeres, 25 of which showed amplification at the FGFR2 locus. All of the cells showed expected genotypes at the D21S11 locus with only one incidence of allele drop-out. A total of five embryos were transferred, two in the first cycle and three in the second, resulting in a singleton pregnancy.     

Successful application of preimplantation genetic diagnosis for beta-thalassaemia and sickle cell anaemia in Italy

BACKGROUND: In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. METHODS AND RESULTS: The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. CONCLUSIONS: These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy.