Analysis of 31 CFTR mutations by polymerase chain reaction/oligonucleotide ligation assay in a pilot screening of 4476 newborns for cystic fibrosis.

OBJECTIVES: Molecular biological testing for genetic diseases has grown rapidly, but speed, accuracy, specificity, sensitivity, throughput, and cost become more important as large scale screening is considered. This is a pilot study of an assay for the simultaneous detection of up to 31 cystic fibrosis mutations in a multicentre population based screening of 4476 Italian newborns. METHODS: The assay is a polymerase chain reaction, followed by an oligonucleotide ligation assay (PCR/OLA) and finally a sequence coded separation. It allows the detection of up to 31 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Guthrie spots were used as a source of template DNA. RESULTS: 144 carriers were detected during the analysis of 4476 samples, which translates into a carrier frequency of 1/31.1. Forty two carriers were detected from 1341 samples in Pavia (1/31.9), 53 from 1574 in Turin (1/29.7), and 49 from 1561 in San Giovanni Rotondo (1/31.8). Fifteen different mutations were detected, the most common being delta F508 (0.625). Other common mutations included G542X (16 of 144), which was particularly common in southern Italy (14 of 49), N1303K (8 of 144), and R117H (8 of 144), detected only in the northern centres. CONCLUSIONS: PCR/OLA is a robust, accurate, user friendly method for cystic fibrosis screening of newborns using blood spots in a semiautomated way at a low cost per mutation (0.8 Euro).

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.

Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations.

Isolation of the gene for cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator (CFTR), provided a basis for analyzing its molecular pathology and resulted in the identification of > 400 mutations associated with disease. Except for the delta F508 mutation, no other single mutation accounts for > 5% of CF chromosomes in most populations, and most mutation frequencies are < 1%. A strategy based on multiplex PCR followed by multiplex allele-specific oligonucleotide probe ligation was used to detect 30 mutations, distributed throughout ten exons and seven introns of the CFTR gene, that together account for > 96% of CF mutant chromosomes worldwide. Mutations were detected by competitive oligonucleotide probe ligation to detect normal and/or mutant genotypes in one reaction. Three probes (one common and two allelic probes) were needed for analysis of each mutation. Probes hybridized to target DNA were joined by a thermostable ligase if there were no mismatches at their junctions; temperature cycling resulted in a linear increase in product. Common probes were labeled with fluorochromes, and allelic probes each had different lengths. Ligation products were analyzed electrophoretically on a fluorescent DNA sequencer. The results show that combined PCR and probe ligation amplification rapidly and reliably screen for CF homozygotes and carriers.

High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.

We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.

Characterization of in vivo somatic mutations at the hypoxanthine phosphoribosyltransferase gene of a human control population.

The ability to recognize a change in mutation spectrum after an exposure to a toxic substance and then relate that exposure to health risk depends on the knowledge of mutations that occur in the absence of exposure. Toward this end, we have been studying both the frequency and molecular nature of mutations of the hypoxanthine phosphoribosyltransferase (hprt) gene in peripheral blood lymphocytes as surrogate reporters of genetic damage. We have analyzed mutants, one per donor to ensure independence, from a control population in which the quantitative effects of smoking and age on mutant frequency have been well defined. Analyses of cDNA and genomic DNA by polymerase chain reaction and sequencing have identified the mutations in 63 mutants, 45 from males and 18 from females, of which 34 were smokers and 29 were nonsmokers. Slightly less than half of the mutations were base substitutions; they were predominantly at GC base pairs. Different mutations at the same site indicated that there are features of the hprt polypeptide that affect the mutation spectrum. Two pairs of identical mutations indicated that there may also be hot spots. Mutations not previously reported have been detected, indicating that the mutation spectrum is only partly defined. The remainder of the mutations were deletions or insertions/duplications; deletions ranged from one base pair to complete loss of the locus. Despite a small average increase in mutant frequency for smokers, an increased proportion of base substitutions at AT base pairs in smokers (p = 0.2) hinted at a smoking-associated shift in the mutation spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Cloning and expression of a human endothelin receptor: subtype A.

The polymerase chain reaction, employing degenerate primers specific for the intramembrane domains III and VI of G-coupled receptors, was used to generate partial clones encoding those receptors carried by cultured rat aorta smooth muscle cells. One clone, spanning the intramembrane domains IV-VI of a receptor specific for endothelin-1 (ET-R[A]), was used as a probe to screen a human placental cDNA library. The clone pL4-3, encoding a selective type of human endothelin receptor (ET-R[A]), has an open reading frame encoding a protein 427 amino acids in length, with a relative molecular weight of 48,625 daltons. The sequence analysis suggests the presence of a signal peptide, two potential sites for glycosylation in the N terminal extracellular domain, the seven transmembrane domains typical of G-protein receptors, and several potential sites for phosphorylation in the C terminal cytoplasmic domain. At the amino acid level, the human ET-R(A) shows 91% and 94% identity with the rat and bovine ET-R(A)s, respectively, and 59% similarity with the human ET-R(B). Xenopus laevis oocytes injected with the cloned cDNA express binding sites specific for endothelin-1. Expression of the message in COS 7 cells gave a membrane-bound product to which binding of the [125I]-ET-1 was inhibited by peptide analogues specific for ET-R(A).

A rat beta-interferon-induced mRNA: sequence characterization.

Polymerase chain reaction amplification of a cDNA derived from rat aortic smooth muscle cells, using sequences from conserved regions of the intramembrane domains of adrenergic receptors as primers, yielded the clone, rat8. This clone possesses a high degree of sequence similarity to a series of human interferon (IFN)-inducible genes. The rat8 sequence is 70% similar to that derived from the human alpha-IFN-induced gene, 9-27; there is 66% similarity between the deduced amino acid sequences encoded by the rat and the human genes. The rat homologue hybridizes with many bands in Southern analysis of rat DNA, suggesting that it is a member of a large multigene family.

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)