Molecular prenatal diagnosis: the impact of modern technologies

Originally prenatal diagnosis was confined to the diagnosis of metabolic disorders and depended on assaying enzyme levels in amniotic fluid. With the development of recombinant DNA technology, molecular diagnosis became possible for some genetic conditions late in the 1970s. Here we briefly review the history of molecular prenatal diagnostic testing, using Duchenne muscular dystrophy as an example, and describe how over the last 30 years we have moved from offering testing to a few affected individuals using techniques, such as Southern blotting to identify deletions, to more rapid and accurate PCR-based testing which identifies the precise change in dystrophin for a greater number of families. We discuss the potential for safer, earlier prenatal genetic diagnosis using cell free fetal DNA in maternal blood before concluding by speculating on how more recent techniques, such as next generation sequencing, might further impact on the potential for molecular prenatal testing. Progress is not without its challenges, and as cytogenetics and molecular genetics begin to unite into one, we foresee the main challenge will not be in identifying the genetic change, but rather in interpreting its significance, particularly in the prenatal setting where we frequently have no phenotype on which to base interpretation. Copyright © 2010 John Wiley & Sons, Ltd.     

Parentage and sex allocation in the facultatively polygynous ant Myrmicatahoensis

Most social groups have the potential for reproductive conflict among group members. Within insect societies, reproduction can be divided among multiple fertile individuals, leading to potential conflicts between these individuals over the parentage of sexual offspring. Colonies of the facultatively polygynous ant Myrmicatahoensis contain from one to several mated queens. In this species, female sexuals were produced almost exclusively by one queen. The parentage of male sexuals was more complex. In accordance with predictions based on worker sex-allocation preferences, male-producing colonies tended to have low levels of genetic relatedness (i.e., high queen numbers). Correspondingly, males were often reared from the eggs of two or more queens in the nest. Further, over half of the males produced appeared to be the progeny of fertile workers, not of queens. Overall investment ratios were substantially more male biased than those predicted by genetic relatedness, suggesting hidden costs associated with the production of female sexuals. These costs are likely to include local resource competition among females, most notably when these individuals are adopted by their maternal nest. Received: 3 March 1998 / Accepted after revision: 20 June 1998     

Paternity and paternal care in the polygynandrous Smith's longspur

In species where females copulate with more than one male during a single breeding attempt, males risk investing in offspring that are not their own. In the polygynandrous Smith's longspur (Calcarius pictus), females copulate sequentially with one to three males for each clutch of eggs and most of these males later assist in feeding the young. Using multilocus DNA profiling, we determined that there was mixed paternity in >75% of broods (n=31) but that few offspring (<1% of 114 nestlings) were sired by males outside the polygynandrous group. Male feeding rate increased significantly with the number of young sired, with males siring four nestlings feeding the brood at double the frequency of males siring only a single nestling. However, male Smith's longspurs appear to show a graded adjustment of paternal care in response to paternity only when other males are available to compensate for reduced care: feeding rate did not vary in relation to paternity when only one male provisioned young at the nest. There was no evidence that males could recognise their own offspring within a brood and feed them preferentially. The number of offspring sired by each male was significantly correlated with the number of days spent copulating with the attending female: on average, a male sired one offspring for every 2 days of copulatory access. If males use their access to females to estimate paternity (and thereby decide on their subsequent level of parental investment), a positive relationship is expected between the amount of female access and the subsequent feeding rate to the nestlings. Nonetheless, male feeding effort was only weakly correlated with female access and more study is needed to determine how males estimate their paternity in a brood. Received: 1 June 1997 / Accepted after revision: 1 April 1998     

Genotoxicity detected in wild mice living in a highly polluted wetland area in south western Spain

A field study was carried out in the south of the Iberian Peninsula in an industrial area in the neighbourhood of Huelva city, SW Spain, and in a natural area (Do?ana National Park) for comparison, to estimate the genetic risk induced by environmental pollution in wild mice. Genotoxic effects in a sentinel organism, the Algerian mice (Mus spretus) free living in the industrial area were compared with animals of the same species living in the natural protected area. The single cell gel electrophoresis, or Comet assay, was performed as a genotoxicity test in peripheral blood of mice. Our results clearly show that mice free living in the contaminated area bear a high burden of genetic damage as compared with control individuals. The results suggest that the assessing of genotoxicity levels by the Comet assay in wild mice can be used as a valuable test in pollution monitoring and environmental conservation.     

芹菜素对丙烯腈引起大鼠精子脂质过氧化和DNA损伤的影响

分析芹菜素(apigenin,AP)对丙烯腈(acrylonitrile,ACN)引起的大鼠精子脂质过氧化和DNA损伤的影响,并探讨其可能的机制。将50只SPF级SD成年雄性大鼠随机分为阴性对照组(玉米油)、ACN组(50 mg·kg~(-1)ACN)、低AP组(50 mg·kg~(-1)ACN+234 mg·kg~(-1)AP)、高AP组(50 mg·kg~(-1)ACN+468 mg·kg~(-1)AP)、N-乙酰半胱氨酸(N-acetylcysteine,NAC)组(50 mg·kg~(-1)ACN+300mg·kg~(-1)NAC),以5 m L·(kg bw)~(-1)灌胃染毒,1次·d~(-1),6 d·周~(-1),连续13周。检测大鼠精子活性氧(ROS)、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性以及精子DNA损伤情况。结果发现,ACN组、低AP组、高AP组、NAC组精子ROS、MDA含量显著升高,SOD活力显著降低,精子尾部DNA含量百分比、尾长、尾距、Olive尾距均显著增高于对照组(均P0.05);而低AP组、高AP组、NAC组精子ROS、MDA含量、SOD活性和精子DNA损伤情况与ACN组相比差异均无统计学意义(P0.05)。提示ACN可引起大鼠精子脂质过氧化和DNA损伤,而AP、NAC对其无干预作用。     

检测细胞DNA断裂损伤效应的彗星实验法的改良

为了解决彗星实验过程中常出现的脱胶、细胞核分离操作繁琐、重复性低等问题,对彗星实验方法进行了改良,初步建立了彗星实验的快速操作流程。结果显示,通过对载玻片进行预处理,可确保凝胶悬挂均匀;采用改良机械法分离的细胞核浓度适中;以0.5%(w/v)涂层琼脂糖作为基层、以1.5%(w/v)低熔点包埋琼脂糖作为叠加层的"双层凝胶法",辅以"推片法"铺胶,操作便捷且不发生脱胶现象;细胞核膜经裂解处理后再进行电泳和荧光观察,彗星图像清晰,杂质少。应用改良后的彗星实验方法,操作简便,耗时更短,实验效果良好,可快速检测出细胞DNA损伤效应。     

植物活性物质对PM_(2.5)致细胞损伤的抗性作用

为研究植物活性物质对PM2.5致细胞损伤的抗性作用,研究了北京城区PM2.5对CHO(Chinese hamster ovary,中国仓鼠卵巢)细胞的损伤作用,并选择植物活性物质绿原酸、阿魏酸和荭草素探讨其对CHO细胞的保护作用.以CCK-8法确定PM2.5的试验剂量和3种活性物质的最佳保护剂量,然后通过彗星试验、微核试验、流式细胞术及Western Blotting分别检测PM2.5的损伤作用及植物活性物质的保护作用.结果表明:1由CHO细胞存活率可知,PM2.5的有效试验剂量为15μg/m L,绿原酸、阿魏酸和荭草素的最佳保护剂量分别为50、20、10μmol/L.2PM2.5可引起CHO细胞明显的DNA损伤、微核形成增加、细胞周期阻滞所致的细胞增殖指数降低,而经阿魏酸、绿原酸和荭草素预处理后,均可减轻PM2.5对细胞的损伤作用,起到了一定的保护作用.3PM2.5能引起CHO细胞中p53和caspase-3蛋白的表达增加,而阿魏酸、绿原酸和荭草素均可使其表达量有所降低.由PM2.5引起的细胞损伤经绿原酸、阿魏酸、荭草素作用后表达量降低.在3种植物活性物质中,以阿魏酸拮抗PM2.5损伤的效果最佳,绿原酸次之,荭草素相对较差.     

Cytogenotoxicity and histopathological assessment of Lekki Lagoon and Ogun River in Synodontis clarias (Linnaeus, 1758)

The cytogenotoxicity and histopathological alterations induced by xenobiotics in Lekki Lagoon and Ogun River on Synodontis clarias were investigated. Fish from these water bodies and a fish farm (control) were examined for micronucleated, binucleated, and immature erythrocytes in both gill and peripheral blood. Also gill, liver, kidney, and ovary were processed for histopathology using hematoxylin-eosin staining. Concentrations of cadmium, zinc, lead and copper in the water were determined. There was significant (p < 0.05) increase in micronucleated, binucleated, and immature erythrocytes in both gill and peripheral blood of S. clarias from the lagoon and river compared to the reference site. Loss and disorganization of the primary and secondary lamellae, multifocal degeneration, hemorrhages, cellular infiltration, congestions, vacuolations, atresia, and necrosis were common lesions in the examined tissues of fish from the lagoon and river. Cd, Zn, Pb, and Cu in water samples from the lagoon and river were higher than the reference site. Xenobiotics in Lekki Lagoon and Ogun River, mostly metals, induced deoxyribonucleic acid (DNA) and pathological damage in S. clarias.