Insertion of disease-causing mutations in BACs by homologous recombination in Escherichia coli.

We have used GET Recombination, an inducible homologous recombination system for Escherichia coli, to insert one of the most common thalassaemia mutations into the intact beta-globin locus in a second generation BAC vector. We first inserted a PCR fragment carrying the tetracycline resistance gene (TetR) into the beta-globin gene. All recombinant clones examined contained the TetR gene at the correct target site. Next, a PCR fragment with the IVS I-110 G-->A splicing mutation but no selectable marker was used to replace the TetR gene in a second round of GET Recombination. Recombinant clones were selected by plating on medium containing chlorotetracycline and fusaric acid. Although counterselection for the TetR gene is not very efficient, four recombinant colonies with the IVS I-110 mutation were identified among 480 clones screened. Analysis of the recombinant clones did not show any other modifications or rearrangements. Thus the TetR gene can be used in combination with GET Recombination to introduce point mutations and other modifications in BACs without leaving behind any operational sequences, in order to generate accurate cell and transgenic mouse models for various diseases.

Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET Recombination.

GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the beta-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the beta- and delta-globin genes also allowed the simultaneous generation of a delta-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the gamma-globin genes and the termination codon of the beta-globin gene enabled the generation of reporter constructs for both (A)gamma- and (G)gamma-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the epsilon- and beta-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.

[The genetics of breast cancer, population and familial research and segregation analysis]. / Genetika raka molochnoi zhelezy, populiatsionnye i semeinye issledovaniia, segregatsionnyi analiz.

The data on clinico-genealogy studies of 1046 probands with breast cancer and their relatives are presented. The nature of inheritance corresponded to the Mendelian model. As to other families, there is no strong evidence for the monogene model both with complete and incomplete penetrance of mutant homo- and heterozygotes. Penetrance of homozygotes was 7.9-30.5%, this being 2.0-7.3% for heterozygotes. The conclusion is drawn that it is necessary to consider the regularities of inheritance of breast cancer in the light of the multifactorial model.

[The genetics of breast cancer. A genetic dispersion analysis and the genetic heterogeneity of breast cancer]. / Genetika raka molochnoi zhelezy. Genetiko-dispersionnyi analiz i geneticheskaia geterogennost' raka molochnoi zhelezy.

The multifactorial nature of breast cancer was established based on population and family study, the contribution of genetic factors being 52% (premenopausal--62 and postmenopausal--39%). Genetic heterogeneity of different coefficients of inheritance of breast cancer with the portion of common genes was shown to be 53%. The analysis of breast cancer interaction with other malignant neoplasms revealed that the development of other malignant neoplasms was the result of the influence of partially common genes. On the basis of data obtained in this study, the tables of repeated risk for the relatives have been worked out which may be used for medico-genetic consultations.

[Analysis of the association of inherited predisposition to breast cancer with c-Ha-ras-1 oncogene alleles]. / Analiz assotsiatsii nasledstvennoi predraspolozhennosti k raku molochnoi zhelezy s allel'nymi variantami c-Ha-ras-1 onkogena.

Polymorphism of the human c-Ha-ras-1 gene has been analysed in 66 BamHI restricted DNAs from blood of 35 patients with "inherited" breast cancer, 7 fibroadenoma patients, 13 healthy first-degree relatives and 11 unaffected controls. Two "common" and four "unusual" alleles were detected. The frequency of "common" (6.6 and 7.4 kb) and "unusual" (6.9 kb) alleles was identical to that in the control and unaffected groups (65.8, 17.1 and 7.1%). Rare alleles (7.6 and 7.8 kb) were only detected in breast cancer patients and in healthy first-degree relatives. A 8.0 kb allele specific for control patients was also detected. No absolute relationship between the genetic predisposition to breast cancer and the Ha-ras genotype was assumed.

[Melanoma of the skin: clinico-genetic studies]. / Melanoma kozhi: kliniko-geneticheskie issledovaniia.

As a result of clinico-genealogical analysis of data on 691 skin melanoma patients, a classification of skin melanoma was elaborated which reflected the etiologic heterogeneity of the disease. Inheritable and non-inheritable forms of skin melanoma were identified. The inheritable tumor group included familial disease (2%) and tumors developing against the background of hereditary diseases and syndromes (32.7%). The data obtained served as basis for the identification of families with high genetic predisposition to skin melanoma development and for the assessment of individual risk of the disease in patients' relatives.

[Familial-populational studies of endometrial cancer: segregation and genetic dispersion analyses]. / Semeino-populiatsionnye issledovaniia raka éndometriia: segregatsionnyi i genetiko-dispersionnyi analiz.

A clinico-genealogic investigation was carried out in 216 patients with endometrial cancer. Familial accumulation of endometrial and other cancer incidence was established. The segregation rates appeared to be lower than those expected from simple Mendelian models (2-11%). A multifactorial nature of endometrial cancer in overall susceptibility to the disease was found to be at 61%. A genetic correlation analysis showed endometrial cancer to share common genes with breast and gastric cancer in females. Tables of recurrent risk of the disease for relatives were prepared to be used in medico-genetic counseling.

[Chromosome fragile sites in patients with multiple primary tumors and familial forms of breast cancer]. / Izuchenie khromosomnoi sait-lomkosti u bol'ynkh s pervichno-mnozhestvennymi opukholiami i semeinymi formami raka molochnoi zhelezy.

An analysis was carried out of chromosomal site-fragility in patients with primary multiple tumors and familial breast cancer. A possible correlation is discussed between fragile sites, breakpoints in chromosome rearrangements in cancer patients and the localization of oncogenes mapped in chromosomal regions involved in said rearrangements.

[Clinico-genetic analysis of the incidence of neoplasms]. / Kliniko-geneticheskii analiz rasprostranennykh onkologicheskikh zabolevanii.

The authors sum up the results of clinical and genealogic examinations of 2460 patients with most prevalent tumors, i.e. gastric and mammary carcinomas, melanomas. The obtained values of segregation frequencies for these tumors have proved to be lower than the theoretically expected values for monogenic types of inheritance. A genetic and epidemiologic approach, employed in the tumors analysis, has demonstrated the multifactorial nature of these tumors: the contribution of the genetic factors in mammary carcinoma has made up 52%, in gastric carcinoma 22% for male and 41.1% for female subjects, with the X-chromosome-linked genetic components making up 19%. The studies have shown the possibility of genetic heterogeneity of the tumor forms, identically localized. Basing on these data, the authors have plotted 'repeated risk tables' to assess the potentiality of new cases of the disease in the patients' families; such tables may be useful for practical medicogenetic counselling.