Systematic screening for PRKAR1A gene rearrangement in Carney complex: identification and functional characterization of a new in-frame deletion.

BACKGROUND: Point mutations of the PRKAR1A gene are a genetic cause of Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD), but in 30% of the patients no mutation is detected. OBJECTIVE: Set up a routine-based technique for systematic detection of large deletions or duplications of this gene and functionally characterize these mutations. METHODS: Multiplex ligation-dependent probe amplification (MLPA) of the 12 exons of the PRKAR1A gene was validated and used to detect large rearrangements in 13 typical CNC and 39 confirmed or putative PPNAD without any mutations of the gene. An in-frame deletion was characterized by western blot and bioluminescence resonant energy transfer technique for its interaction with the catalytic subunit. RESULTS: MLPA allowed identification of exons 3-6 deletion in three patients of a family with typical CNC. The truncated protein is expressed, but rapidly degraded, and does not interact with the protein kinase A catalytic subunit. CONCLUSIONS: MLPA is a powerful technique that may be used following the lack of mutations detected by direct sequencing in patients with bona fide CNC or PPNAD. We report here one such new deletion, as an example. However, these gene defects are not a frequent cause of CNC or PPNAD.

Description of a new DRB1*11 allele (DRB1*1132).

We describe a new DRB1*11 allele which is similar to DRB1*11011 except at codon 74, where a GCG is changed for a GTG leading to an alanine/valine substitution. This new allele was carried by a Caucasian patient suffering from rheumatoid arthritis and by her healthy daughter. The motif at codon 74 of the new DRB1*11 is not found in any other known DRB alleles, nor among the published DQA1, DQB1, DPA1 or DPB1 alleles, and therefore suggests a mechanism of point mutation.

Detection and quantification of the A3243G mutation of mitochondrial DNA by ligation detection reaction.

The A3243G mutation of mitochondrial DNA is associated to the MELAS syndrome and to transmitted forms of diabetes mellitus. This mutation exists in a heteroplasmic state and can be present at a minor and hardly detectable level. The aim was to design a method which could be applied to large series of samples and could provide rapid, sensitive and quantitative detection of this mutation in the wild-type mitochondrial DNA background. The ability of ligation detection reaction (LDR) to satisfy these objectives was evaluated. Ligation detection reaction was performed on a model template composed of mixtures of various proportions of plasmids bearing the wild-type or mutant mitochondrial DNA sequence. Radiolabelled or fluorescent primers and the wild-type and mutant LDR products were separated by electrophoresis on conventional denaturating gel or on an Applied Biosystem 373. The ratios of mutant/wild-type products were consistent with the initial ratios of the plasmids in the template. The sensitivity and accuracy of the fluorescence and isotopic detection methods were similar. The detection limit of mutant DNA was 10% of total mitochondrial DNA. The percentage of mutant DNA in DNA samples extracted from leukocytes of 19 patients having the mutation at different levels, was evaluated by fluorescent or isotopic LDR.

Biodiversity of Pneumocystis carinii hominis: typing with different DNA regions.

The purpose of this study was to identify the most useful gene for the detection of biodiversity of Pneumocystis carinii hominis isolates and to compare samples from French and Italian subjects. We studied 20 bronchoalveolar lavage fluid specimens from 20 human immunodeficiency virus-infected patients (10 French and 10 Italian patients) with Pneumocystis carinii pneumonia by DNA sequencing of the thymidylate synthase (TS), 5S rRNA, large-subunit mitochondrial rRNA (mt LSU rRNA), and internal transcribed spacer (ITS1 and ITS2) genes. Thirteen of the 20 sequenced samples had the prototype TS gene sequence. Fourteen of the 20 samples showed the prototype sequence of the 5S rRNA gene, and 6 had variant sequences of the 5S rRNA gene. The mt LSU rRNA gene was sequenced for 18 of the 20 samples; all sequences were different from the prototype sequence and were classified into four groups. Thirteen of the 20 ITS1 and ITS2 sequences were analyzed, and all the sequences were found to be different from the prototype sequence and were classified into 10 groups. The internal transcribed spacer regions thus appear to be the most discriminatory region of DNA for analysis of the biodiversity of P. carinii hominis isolates.

Usefulness of PCR for detection of Pneumocystis carinii DNA.

Diagnosis of Pneumocystis carinii pneumonia is based on the identification of the various stages of the parasite in lung samples by standard staining techniques. We therefore assessed the value of the PCR for detection of P. carinii in bronchoalveolar lavage, induced sputum, and blood samples relative to that of standard staining techniques.

Extended haplotypes and linkage disequilibrium between 11 markers at the APOA1-C3-A4 gene cluster on chromosome 11.

One hundred fifty-four unrelated French Caucasian subjects were typed for 11 RFLPs at or near the APOA1-C3-A4 gene cluster on the long arm of chromosome 11. All subjects belonged to families having lived in the Toulouse area (in the southwest of France) for over three generations. Allele frequencies for each RFLP were in agreement with previous studies in Caucasian populations for the APOA1/SstI marker. Pairwise linkage disequilibrium was determined. Among the 55 pairs studied, 30 are newly reported. Only three significant nonrandom associations were found: APOA1/MspI-3'APOC3/SstI, APOA1/MspI-3'APOA4/XbaI, and APOA4/DraI-APOA4/XbaI. Extended 11-marker haplotypes were constructed. Haplotype frequencies were estimated by the maximum-likelihood procedure and compared to expected frequencies calculated under the assumption of equilibrium. Among the 37 estimated haplotypes, seven containing at least four nonrandomly associated alleles showed markedly increased frequencies. These results, obtained in a geographically homogeneous population, confirm the existence of disequilibrium in the apolipoprotein cluster, but to a lower extent than previously reported in Caucasian populations, which were geographically more heterogeneous.