应用荧光斑点法和比值法检测G6PD

目的建立适合于G6PD缺乏的新生儿筛查及确诊方法。方法应用荧光斑点法(FST)对新生儿筛查滤纸干血片进行检测，对可疑阳性者召回，抽静脉血以D6PD／6PGD比值法进行确诊，同时结合新生儿父母亲的G6PD结果，根据遗传关系综合分析。结果FST筛查25000例新生儿，G6PD缺乏阳性率为4．56％，确诊检出率为4．09％。与比值法的符合率为90．4％，G6PD重度缺乏者的符合率为100％，G6PD中间缺乏者的符合率为78．5％，室间质量控制结果与反馈结果符合率为100％。结论FST汀具有高的敏感性、特异性和准确性，方法简便、快捷、费用低廉，可对滤纸干血片标本进行大规模的筛查检测，同时利用比值法进行确诊，可减少假阳性及假阴性，有利于早期诊断和防治G6PD缺乏所致的新生儿黄疸和急性溶血。     

Knowledge and attitudes towards preimplantation genetic diagnosis in Germany

BACKGROUND: Preimplantation genetic diagnosis (PGD) is a technique which is often related to emotional debates because of its ethical and social implications. Worldwide there are different forms of legislation; Germany constitutes an interesting case because of the historical background concerning eugenics and dealing with handicapped persons at the time of national socialism. PGD is currently not legal but there are still polarized positions and legalization remains an issue. Studies about the attitudes of the general population towards PGD are rare. METHODS: Data were collected in a representative survey carried out in November 2003. Subjects were 2110 persons in Germany aged 18-50 years. RESULTS AND CONCLUSIONS: Respondents had little knowledge about PGD. There were incorrect assumptions about the diagnostic possibilities and a lack of basic genetic knowledge. A tendency towards a general acceptance of PGD for medical indications was found. Non-medical indications such as sex selection were generally not accepted. It could be observed that respondents who already had a notion about PGD overestimated the diagnostic possibilities and would eventually use PGD in the future more than respondents who had never heard about PGD before.     

运用全染色体探针FISH技术进行罗伯逊易位携带者胚胎植入前诊断

目的运用全染色体探针荧光原位杂交技术(fluorescent in situ hybridization ,FISH)对14/21罗伯逊易位携带者的胚胎进行植入前遗传学诊断,达到优生目的.方法应用FITC、Texas标记的21/14全染色体探针对14/21罗伯逊易位携带者的胚胎活检后的单个卵裂球进行遗传学检测,选择正常信号或携带者信号核型胚胎进行移植.结果常规单精子显微注射-胚胎移植(intracytoplasmic sperm injection,ICSI)后获取胚胎12个,活检优质胚胎11个,杂交后有信号胚胎7个,其中1个正常胚胎,1个携带者胚胎;1个14单体胚胎,1个21单体胚胎,1个21三体胚胎,另2个胚胎活检后均为21号二体,无14号信号.结论运用14/21全染色体探针荧光原位杂交技术对14/21罗伯逊易位携带者的胚胎进行植入前遗传学诊断是能够将正常核型胚胎挑选出来进行移植,从而达到优生目的.     

Birth of a healthy infant following trophectoderm biopsy from blastocysts for PGD of beta-thalassaemia major

PGD is a well accepted reproductive choice for couples at genetic risk and involves the diagnosis and transfer of unaffected IVF embryos. PGD for monogenetic diseases is most commonly accomplished by the biopsy of one or two blastomeres from cleavage stage embryos, followed by PCR-based protocols. However, PCR-based DNA analysis of one or two cells is subject to several problems, including total PCR failure, or failure of one allele to amplify. Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development. The aim of this study was to develop a safe, reliable technique for the biopsy of trophectoderm cells from human blastocysts. This case report demonstrates that removal of trophectoderm cells prior to blastocyst transfer is compatible with implantation and development to term. Here we report successful PGD for beta-thalassaemia following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant.     

Preimplantation genetic diagnosis for an insertional translocation carrier

BACKGROUND: While preimplantation genetic diagnosis (PGD) is well established for carriers of reciprocal terminal translocations, reports on PGD for insertional translocation carriers are lacking. Here, we report on the PGD of an insertional translocation carrier with karyotype 46,XX,ins(14;2)(q21;q31q35). Due to the possibility of crossovers within the inserted region, rather than a single probe, four probes are required for proper embryo selection. METHODS: Probes were generated for PGD using fluorescence in situ hybridization and two PGD cycles. RESULTS: Analysis of 10 embryos revealed four embryos to be normal diploid. Two embryos were consistent with 3:1 segregation of the theoretical quadrivalent and one was consistent with 2:2 or 1:1 segregation. Furthermore, one embryo was mosaic abnormal and one remained without diagnosis. CONCLUSIONS: With increased acceptance of PGD, it is likely that more carriers of complex translocations will enter PGD programmes. The present results suggest that a careful genetic work-up of complex translocations is essential for proper embryo selection. While theoretical modelling may predict that quadrivalents will form during the meiosis of insertional translocations, experimental proof for the occurrence of quadrivalents is still lacking and more research on the meiotic process of both female and male insertional translocation carriers is warranted.     

PGD in 47,XXY Klinefelter's syndrome patients

The use of ICSI has been a major breakthrough in the treatment of male infertility. Even azoospermic patients with focal spermatogenesis in the testis, may benefit from the ICSI technique in order to father a child. As ICSI use has become more common, centres have introduced infertility treatment for Klinefelter patients. To date, 34 healthy children have been born using ICSI without PGD, and the conception of one 47,XXY fetus has been reported. In view of the possible risk of an increased gonosome number in the spermatozoa of Klinefelter patients, a safer approach--offering these couples ICSI combined with PGD--has been used, and has resulted in the birth of three healthy children. Couples in which the male suffered from Klinefelter's syndrome were first treated in 1995; these patients were offered ICSI + PGD using FISH technology, notably to enumerate the X and Y chromosomes. ICSI + PGD was performed in 32 cycles of 20 couples with spermatozoa originating from a fresh ejaculate (n = 1), testicular biopsy (n = 21) or frozen-thawed testicular biopsy (n = 10). Normal fertilization occurred in 56.0 +/- 22.4% of the successfully injected oocytes. On day 3 of development, 119 embryos from 29 cycles were of sufficient quality to undergo biopsy and subsequent PGD; a positive result was obtained in 113 embryos. Embryos were available for transfer in 26 cycles, with a mean of 1.6 +/- 0.6 embryos per transfer. Eight pregnancies were obtained, and five resulted in a delivery. A total of 113 embryos from couples with Klinefelter's syndrome was compared with 578 embryos from control couples with X-linked disease where PGD was used to determine gender. A significant fall occurred in the rate of normal embryos for couples with Klinefelter's syndrome (54.0%) compared with controls (77.2%). Moreover, a significantly increased risk of abnormalities was observed for sex chromosomes and autosomes; for each autosome separately, this reached significance level for chromosomes 18 and 21 only. Hence, a cautious approach is warranted in advising couples with non-mosaic Klinefelter's syndrome. Moreover, the use of ICSI + PGD or prenatal diagnosis should be carefully considered.     

分子生物学技术在植入前诊断中的应用

植入前诊断是产前诊断非常早的一种方法，目的是放弃携带严重遗传病的胚胎，将健康胚胎植入母体。两种主要的方法是聚合酶链反应（PCR）和荧光原位杂交（FISH）。PCR用于单基因病诊断，FISH用于染色体异常诊断。临床主要应用于存在遗传风险的患者如携带单基因病和染色体易位的患者。随着分子生物学技术的飞速发展，如比较基因组（CGH），全基因组扩增技术（WGA），引物延伸预扩增（PEP），间期核转换技术及DNA芯片技术（DNAchip）等PGD先进检测手段的应用，单细胞用于诊断单基因或多基因突变及染色体疾病，为期不远。     

双侧输精管缺如等三种不育患者CFTR基因突变筛查

目的探讨国人先天性双侧输精管缺如（CBAvD）、特发性少精子症（idiopathic oligospennia，IO）及特发性无精子症（idiopathic azoospennia，IA）患者CFFR基因有无突变，从而为患者行辅助生殖胚胎种植前是否需行常规的遗传学诊断提供依据。方法运用银染聚合酶链一单链构象多态性技术对25例CBAVD，40例IO，25例n患者周围静脉血白细胞DNA的CFTR基因的2、3、4、5、7A、8、9、10、11、12、13A、14a、14b、15B、19B、20、21、23共18个外显子进行检测。结果上述患者CFTR基因的18个外显子均未发现异常单链条带或位移。结论本组所检测的CFTR基因突变与国人精子发生尚无明确的相关性，国人在实施辅助生殖胚胎种植前是否需常规行CFFR基因突变检测有待进一步研究。     

植入前遗传学诊断"知情同意"的影响因素与对策

植入前遗传学诊断（Preimplantation Genetic Diagnosis PGD）是辅助生育技术与分子生物学技术相结合而发展的孕前遗传学诊断技术,在植入子宫前淘汰了遗传异常的胚胎,是产前诊断技术的重大进展.但是,由于技术本身存在着一定局限性和不确定性,同时,受到病人认知能力等因素的影响,由此引发了系列伦理学争议.在进行PGD前,一个明了、详尽的患者知情同意过程是必须的.包括通俗全面告知PGD有关信息、手术和检测的局限性和可能结果;充分告知通过PGD所获得的利益和风险.在此基础上针对不同的遗传病检测签署详细的书面知情同意书.