Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.

Trigonocephaly is a rare form of craniosynostosis characterized by the premature closure of the metopic suture. To contribute to a better understanding of the genetic basis of metopic synostosis and in an attempt to restrict the candidate regions related to metopic suture fusion, we studied 76 unrelated patients with syndromic and non-syndromic trigonocephaly. We found a larger proportion of syndromic cases in our population and the ratio of affected male to female was 1.8 : 1 and 5 : 1 in the non-syndromic and syndromic groups, respectively. A microdeletion screening at 9p22-p24 and 11q23-q24 was carried out for all patients and deletions in seven of them were detected, corresponding to 19.4% of all syndromic cases. Deletions were not found in non-syndromic patients. We suggest that a molecular screening for microdeletions at 9p22-p24 and 11q23-q24 should be offered to all syndromic cases with an apparently normal karyotype because it can potentially elucidate the cause of trigonocephaly in this subset of patients. We also suggest that genes on the X-chromosome play a major role in syndromic trigonocephaly.

Systemic lupus erythematosus (SLE) is characterized by several T lymphocyte abnormalities. An indirect assessment of recent thymus emigrants (RTE) has been recently been made available by measuring the number of TCR recombination excision circles (TREC) in peripheral T cells. We studied TREC levels in peripheral blood mononuclear cells (PBMC) of 32 SLE patients with active disease and 32 normal age- and sex-matched controls. Signal-joint TREC concentration was determined by real-time quantitative-PCR as the number of TREC copies/μg PBMC DNA. SLE patients had lower TREC levels (4.1 ±3.9 ×104 TREC/μg DNA) than controls (8.9 ±7.9 ×104/μg DNA) ( P = 0.004). There was an inverse correlation between age and TREC levels in controls ( r = 20.41, P = 0.02) but not in SLE patients. No clinical association was observed between TREC levels and clinical and laboratory SLE manifestations. TREC levels tended to be lower in patients with SLEDAI above 20 than in the rest of the patients ( P = 0.08). The decreased PBMC TREC levels is indicative of a low proportion of RTE in SLE and could be caused by decreased RTE output and/or by increased peripheral T cell proliferation in this disease. The under-representation of RTE in the peripheral T cell pool may play a role in the immune tolerance abnormalities observed in SLE.

BACKGROUND:Polymorphisms of platelet membrane glycoproteins such as human platelet antigen (HPA)-1b, HPA-2b, the -5T/C Kozak sequence and C807T have been described as risk factors for vascular disease. Vaso-occlusion episodes are a common feature of sickle cell anaemia (SCA), leading to complications such as stroke, acute chest syndrome, avascular head femur necrosis and priapism. Complex interactions are involved in vaso-occlusion, and activated platelets may play an important role. These data raised the question of whether platelet polymorphisms could be implicated in occlusive vascular complications (OVC) of SCA.MATERIALS AND METHODS:In this study, 97 patients with SCA were analysed in two groups: 34 patients presenting with OVC (SCA-VC) and 63 without these complications (SCA-N). The distribution of the HPA-1, -2 and -5 systems, as well as C807T dimorphism and -5T/C Kozak sequence alleles, was evaluated using DNA-based methods.RESULTS:Patients of the SCA-VC group showed a higher frequency of the HPA-5b allele (0.324) compared with those of the SCA-N group (0.111) (chi2 = 13.19, P = 0.0002). None of the other polymorphisms, isolated or associated as haplotypes, demonstrated any correlation with the development of OVC in these patients.CONCLUSIONS:The findings of this study suggest that the HPA-5b allele is a genetic risk factor for the development of OVC in patients with SCA. This allele could be explored as a target for the development of new therapeutic approaches.

Objective: The study aimed to investigate the use of hydroxyurea (HU) for the treatment of beta-thalassaemia (beta -thal) patients. Methods: We examined the haematological effects of orally administered HU (10-20 mg/kg/d) in 11 patients, including four beta -thal major and seven beta -thal intermedia patients. Complete blood count and levels of foetal haemoglobin (HbF), liver enzymes and serum creatinine were evaluated before and during HU. Response to therapy was evaluated at 6 months of treatment. Results: A substantial increase in haemoglobin (Hb) level (4.1 g/dL), leading to complete withdrawal from a regular transfusion programme, was observed in one unique beta -thal major patient. In the beta -thal intermedia patients, increases in Hb level of 1.3, 1.9 and 2.0 g/dL were observed in three of seven (42.9%) patients during HU therapy. The mean values of Hb, mean corpuscular haemoglobin (MCH), and HbF were higher during HU treatment than baseline values (8.7 vs. 7.7 g/dL, P = 0.05; 26.7 vs. 22.9 pg, P = 0.05; 57.2 vs. 44.9%, P = 0.04; respectively). In contrast, the mean reticulocyte count measured during therapy decreased (97.0 x 10(9) vs. 632.0 x 10(9) /L, P = 0.03). No correlations were observed between levels of Hb and HbF (r = 0.77, P = 0.10), and levels of Hb and reticulocyte counts (r = 0.26, P = 0.31). No significant toxicity was observed in our patients. Conclusion: These results suggest that HU may improve Hb levels in beta -thal. Thus, we may conclude that a large trial concerning the response to HU in these patients should be carried out to clarify this issue.

AIMTo assess the utility of a volumetric low-dose computed tomography (CT) thorax (LDCTT) protocol at a dose equivalent to a posteroanterior (PA) and lateral chest radiograph for surveillance of cystic fibrosis (CF) patients.MATERIALS AND METHODSA prospective study was undertaken of 19 adult patients with CF that proceeded to LDCTT at 12 and 24 months following initiation of ivacaftor. A previously validated seven-section, low-dose axial CT protocol was used for the 12-month study. A volumetric LDCTT protocol was developed for the 24-month study and reconstructed with hybrid iterative reconstruction (LD-ASIR) and pure iterative reconstruction (model-based IR [LD-MBIR]). Radiation dose was recorded for each scan. Image quality was assessed quantitatively and qualitatively, and disease severity was assessed using a modified Bhalla score. Statistical analysis was performed and p-values of <0.05 were considered statistically significant.RESULTSVolumetric LD-MBIR studies were acquired at a lower radiation dose than the seven-section studies (0.08 ± 0.01 versus 0.10 ± 0.02 mSv; p=0.02). LD-MBIR and seven-section ASIR images had significantly lower levels of image noise compared with LD-ASIR images (p<0.0001). Diagnostic acceptability scores and depiction of bronchovascular structures were found to be acceptable for axial and coronal LD-MBIR images. LD-MBIR images were superior to LD-ASIR images for all qualitative parameters assessed (p<0.0001). No significant change was observed in mean Bhalla score between 1-year and 2-year studies (p=0.84).CONCLUSIONSThe use of a volumetric LDCTT protocol (reconstructed with pure IR) enabled acquisition of diagnostic quality CT images, which were considered extremely useful for surveillance of CF patients, at a dose equivalent to a PA and lateral chest radiograph.

We evaluated the usefulness of blood group genotyping as a supplement to hemagglutination to determine the red blood cell (RBC) antigen profile of polytransfused patients with beta-thalassemia. We selected 10 alloimmunized patients who were receiving antigen-matched RBCs based on phenotype, and had clinical evidence of delayed hemolytic transfusion reaction. DNA was prepared from blood samples and RH E/e, K1/K2, FY A/FY B, and X A/JK B alleles were determined by PCR-RFLP. RH D/non-D was determined according to the PCR product size associated with the RHD gene sequence in intron 4 and exon 10/3'UTR. RH C/c was tested by multiplex PCR. The phenotypes and genotypes of nine of the 10 samples were discrepant. Five of the discrepancies occurred in the Rh system. One sample was phenotyped as Rhcc and genotyped as RH C/C, and two samples were phenotyped as RhCc and genotyped as RH C/C. Two other samples were phenotyped as RhEe and genotyped as RH e/e. Three samples had discrepancies in the Kidd system with phenotype Jk(a+b+) and were genotyped as homozygous for JK B. One sample had a discrepancy in the Duffy system: it was phenotyped as Fy(a+b-) and homozygous for FY B. Genotyping was very important in determining the true blood groups of many polytransfused patients with beta-thalassemia, and it assisted in the identification of suspected alloantibodies and the selection of antigen-negative RBCs for transfusion. (C) 2002 Wiley-Liss, Inc.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Abstract: Objectives: Glutathione S‐transferases (GST) modulate the effects of exposure to various cytotoxic and genotoxic agents, including those associated with increased risks of the myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML) and aplastic anemia (AA). Both the GST mu 1 (GSTM1) and GST theta 1 (GSTT1) genes have a null variant allele in which the entire gene is absent. In this study, we tested whether null genotypes for the GSTM1 and GSTT1 genes altered the risks for MDS, AML and AA. Methods: Genomic DNA from 49 MDS, 38 AML and 37 AA patients and 276 controls was analysed using the polymerase chain reaction (PCR). Results: The frequencies of GSTM1 (73.6%) and GSTT1 (34.2%) null genotypes were significantly higher in AML patients than in the controls (36.9 and 18.1%, respectively). A higher frequency of the combined null genotype for both genes was also observed in patients with AML (26.3% compared with 5.0% in the controls). In contrast, no differences in the frequencies of the null genotypes were found among MDS patients, AA patients and the controls. Conclusion: Our observation of a 4.7‐fold (95% CI: 2.1–11.0) and 2.3‐fold (95% CI: 1.0–5.2) increased risk associated with the GSTM1 and GSTT1 null genotypes, respectively, and a 6.6‐fold (95% CI: 2.4–7.9) increased risk associated with the combined null genotype presents preliminary evidence that the inherited absence of this carcinogen detoxification pathway may be an important determinant of AML.

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).

Familial hypercholesterolemia (FH) is a common autosomal disorder that affects about one in 500 individuals in most Western populations and is caused by a defect in the low-density-lipoprotein receptor (LDLr) gene. In this report we determined the molecular basis of FH in 59 patients from 31 unrelated Brazilian families. All patients were screened for the Lebanese mutation, gross abnormalities of the LDLr gene, and the point mutation in the codon 3500 of the apolipoprotein B-100 gene. None of the 59 patients presented the apoB-3500 mutation, suggesting that familial defective ApoB-100 (FDB) is not a major cause of inherited hypercholesterolemia in Brazil. A novel 4-kb deletion in the LDLr gene, spanning from intron 12 to intron 14, was characterized in one family. Both 5' and 3' breakpoint regions were located within Alu repetitive sequences, which are probably involved in the crossing over that generated this rearrangement. The Lebanese mutation was detected in 9 of the 31 families, always associated with Arab ancestry. Two different LDLr gene haplotypes were demonstrated in association with the Lebanese mutation. Our results suggest the importance of the Lebanese mutation as a cause of FH in Brazil and by analogy the same feature may be expected in other countries with a large Arab population, such as North American and Western European countries.