Introduction to PCR/OLA/SCS, a multiplex DNA test, and its application to cystic fibrosis.

The field of medical, molecular diagnostics has grown rapidly over the last few years, becoming increasingly informative to both clinician and patient. As genes associated with specific diseases have been discovered and sequenced, many genotype-phenotype relationships have been defined. For those genetic diseases with associated, defined, gene mutations, sophisticated DNA diagnostic tests are being developed. As an example, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, is associated with Cystic Fibrosis (CF). We have developed a new molecular diagnostic technology, PCR/OLA/SCS, and applied it first to the diagnosis of CF. Test design in the field of molecular diagnostics must consider such characteristics as specificity, sensitivity, ease and speed of protocol, multiplex capacity, and cost. PCR/OLA/SCS addresses these requirements. Polymerase Chain Reaction (PCR) is widely used in both diagnostics and research. We have combined well established PCR technology with Oligonucleotide Ligation Assay (OLA) and Sequence-Coded Separation (SCS), two relatively new technologies.

The 32.6 kb Indian delta beta-thalassaemia deletion ends in a 3.4 kb L1 element downstream of the beta-globin gene.

The Indian delta beta-thalassaemia, with elevated fetal gamma globin gene expression, was previously found to have a large deletion beginning 1 kb 3' of the (A) gamma globin gene at GenBank HUMHBB coordinate 42151, and extending into a new L1 sequence. We have now determined the 3' breakpoint of this deletion, and in doing so we have extended the known beta-globin gene cluster DNA sequence from its end at 73326 to projected GenBank coordinate 79016. These data show that the deletion is 32.6 kb long, terminating 11 kb 3' of the beta-globin gene. This 3' breakpoint is at 74772, within a 3.4 kb partial L1 repeat at 74263-77665; the Black ((A) gamma delta beta)(0)-thalassaemia also terminates in this L1, at 76508. In addition, two Alu sequences were found, at 73692-73816 and 78171-78441. Among the protein-binding DNA sequence motifs 3' to the Indian delta beta-thalassaemia breakpoint, at 76581/76607 there is a TGATAA/ACACCC pair that binds the erythroid-specific GATA-1 and ubiquitous CACCC-box binding proteins. We hypothesize that elevated fetal haemoglobin may be due to an enhancer or enhancers 3' to the deletion breakpoints and may involve the TGATAA/ACACCC pair.

G gamma and A gamma globin genes are identical from -471 of the promoter midway through gamma IVSII in a Benin beta s haplotype associated with elevated fetal hemoglobin.

In seven kindreds in which sickle cell (SS) patients had elevated (greater than 12%) fetal hemoglobin (Hb F), Milner and colleagues reported that a determinant for elevated Hb F and elevated F cells was linked to the beta s gene. Independently, the Senegal (SEN) beta s haplotype has been found in association with elevated Hb F in SS and beta-thalassemia patients. We have used the kindreds of Milner and colleagues to characterize further the association of haplotype and gamma gene DNA sequence variation with Hb F expression. For the largest kindred, Wi, all four SS had high (greater than 14%) Hb F and both SEN and Benin (BEN) haplotypes. Two AS cases carrying SEN had low Hb F and low F cells, while three AS and one CS carrying BEN had elevated Hb F and elevated F cells; only one AS carrying BEN had low Hb F and low F cells. In order to look for genetic alterations that could account for the elevated Hb F of kindred Wi, we sequenced both the G gamma and A gamma genes of the Wi BEN haplotype. The data showed largely identical G gamma and A gamma genes which may have been generated by two gene conversions: the A gamma promoter was like that of G gamma 3' to -471, while the G gamma IVSII was like that of A gamma in its 5' half. In addition, three new mutations were found in gamma IVSII.(ABSTRACT TRUNCATED AT 250 WORDS)