Manipulating the mammalian genome by homologous recombination.

Gene targeting in mammalian cells has proven invaluable in biotechnology, in studies of gene structure and function, and in understanding chromosome dynamics. It also offers a potential tool for gene-therapeutic applications. Two limitations constrain the current technology: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Here we consider possible ways to overcome these limitations within the framework of our present understanding of recombination mechanisms and machinery. Several studies suggest that transient alteration of the levels of recombination proteins, by overexpression or interference with expression, may be able to increase homologous recombination or decrease random integration, and we present a list of candidate genes. We consider potentially beneficial modifications to the vector DNA and discuss the effects of methods of DNA delivery on targeting efficiency. Finally, we present work showing that gene-specific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies--chimeric nucleases and triplex-forming oligonucleotides--for stimulating recombination in cells.

Blocking transcription of the human rhodopsin gene by triplex-mediated DNA photocrosslinking.

To explore the ability of triplex-forming oligodeoxyribonucleotides (TFOs) to inhibit genes responsible for dominant genetic disorders, we used two TFOs to block expression of the human rhodopsin gene, which encodes a G protein-coupled receptor involved in the blinding disorder autosomal dominant retinitis pigmentosa. Psoralen-modified TFOs and UVA irradiation were used to form photoadducts at two target sites in a plasmid expressing a rhodopsin-EGFP fusion, which was then transfected into HT1080 cells. Each TFO reduced rhodopsin-GFP expression by 70-80%, whereas treatment with both reduced expression by 90%. Expression levels of control genes on either the same plasmid or one co-transfected were not affected by the treatment. Mutations at one TFO target eliminated its effect on transcription, without diminishing inhibition by the other TFO. Northern blots indicated that TFO-directed psoralen photoadducts blocked progression of RNA polymerase, resulting in truncated transcripts. Inhibition of gene expression was not relieved over a 72 h period, suggesting that TFO-induced psoralen lesions are not repaired on this time scale. Irradiation of cells after transfection with plasmid and psoralen-TFOs produced photoadducts inside the cells and also inhibited expression of rhodopsin-EGFP. We conclude that directing DNA damage with psoralen-TFOs is an efficient and specific means for blocking transcription from the human rhodopsin gene.

Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells.

Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1(-) cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3' tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3' tails after treatment with the rare-cutting endonuclease I-SCE:I. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3' tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3' tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SCE:I-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SCE:I-induced double-strand breaks.

[Intrauterine management of Rh-alloimmunization]. / Az Rh-alloimmunizáció intrauterin kezelése.

The authors report the strategy of invasive management of Rh alloimmunisation in pregnancy. From the 34 pregnancies 6 were monitored by amniocenteses, 11 by fetal blood sampling, and 4 with combination of the two above mentioned diagnostic procedures. In 13 cases the fetuses were treated with intrauterine intravascular blood transfusions. All the procedures were ultrasound guided. The fetal blood sampling and the transfusions were carried out by puncturing the umbilical vein or artery. For transfusions, maternal blood was used in case of identical blood type, otherwise adult Rh negative, filtered, washed, irradiated blood was transfused. They report the complications as well, giving the cause of their fetal losses in details. There were no maternal complications observed. Out of the 34 pregnant women 25 had healthy newborns, which number is acceptable in this disease with a very high mortality rate. The authors underline that the technique of fetal blood sampling and intrauterine transfusion if needed is necessary in the management of Rh alloimmunised pregnancies.

What proportion of congenital abnormalities can be prevented?

OBJECTIVE: To estimate the proportion of preventable congenital abnormalities in Hungary. DESIGN: Analysis of available Hungarian data-bases and of the effectiveness of primary, secondary, and tertiary preventive methods. SETTING: Databases of ad hoc epidemiological studies and of the Hungarian congenital abnormality registry. MAIN OUTCOME MEASURES: Prevalence at birth and prevalence after prevention in 73 congenital abnormality types or groups. RESULTS: Preventive methods are available for 51 (70%) of the 73 congenital abnormality types or groups evaluated. The birth prevalence of all congenital abnormalities could be reduced from 65 to 26 per 1000; thus 39 per 1000 (60%) are preventable. Without congenital dislocation of the hip, which is unusually common in Hungary, the preventable proportion of congenital abnormalities is 52%. CONCLUSION: Many congenital abnormalities can be prevented, but as they do not represent a single pathological category there is no single strategy for their prevention.

[Results of prenatal cytogenetic screening at the Prenatal Genetic Center of the Postgraduate Medical University between 1980-1990]. / Az Orvostovábbképzö Egyetem Prenatális Genetikai Központjának eredményei a terhesség alatti citogenetikai szürés terén 1980 és 1990 között.

Evaluation of prenatal cytogenetic diagnosis by Genetic Center of Postgraduate Medical University in 1980 and 1990. Between 1980 and 1990, 1039 amniocenteses (AC), 1263 chorionic villus samples (CVS), and 30 fetal blood sampling were performed for cytogenetic reasons. The rate of chromosome abnormalities were 5.5 per cent in the first trimester CVS, 5.2 per cent in the second trimester CVS, and 3.1 per cent in AC. The Down syndrome was the most frequent abnormality (46 fetuses) and the next was the Edwards syndrome (15 cases). It was established that though the case number is fourteen times more than the beginning of this decade, this was enough only for screening women 39 or over. During this period several new methods were introduced making possible the diagnosis from 9th week of pregnancy until term. Among these methods the CVS has not only become an alternative to the AC but now it is the most frequent procedure in our laboratory. Though most pregnants are still referred for prenatal cytogenetic investigation because of their advanced age, the authors search for other risk factors which would make possible screening in younger women, too.

[Transabdominal chorion aspiration in the second pregnancy trimester]. / Transabdominalis chorion-aspiráció a terhesség második trimesterében.

The authors report their experiences with 377 transabdominal chorionic villi samplings performed in the second trimester of pregnancy, between 1987-1989. They used the double needle technique with continuous ultrasound guidance. In every case they could get a sufficient amount of villi from one puncture, and there was no unsuccessful direct chromosome-preparation. The obstetrical complications of the procedure were measured by the analysis of the outcome of the first 300 pregnancies intended to continue: the abortion rate after the transabdominal chorionic villi sampling seems to be lower, than after amniocentesis.

Prenatal cytogenetic study of translocation carriers.

A total of 37 prenatal diagnoses were analysed: 10 observations in which one of the parents carried a Robertsonian translocation and 27 observations in which one a reciprocal translocation was carried by one of the parents. The segregations of the inherited chromosome structural rearrangements were analysed in relation to the methods of ascertainment of the anomaly in the family, and the types of rearrangement. The mode of ascertainment proved to be a very useful indicator of the risk: those cases ascertained through abnormal livebirths had a 44% risk in our series, but there was no unbalanced fetus in the group ascertained through recurrent abortions.

[Fetal diagnosis of Edwards syndrome]. / Az Edwards-syndroma fetális diagnosztikája.

Eight cases of Edward's syndrome were found prenatally by cytogenetical analysis of 1680 pregnant women. It has been estimated that after Down's syndrome Edwards's syndrome is the most frequently encountered chromosomal abnormality. This syndrome is associated with high rate of anomalies detectable by ultrasound (e.g. omphalocele, polyhydramnion, growth retardation). Here it is discussed in relation with sonographical findings related to Edwards's syndrome and representing clear indications for chromosomal analysis. The authors call attention to the importance of the diagnosis of Edward's syndrome at each gestational age.

[Prenatal diagnosis of thoracopagus in the 19th week of pregnancy]. / Pränatale Diagnostik eines Thoracopagus in der 19. Schwangerschaftswoche.

A pair of conjoined twins (thoracopagus) was detected in the 19th week of pregnancy, by ultrasound. Serumalphafetoprotein was normal. Since the fetuses had only one heart, the pregnancy was terminated. The embryopathological investigation confirmed the prenatal diagnosis. Authors recommend the ultrasound screening of all pregnant women between the 16-20th week of gestation. This case is a good example of progressive care in the field of prenatal genetics.

[Ultrasound in the prenatal diagnosis of heart and lung abnormalities--Down's syndrome]. / Mittels Ultraschall pränatal festgestellte Herzentwicklungsanomalie--Down-Syndrom.

The authors describe a 21 year old gravida 2, para 2 patient in whom cardiac malformation (endocardial cushion defect) and secondary non-immune hydrops fetalis were detected by ultrasound in the 27th week of pregnancy. Because of the serious fetal demise premature delivery was induced. The on the basis of the prenatal ultrasound scan suspected Down-syndrome was proved by karyotyping from the amniotic fluid and fetal fibroblasts.

[Aspiration of chorionic tissue: diagnostic potential in prenatal (fetal) diagnosis]. / Aspiration des Choriongewebes: Eine neue Möglichkeit in der pränatalen (fetalen) Diagnostik.

The authors have investigated the possibilities of obtaining chorionic villi from legal pregnancy termination patients between the 7th and 11th week and the method of direct chromosome preparation from the villi as well. According to their investigations under continuous "real time" ultrasound guidance and with the immediate microscopic checking of the obtained material it is possible to receive chorionic villi of necessary quantity in nearly 100% of the cases, and after 2 or 3 hours the evaluation in a great deal of mitosis of good quality can be carried out. The method may revolutionize the prenatal (fetal) diagnosis, but its routine application may not be proposed until the rate of fetal risk is exactly known.