Fetal nuchal translucency: ultrasound screening for chromosomal defects in first trimester of pregnancy.

OBJECTIVE--To examine the significance of fetal nuchal translucency at 10-14 weeks'' gestation in the prediction of abnormal fetal karyotype. DESIGN--Prospective screening study. SETTING--The Harris Birthright Research Centre for Fetal Medicine, King''s College Hospital, London. SUBJECTS--827 fetuses undergoing first trimester karyotyping by amniocentesis or chorionic villus sampling. MAIN OUTCOME MEASURE--Incidence of chromosomal defects. RESULTS--The incidence of chromosomal defects was 3% (28 of 827 cases). In the 51 (6%) fetuses with nuchal translucency 3-8 mm thick the incidence of chromosomal defects was 35% (18 cases). In contrast, only 10 of the remaining 776 (1%) fetuses were chromosomally abnormal. CONCLUSION--Fetal nuchal translucency > or = 3 mm is a useful first trimester marker for fetal chromosomal abnormalities.     

Fetal nuchal translucency thickness

In the early 1990s Nicolaides introduced screening for trisomy 21 by fetal nuchal translucency thickness measurement with ultrasound between 11-13(+6) weeks. Already in 1866 L. Down noted that common features of patients with trisomy 21 are a skin being too large for the body and a flat face with a small nose. While detection rates for trisomy 21, given an invasive testing rate of 5%, were only 30% for screening by maternal age and 65% for screening by maternal serum triple test, the detection rate for screening by nuchal translucency combined with maternal age was 75% and this could be increased to 90% in combination with maternal serum screening (serum B-human chorionic gonadotropin and pregnancy-associated plasma protein-A) at 11-13(+6) weeks. The additional soft markers in the first trimester are the fetal nasal bone, the Doppler velocity waveform in the ductus venosus and tricuspid regurgitation and these markers can be used to further increase the detection rate of trisomy 21. In addition increased nuchal translucency thickness can also identify other chromosomal defects (mainly trisomy 13 and 18 and monosomy X) and major congenital malformations (mainly cardiac defects) and genetic syndromes.     

Interphase M-FISH applications using commercial probes in prenatal and PGD diagnostics

Early, rapid and reliable diagnosis is of first priority in prenatal medicine. The combination of specific sonographic markers (e.g. nuchal translucency) and biochemical parameters in maternal serum (e.g. free beta-human chorionic gonadotropin, pregnancy-associated plasma protein A), has already dramatically improved the sensitivity of non-invasive first trimester risk screening in pregnancy. In invasive prenatal diagnosis, in addition to well-established chorionic villi short-term culture, interphase multi-colour-fluorescence in situ hybridisation (M-FISH) on uncultured amnion cells has become a reliable tool for the rapid detection of fetal aneuploidies. Interphase M-FISH applications have enabled the diagnosis of selected chromosomal abnormalities in single cells and, therefore, have also become an important diagnostic tool for preimplantation diagnosis (PGD). The development of commercially available probe sets, in particular, has led to a broad use of interphase M-FISH in prenatal and PGD diagnosis.     

Anticipating twin-twin transfusion syndrome in monochorionic twin pregnancy. Is there a role for nuchal translucency and ductus venosus blood flow evaluation at 11-14 weeks?

Twin-twin transfusion syndrome is a major complication of monochorionic twin pregnancies. In foetuses from monochorionic twinning the presence of increased nuchal translucency thickness (NT) has been associated with an increased risk of developing this syndrome. One of the presumed mechanisms of increased NT is early cardiac failure, indirectly indicated by abnormal blood flow in the ductus venosus. We present eleven cases of monochorionic twin pregnancies in which nuchal translucency thickness and ductus venosus blood flow evaluation was performed at 11-14 weeks. In the two cases presenting with nuchal translucency discrepancy between the two foetuses along with anomalous ductus venosus blood flow in the foetus with increased nuchal translucency, twin-twin transfusion syndrome (TTTS) eventually developed. In none of the twins displaying no inter-twin difference in NT measurements and in those with discrepant NT but normal flow in both ductus venosus, was the progression to TTTS observed. In the two cases which developed TTTS, foetoscopic laser coagulation of the vascular anastomosis was successfully carried out at 18 weeks and normalisation of the venous return was registered. These findings suggest that the association of increased NT and abnormal flow in the ductus venosus in monochorionic twins may be an early manifestation of haemodynamic imbalance between the donor and the recipient eventually manifested as twin-twin transfusion syndrome. Further studies, however, are necessary to establish the potential role of the combination of NT and ductus venosus blood flow assessment as a screening method for TTTS.     

Ultrasound diagnostic schema for the determination of increased risk for chromosomal fetal aneuploidies in the first half of pregnancy

The aim of the study was to develop an early ultrasound diagnostic schema for the determination of increased risk for fetal chromosomal aneuploidies. The study was conducted on a population of 1318 pregnant women divided into 2 groups: 1255 women with the normal course of pregnancy and 63 women with diagnosed fetal abnormalities. There were 34 cases of chromosomal abnormalities (trisomy 21, 18, 13; triploidy; unbalanced inversion 9; deletion 16) and 29 cases of structural malformations. The estimation of the range of normal values was performed for the nuchal translucency (NT) measurement between 11 and 13 weeks and the nasal bone length (NB) measurement between 12 and 20 week. The results obtained in the collective set of normal pregnancies constituted the basis for the calculation of the range of normal values. The measurements of NB and NT showed a linear value increase with the pregnancy course. The following test characteristics (correlation to CRL) were recorded: NB - sensitivity 60%, specificity 98%, positive predictive value (PPV+) 43%, negative predictive value (NPV-) 98.9%. For the assumption that the test outcome means the presence or absence of the nasal bone in the ultrasound scan the sensitivity was 40%, but specificity 100%; NT - sensitivity 63.6%, specificity 98.2%, PPV+ 38.9%, NPV - 98.2%; NT + NB - presents similar characteristic to the NB or NT alone - sensitivity 55.6%, specificity 98.6%, PPV+ 50%, NPV - 98.9%. The following test characteristics for chromosomal aberration markers (correlation to BPD) were observed: NB - sensitivity 68.4%, specificity 97.4%, PPV+ 56.5%, NPV - 98.4%; NT - sensitivity 73.9%, specificity 97.9%, PPV+ 54.8%, NPV- 99.2%; NT + NB - sensitivity 94.7%, specificity 98.9%, PPV+ 90%, NPV - 99.7%, respectively. The "genetic sonogram" protocol for the structural defect detection was analysed: sensitivity was 80%, specificity 100%, PPV+ 100%, NPV - 99.7%. It is concluded that the new biometric parameter--nasal bone length (NB) and the corrected one--nuchal translucency thickness (NT) are useful markers for fetal abnormalities, especially for chromosomal aberrations. High predictive values of the diagnostic schema for the detection of aneuploidies and structural defects indicate that its application in correlation with the biparietal diameter (BPD) is highly recommended. The proposed schema is an effective algorithm for prenatal diagnostics characterised by high prognostic values. The possible introduction of the schema could result in a decrease of the invasive procedure rates, which could minimise the rate of miscarriages as a complication of amniocenteses.     

The characteristics of combined genetic screening of the first trimester of pregnancy after use of assisted reproductive technology programs

Results of comparison of nuchal translucency measurments (NT) and parameters of the biochemical analysis of blood serum of pregnant women in the first trimester and after use of assisted reproductive technologies (in vitro fertilization and cytoplasmic sperm injection (ICSI)) are submitted. It was shown that NT does not differ from those of women with spontaneous pregnancy, and parameters of biochemical markers tend only to change. After ICSI use concentration of free beta-HCG authentically increases.     

Ultrasound markers of genetic pathology and early hemodynamic changes in human embryo

An increase in the nuchal translucency (NT) by more than 3 mm and ductus venosus reverse blood flow (DVRBF) in pregnancy weeks 11–14 are used as ultrasound markers (UMs) to determine the risk of genetic pathology and/or high probability of heart defects or severe maternal preeclampsia. A pathophysiological explanation for this phenomenon is the development of transient heart failure and increased afterload on the left myocardial ventricle in the embryo in this period. Earlier, it was shown that an increased left ventricular isovolumic relaxation time (LV IRT) reflects an increased systolic arterial pressure in fetal developmental delay. The mean LV IRT measured during an ultrasound study of 122 normally developing human embryos was 30 ± 2 ms. In 27 embryos with an increased NT and DVRBF, the mean LV IRT was 41 ± 3 ms (p < 0.01). This difference confirms the probable increased afterload on human embryo heart in this period of development.     

Isolation of Deficiencies in the Arabidopsis Genome by γ-Irradiation of Pollen

Chromosomal deficiencies are a useful genetic tool in fine-scale genetic mapping and the integration of physical and visible marker genetic maps. Viable overlapping deficiencies may permit gene cloning by subtractive procedures and provide a means of analyzing the functional importance of different chromosomal regions. A method is described for isolation of deficiencies in the Arabidopsis genome which encompass specific loci and other extended chromosomal regions. The technique employs pollen mutagenized by γ-irradiation to pollinate marker lines homozygous for recessive mutations. Deficiencies at specific loci were detected by screening for marker phenotypes in the F(1). Screening for lethal mutations in the F(1)/F(2) confirmed specific deficiencies and revealed other deficiencies that did not overlap the marker loci. Further evidence for such mutations was provided by distorted F(2) segregation of the chromosomal markers linked to putative deficiencies. Maintainable (transmissible) and non-transmissible deficiencies were demonstrated by their pattern of inheritance in subsequent generations.     

Plasmid RP4, with Escherichia coli DNA inserted in vitro, mediates chromosomal transfer.

The wide host-range plasmid RP4 is generally poor at mobilizing chromosomal markers. Insertion in vitro of Escherichia coli chromosomal DNA into its HindIII site (recognized by loss of its kanamycin resistance marker) or its EcoRI site generated RP4-primes that efficiently mobilized chromosomal markers. These RP4-primes behaved analogously to F-primes, each had a particular transfer origin and direction of marker mobilization, and this was abolished by a recA allele in the donor. Insertion sequences on the RP4-primes do not appear to be necessary for mobilization, it seems very unlikely that each of the RP4-primes generated contained an insertion sequence. The construction and use of such plasmids should facilitate inter- and intrageneric genetic analyses and manipulations.     

Genetic Mapping in Escherichia coli K-12 by Radiation-Induced Crossing-Over

Conventional methods for chromosomal mapping in Escherichia coli are (i) interruption of matings to obtain minimum marker entry times, (ii) linkage analysis of recombinants, and (iii) cotransduction. Method (i) has a resolution of about 0.5 min (5 x 10(4) nucleotides) and is not useful for distances less than about 1 min; methods (ii) and (iii) are capable of better resolution but are generally not very reproducible and no general theory is available for translating crossing-over and cotransduction frequencies into physical chromosomal distances. We found that when merozygotes are irradiated (X rays or ultraviolet light) soon after marker transfer, high linkage values (0.8 to 1.0) between nearby marker pairs decrease with radiation dose to 0.5. Our results are quantitatively consistent with the idea that radiations induce crossing-over lesions proportional to dose, and the number of such lesions between two markers is proportional to the physical separation of the markers in the range that can also be measured by interruption of mating (0.5 to 4.0 min). Additivity relations among markers are also satisfied. We used this technique to measure the distances (0.1 to 1.0 min) between several pairs of closely linked markers.     

Mapping Approaches to Gene Identification in Humans

Although a number of human genes that cause disease have been traced through the defective product, most genetic defects are recognized only by phenotype. When the biochemical defect is unknown, a gene can be located only through molecular approaches based on coinheritance (genetic linkage) of the disease phenotype with a particular allele of a polymorphic DNA marker that has already been mapped to a specific chromosomal region. Linkage studies in affected families have already localized genes for several important diseases, including cystic fibrosis. Finding a genetic linkage in families in which a disease segregates requires that the human genetic map have a large number of polymorphic markers; when the map is dense enough, any disease gene can be located by linkage to a known marker. Many DNA segments with a high degree of polymorphism are being found and mapped as markers in normal reference pedigrees. Genetic linkage mapping has implications even broader than its application to prenatal diagnosis or therapeutic strategy; analyzing mutations in important genes will illuminate basic mechanisms in molecular biology and the early events that lead to cancer and other disorders.     

Factors Affecting Competence for Transformation in Staphylococcus aureus

A chemically defined medium has been developed for isolation of amino acid-requiring mutants of Staphylococcus aureus strain 8325, and for use as a selective medium in transformation assays. Variables affecting transformation of both plasmid and chromosomal markers have been studied. The optimal pH and temperature for transformation are 6.75 to 7.0 and 30 C, respectively. Ca ions are required for transformation, and only cells lysogenic for the phage phi11 can be transformed. Superinfection of competent cells with phi11 does not increase the transformation frequency. Maximal number of transformants is obtained after 20 min of contact between cells and deoxyribonucleic acid. The transformation frequencies for the plasmid marker erythromycin resistance (ero) and the chromosomal markers trp, thy, and cyt are of the same order of magnitude, whereas the frequency for the chromosomal marker tyr is approximately one order of magnitude lower.     

Linkage maps of human chromosomes

Finding the chromosomal location of human genes that heretofore have been defined solely by phenotypes, in particular clinical phenotypes that are transmitted in Mendelian fashion in families, is an early and often crucial step in the process of identifying the molecular basis of a disease. Recent progress in construction of chromosomal maps of genetically linked DNA markers has made almost the entire human genome accessible to linkage studies in families that are segregating genetic defects. Construction of linkage maps requires a panel of three-generation families for genotyping, a large number of polymorphic markers, and sophisticated computer programs for analysis of genotypic data. After a locus harboring a deleterious mutation has been identified by linkage to a mapped marker, a high-resolution map of the region can be constructed with new markers derived from cosmid libraries, to narrow the search for the gene in question. For example, this strategy has been pursued in the effort to characterize the gene responsible for familial adenomatous polyposis. When a target region has been narrowed to about 1 centiMorgan, corresponding to roughly a million base pairs in physical distance, other techniques of molecular biology can be brought to bear to isolate and clone the actual gene.     

Towards a better recording of microtubule cytoskeletal spatial organization and dynamics in plant cells

Numerous fluorescent marker lines are currently available to visualize microtubule(MT)architecture and dynamics in living plant cells, such as markers expressing p35S::GFP-MBD or p35S::GFP-TUB6.However, these MT marker lines display obvious defects that affect plant growth or produce unstable fluorescent signals. Here, a series of new marker lines were developed, including the pTUB6::VisGreen-TUB6-expressing line in which TUB6 is under the control of its endogenous regulatory elements and e GFP is replaced with VisGreen, a brighter fluorescent protein. Moreover, two different markers were combined into one expression vector and developed two dual-marker lines.These marker lines produce bright, stable fluorescent signals in various tissues, and greatly shorten the screening process for generating dual-marker lines.These new marker lines provide a novel resource for MT research.     

Comparative genomics, marker density and statistical analysis of chromosome rearrangements

Estimates of the number of chromosomal breakpoints that have arisen (e.g., by translocation and inversion) in the evolutionary past between two species and their common ancestor can be made by comparing map positions of marker loci. Statistical methods for doing so are based on a random-breakage model of chromosomal rearrangement. The model treats all modes of chromosome rearrangement alike, and it assumes that chromosome boundaries and breakpoints are distributed randomly along a single genomic interval. Here we use simulation and numerical analysis to test the validity of these model assumptions. Mean estimates of numbers of breakpoints are close to those expected under the random-breakage model when marker density is high relative to the amount of chromosomal rearrangement and when rearrangements occur by translocation alone. But when marker density is low relative to the number of chromosomes, and when rearrangements occur by both translocation and inversion, the number of breakpoints is underestimated. The underestimate arises because rearranged segments may contain markers, yet the rearranged segments may, nevertheless, be undetected. Variances of the estimate of numbers of breakpoints decrease rapidly as markers are added to the comparative maps, but are less influenced by the number or type of chromosomal rearrangement separating the species. Variances obtained with simulated genomes comprised of chromosomes of equal length are substantially lower than those obtained when chromosome size is unconstrained. Statistical power for detecting heterogeneity in the rate of chromosomal rearrangement is also investigated. Results are interpreted with respect to the amount of marker information required to make accurate inferences about chromosomal evolution.     

颈项透明层厚度超声联合无创DNA对孕妇胎儿染色体非整倍体异常诊断效能的影响

摘要 目的：探讨颈项透明层(nuchal translucency,NT)厚度超声联合无创DNA对孕妇胎儿染色体非整倍体异常诊断效能的影响。方法：2018年7月到2020年4月选择在本院进行产前筛查的孕妇120例,所有孕妇都给予NT厚度超声联合无创DNA检查,采用羊水穿刺分析检测结果为阳性的胎儿情况。结果：120例胎儿的NT厚度为0.8~10 mm,平均厚度为1.57±0.41 mm;不同孕妇年龄的NT厚度对比差异无统计学意义(P>0.05)。以羊水穿刺检测结果为金标准,120例胎儿中检出染色体非整倍体异常7例,NT超声检出12例,无创DNA检出13例,联合检出14例。NT超声、无创DNA与联合诊断的染色体非整倍体异常敏感性为57.1%、85.7%和100.0%,特异性为92.9%、93.8%和93.8%。检测结果为阳性的14例胎儿中,还包括3例淋巴水囊瘤,2例单脐动脉伴胎儿宫内发育迟缓,1例胎儿双肾畸形,1例胎儿并腿畸形。结论：颈项透明层厚度超声联合无创DNA在孕妇胎儿染色体非整倍体异常中的诊断具有操作简便、无创伤等特点,诊断敏感性与特异性都比较高,可对临床医生遗传咨询有一定的参考价值。     

Prenatal diagnosis of Pallister-Killian syndrome and literature review

Pallister-Killian syndrome (PKS) is a rare sporadic genetic disorder usually caused by mosaicism of an extra isochromosome of 12p (i(12p)). This retrospective study analysed the prenatal ultrasound manifestations and molecular and cytogenetic results of five PKS foetuses. Samples of amniotic fluid and/or cord blood, skin biopsy and placenta were collected. Conventional karyotyping and single nucleotide polymorphism array (SNP array) were performed on all the amniotic fluid or cord blood samples. Copy number variants sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) were also used for the validation for one foetus. All the five foetuses were from pregnancies with advanced parental age. Two foetuses involved structural abnormalities and one foetus had only soft markers, all of which included increased nuchal translucency. The rest two foetuses had normal ultrasounds in the second trimester, which has rarely been reported before. The karyotype revealed typical i(12p) in four cases and a small supernumerary marker chromosome consisting of 12p and 20p in the remaining one case. The proportion of cells with i(12p) ranged from 0 to 100% in cultural cells, while SNP array results suggested 2−4 copies of 12p. For one foetus, metaphase FISH showed normal results, but the interphase FISH suggested cell lines with two, three and four copies of 12p in the amniotic fluid. Advanced parental age may be an important risk factor for PKS, and there were no typical ultrasound manifestations related to PKS. A combination of karyotype analysis and molecular diagnosis is an effective method for the diagnosis of PKS.     

Primary autosomal recessive microcephaly: MCPH5 maps to 1q25-q32

Primary microcephaly is thought to result from genetic defects of the developmental program that generates large brain hemispheres in humans. Autosomal recessive inheritance is likely in most familial cases, and four loci were recently mapped by homozygosity. We report homozygosity mapping of a new locus, MCPH5, with a maximum multipoint LOD score of 3.51 at marker D1S1723, in a family of Turkish origin. The minimal critical region spans 11.4 cM between markers D1S384 and D1S2655, at 1q25-q32, and encompasses the cytogenetic breakpoints of chromosomal aberrations previously reported in unrelated patients with microcephaly.     

Exchange of genetic markers at extremely high temperatures in the archaeon Sulfolobus acidocaldarius.

When cells of two auxotrophic mutants of Sulfolobus acidocaldarius are mixed and incubated on solid medium, they form stable genetic recombinants which can be selected, enumerated, and characterized. Any of a variety of auxotrophic markers can recombine with each other, and the phenomenon has been observed at temperatures of up to 84 degrees C. The ability to exchange and recombine chromosomal markers appears to be an intrinsic property of S. acidocaldarius strains. It occurs between two cell lines derived from the same parent or from different parents and also between a recombinant and its parent. This is the first observation of chromosomal marker exchange in archaea from geothermal environments and provides the first functional evidence of generalized, homologous recombination at such high temperatures.     

Development of new methods in human gene mapping: selection for fragments of the human Y chromosome after chromosome-mediated gene transfer.

Chromosome-mediated gene transfer (CMGT) can be used to generate fragments of human chromosomes and chromosomal maps can be constructed using these fragments. In previous experiments CMGT techniques have been limited to those regions of the genome which encode biochemically selectable markers. We have extended the regions of the human genome which can be subjected to CMGT methods by employing a cell surface antigen as a selectable marker. These experiments have been facilitated by the discovery that co-transformation of chromosomes with a plasmid bearing a biochemically selectable marker followed by selection for the marker pre-selects for cells which have incorporated chromosomal fragments. The plasmid may also integrate into the donor chromosomes and this provides, in some cases, an additional selectable marker in the chromosome fragment of interest. Using these methods we have isolated for the first time cells containing varying portions of the human Y chromosome.