Localization of the mcf.2 transforming sequence to the X chromosome.

A transforming sequence was identified using co-transfection of DNA from the human mammary carcinoma cell line MCF-7 and of a G418 resistance gene into NIH 3T3 cells, followed by tumor formation in athymic mice. This sequence, named mcf.2, was molecularly cloned. A transforming activity resides in a cosmid clone of 42 kb. mcf.2 did not cross-hybridize with the known oncogenes tested. In situ hybridization localized it on the X chromosome, probably at q27. This localization was confirmed by hybridization to a panel of human--rodent cell line DNAs.     

Carrier detection in haemophilia B using two further intragenic restriction fragment length polymorphisms.

A normal human population has been screened for the existence of further restriction fragment length polymorphisms (RFLPs) in the clotting factor IX gene in addition to the TaqI polymorphism already characterised (1,2). Two polymorphic loci were found, both within 6 Kb of the TaqI polymorphism within the body of the factor IX gene. One of the polymorphisms has been shown to be due to either the presence or absence of a particular recognition site for the restriction enzyme XmnI. The other, visualised as a difference in fragment pattern produced by digestion with either HinfI or DdeI, has two allelic forms differing by a 50 bp element of inserted DNA. Sequence analysis has shown the inserted element to be in a region of Z type DNA sequence, the insertion representing a duplication of flanking sequence on either side. The two polymorphisms are inherited in simple Mendelian fashion and have both been used to diagnose haemophilia B carrier status. It is estimated that the combined use of these polymorphisms in the factor IX gene, despite linkage disequilibrium between the 3 polymorphic loci, should enable carrier status to be determined in approximately 66% of all haemophilia B families.     

Characterization of a common deletion polymorphism of the UGT2B17 gene linked to UGT2B15

Members of the human UDP-glucuronosyltransferase 2B family are located in a cluster on chromosome 4q13 and code for enzymes whose gene products are responsible for the normal catabolism of steroid hormones. Two members of this family, UGT2B15 and UGT2B17, share over 95% sequence identity. However, UGT2B17 exhibits broader substrate specificity due to a single amino acid difference. Using gene-specific primers to explore the genomic organization of these two genes, it was determined that UGT2B17 is absent in some human DNA samples. The gene-specific primers demonstrated the presence or absence of a 150 kb genomic interval spanning the entire UGT2B17 gene, revealing that UGT2B17 is present in the human genome as a deletion polymorphism linked to UGT2B15. Furthermore, it is shown that the UGT2B17 deletion polymorphism shows Mendelian segregation and allele frequencies that differ between African Americans and Caucasians.     

Molecular pathology of haemophilia B.

Direct sequencing of amplified genomic DNA has been used to investigate the molecular basis of haemophilia B and thus identify specific amino acids that are essential for maintenance of structure or function of factor IX. Substitution of Cys 336, Asn 120 results in loss of circulating factor IX antigen and deletion of Arg 37 in gross reduction of circulating protein and loss of activity, while substitution of Arg -4, Arg 333, Asp 64 and Pro 55 cause loss of function without marked reduction in protein serum levels. Frameshift or point mutations resulting in marked loss of coding information are found in patients who develop antibodies to administered factor IX. An enhanced rate of mutation is evident at two CpG dinucleotides in the factor IX gene, which accounts for approximately 25% of all point mutations causing haemophilia B known to date. Direct sequencing of mutations also permits, for the first time, rapid and unequivocal prenatal and carrier diagnoses, in all cases, by eliminating the need for informative segregation of markers.     

Partial deletion 10p syndrome. Report of two patients.

Two patients with partial deletion of the short arm of chromosome 10 are described. They showed most of the features observed in twenty other known patients, including growth retardation, mental deficiency, abnormally shaped skull, distinct facial dysmorphisms, cardiac and genitourinary malformations, and limbs anomalies. One of our patients also had features of the DiGeorge sequence, which has been found in five other cases with this imbalance.     

Cre-mediated gene deletion in the mammary gland.

To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.     

Molecular demonstration of SLC4A1 gene deletion in two Mexican patients with Southeast Asian ovalocytosis

We describe the finding of two Mexican patients with a specific 27-bp deletion in the solute carrier family 4 gene (SLC4A1delta27) (also known as the band 3 gene found on chromosome 17q21-q22), characteristic of Southeast Asian ovalocytosis (SAO). The patients were asymptomatic, and the initial diagnosis was made by microscopic observation of the presence of typical stomatocytic ovalocytes. The gene deletion was confirmed by PCR and DNA sequencing. Both patients were heterozygous for the deletion. One patient is from Tabasco state, in southeastern Mexico, a malaria-endemic zone. The other patient is from Mexico City, which is not a malaria-endemic area. Their families have no non-Mexican ancestors and their previous generations were born in Mexico. Both patients carry the HLA-B*3501 subtype, characteristic of Amerindians and Asian populations. Familial and HLA data led us to conclude that these two patients are the first report of SLC4A1delta27 in Amerindians. The nucleotide analysis showing a perfect match sequence between Southeast Asian and Mexican patients suggests, but does not prove, that the Mexican gene is not a de novo mutation. Instead, this gene might be the result of migration of individuals with Asian ancestry into the Mexican gene pool. We are looking for other families with the mutation to detect, by HLA analysis, the ancient ethnic origin of these patients.     

Insertion and deletion polymorphism in the alpha-2B adrenoceptor gene in pregnant women ripens gestational diabetes mellitus

There are no earlier studies that reported the association of the 12Glu9 polymorphism in the alpha-2B adrenoceptor (ADRA2B) gene with gestational diabetes mellitus (GDM). We examined the potential association between the ADRA2B gene insertion/deletion (I/D) polymorphism in the Saudi population with GDM. Pregnant women with GDM have been reported to exhibit the same susceptibility as that observed in type 2 diabetes mellitus (T2DM). We have selected I/D polymorphism of the ADRA2B gene located in chromosome 2q11.1 that has been extensively related to T2DM and cardiovascular diseases. This case–control study was conducted with 200 GDM and 300 non-GDM pregnant women. Genotyping of I/D polymorphism was performed by conventional PCR method. Biochemical analyses were found to be significantly different between GDM and non-GDM subjects (p < 0.05). Genotype (ID + DD vs II, p = 0.0002) and allele (D vs I, p = 0.0002) frequencies of the 12Glu9 polymorphism were found to be statistically significant. However, a significant difference was found between allele and genotypes of I/D polymorphism of the ADRA2B gene or the clinical characteristics of the subjects. Our results obtained in this study indicate the ADRA2B gene in the Saudi women was associated with the development of GDM.     

Large scale physical mapping in the q27 region of the human X chromosome: the coagulation factor IX gene and the mcf.2 transforming sequence are separated by at most 270 kilobase pairs and are surrounded by several ''HTF islands''.

In spite of the large amount of genetic data obtained on the X chromosome and of the availability of many cloned sequences little is known about the physical map of this chromosome. The construction of large-scale restriction maps is now possible with pulsed field gel methods and data has recently been obtained in the region of band Xq28. We present here results of physical mapping in the Xq27 region, i.e. proximal to the fragile site at Xq27.3 associated with mental retardation, and show physical linkage between the coagulation factor IX gene and the mcf.2 transforming sequence recently localized to Xq27. Our data also indicate partial methylation of some sites in this region, and locate several 'HTF islands', i.e. CpG-rich, unmethylated sequences, containing several sites for 'rare cutter' enzymes, which are believed to be associated with expressed 'housekeeping' genes.     

An insertion deletion polymorphism in the signal peptide of the human apolipoprotein B gene

Summary In this communication we report the genetic properties of an insertion/deletion polymorphism in the signal peptide of the human apolipoprotein B (apo b) gene. There are two alleles of the apo B signal peptide; one codes for a peptide 27 amino acids in length and the other a peptide only 24 amino acids in length. Using the polymerase chain reaction the difference of nine nucleotides between the two alleles is readily detectable after electrophoresis of the amplification products. The relative frequencies of the Ins and Del alleles are 0.655 and 0.345, respectively. The apo B signal peptide genotypes are transmitted in a manner consistent with an autosomal codominant mode of inheritance with two alleles.     

A silent deletion in the beta-globin gene cluster.

A survey of the gamma-globin gene region of over 1000 normal individuals revealed a novel 2.5 kb deletion which removes the 5' end of the A gamma-globin gene. Unusually, this deletion in the beta-globin gene cluster is not associated with increased fetal haemoglobin production. Sequence analysis of the deletion endpoints revealed no significant homology at the breakpoint and failed to support a role for a proposed recombination hotspot in IVS-2 in the generation of this illegitimate recombination event. The existence of small "silent" deletions in the beta-globin gene cluster emphasizes the importance of deletion size in altering expression of the fetal globin genes.     

Nullisomic Tetrahymena. II. a Set of Nullisomics Define the Germinal Chromosomes

Crosses of a diploid Tetrahymena thermophila to a strain with a haploid germinal nucleus result in chromosome loss during meiosis in the haploid. The resulting monosomics can be made nullisomic by a special cross that induces homozygosis of a meiotic product of the germinal nucleus, but retention of the parental somatic nucleus. The creation and testing of single nullisomics for three of the five chromosome pairs and a triple nullisomic missing another pair is presented. Taken together, these strains make possible a series of crosses in which all but one of the chromosomes is missing in one parent. This set of nullisomics can, therefore, be used to map any mutation in Tetrahymena to a specific chromosome.     

A DNA marker closely linked to the factor IX (haemophilia B) gene

Summary We have isolated a DNA segment, pX58dIIIc, from an X-chromosome library which identifies an SstI restriction fragment length polymorphism (RFLP) at locus DXS99. Linkage analysis in six informative families has shown that the DXS99 locus lies close to the factor IX gene (F9). No recombination was detected between these loci in 39 informative meioses (Z=9.79, =0.0). Therefore, DXS99 will be useful as a DNA marker for the assessment of carrier status in families with haemophilia B where intragenic markers are not informative. Heterozygosity at DXS99 is approximately 50% and, in conjunction with the RFLPs at F9, 90% of females at risk for being haemophilia B carriers should be diagnosed.     

Amplification and deletion of the RAPH1 gene in breast cancer patients

Lamellipodin protein (Lpd), encoded by the RAPH1 gene, modulates the assembly of actin cytoskeleton through its binding to the Ena/VASPs proteins, and acts in cellular motility and lamelipodial protrusion. The region where RAPH1 gene is located (2q33) is deleted in various types of cancer and the gene expression changes in tumors when compared to normal tissues. Amplifications and deletions of the RAPH1 gene were investigated in breast carcinoma samples, in order to determine the possible relationship of the gene with breast cancer tumorigenesis and lymph node metastasis. RAPH1 gene alterations were determined by relative quantification, standard curve method using Real-time PCR technique in samples of tumor and peripheral blood from 52 patients. Regression and correlation analyses were conducted using gene alterations and clinicopathological data. All samples analyzed were altered, with 63.5 % deletion cases and 36.5 % amplification cases. The logistic regression and correlation analysis with clinicopathological data did not show significant results. The results suggest that although the RAPH1 gene was deleted or amplified in all samples, the Lpd does not seem to play a major role in tumorigenesis of mammary carcinomas and probably other proteins, also involved in the process of cellular motility and metastasis, are acting more effectively for or against the migration of breast tumor cells.     

Genomic diagnosis of haemophilia A and B in the German Democratic Republic.

Since 1986 the genomic diagnosis of haemophilia A and B in the GDR is realized as a national programme. Until Aug. 1989 56 families at risk of haemophilia A are analysed using RFLPs of different intragenic and intergenic probes (BclI/F8e 16-19, KpnI-XbaI/int 22, TaqI/St 14.1). 117 out of 162 females at risk being heterozygous were identified as carriers, in 40 cases the carrier state was excluded, and in 5 females the data were not informative. Prenatal diagnosis was offered to 93 carriers in reproductive age. Six genomic prenatal diagnoses in haemophilia A were performed. In four patients different partial deletions of factor VIII:C gene were found. 10 families of haemophilia B were analysed using intragenic and intergenic probes (P 1; pX58dIIIc). 14 females were identified as carriers, 11 were excluded. The application of direct and indirect gene diagnosis in haemophilia is discussed.