COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma.

BACKGROUND: Until now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through the detection of a paternally inherited disease allele in cases where maternal and paternal mutated alleles differ. METHODS: Conditions for non-invasive identification of fetal paternally inherited mutations in maternal plasma were developed by two independent approaches: coamplification at lower denaturation temperature-PCR (COLD-PCR) and highly sensitive microarrays. Assays were designed for identifying 14 mutations, 7 causing ß-thalassaemia and 7 cystic fibrosis. RESULTS: In total, 87 non-invasive prenatal diagnoses were performed by COLD-PCR in 75 couples at risk for ß-thalassaemia and 12 for cystic fibrosis. First, to identify the more appropriate methodology for the analysis of minority mutated fetal alleles in maternal plasma, both fast and full COLD-PCR protocols were developed for the most common Italian ß-thalassaemia Cd39 and IVSI.110 mutations. In 5 out of 31 samples, no enrichment was obtained with the fast protocol, while full COLD-PCR provided the correct fetal genotypes. Thus, full COLD-PCR protocols were developed for all the remaining mutations and all analyses confirmed the fetal genotypes obtained by invasive prenatal diagnosis. Microarray analysis was performed on 40 samples from 28 couples at risk for ß-thalassaemia and 12 for cystic fibrosis. Results were in complete concordance with those obtained by both COLD-PCR and invasive procedures. CONCLUSIONS: COLD-PCR and microarray approaches are not expensive, simple to handle, fast and can be easily set up in specialised clinical laboratories where prenatal diagnosis is routinely performed.

Driver somatic mutations identify distinct disease entities within myeloid neoplasms with myelodysplasia.

Our knowledge of the genetic basis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) has considerably improved. To define genotype/phenotype relationships of clinical relevance, we studied 308 patients with MDS, MDS/MPN, or acute myeloid leukemia evolving from MDS. Unsupervised statistical analysis, including the World Health Organization classification criteria and somatic mutations, showed that MDS associated with SF3B1-mutation (51 of 245 patients, 20.8%) is a distinct nosologic entity irrespective of current morphologic classification criteria. Conversely, MDS with ring sideroblasts with nonmutated SF3B1 segregated in different clusters with other MDS subtypes. Mutations of genes involved in DNA methylation, splicing factors other than SF3B1, and genes of the RAS pathway and cohesin complex were independently associated with multilineage dysplasia and identified a distinct subset (51 of 245 patients, 20.8%). No recurrent mutation pattern correlated with unilineage dysplasia without ring sideroblasts. Irrespective of driver somatic mutations, a threshold of 5% bone marrow blasts retained a significant discriminant value for identifying cases with clonal evolution. Comutation of TET2 and SRSF2 was highly predictive of a myeloid neoplasm characterized by myelodysplasia and monocytosis, including but not limited to, chronic myelomonocytic leukemia. These results serve as a proof of concept that a molecular classification of myeloid neoplasms is feasible.

HIF1A and MIF as potential predictive mRNA biomarkers of pre-eclampsia: a longitudinal prospective study in high risk population.

BACKGROUND: Pre-eclampsia (PE) is a hypertensive multisystem disorder, causing significant fetal-maternal mortality and morbidity worldwide. This study aims to define possible longitudinal predictive mRNA markers involved in the main pathogenic pathways of PE: inflammation [macrophage migration inhibitory factor (MIF)], hypoxia and oxidative stress [hypoxia inducible factor 1-α subunit (HIF1A) and ß-site APP-cleaving enzyme-2 (BACE2)] and endothelial dysfunction [endoglin (ENG), fms-related tyrosine kinase-1 (FLT1) and vascular endothelial growth factor (VEGF)]. METHODS: Peripheral blood was collected from 33 singleton pregnancies characterized by a high cardiovascular profile risk sampled consecutively at 6-16; 17-23; 24-30; 31-34; ≥35 weeks followed by the Obstetrics and Gynecology Unit of the San Raffaele Hospital in Milan. A real-time quantitative PCR reaction was performed on plasma RNA. RESULTS: Of the 33 women enrolled, nine developed PE. Until 23 weeks HIF1A was significantly higher in women who later developed PE compared to women who did not (p=0.049 and p=0.012 in the first and second blood collection). In the third time interval MIF (p=0.0005), FLT1 (p=0.024), ENG (p=0.0034) and BACE2 (p=0.044) appeared to be significantly increased while HIF1A was elevated even from 24 week onwards but not reaching the statistical significance. In the fourth time interval ENG mRNA still remained increased (p=0.037). CONCLUSIONS: HIF1A, marker of hypoxia and oxidative stress, and MIF, marker of inflammation, seemed to be the most promising RNA markers, suggesting that hypoxia, principally, and inflammation may play an important role in PE pathogenesis.

Communicating Down syndrome risk according to maternal age: "1-in-X" effect on perceived risk.

OBJECTIVE: The appropriateness of the 1-in-X numerical format to convey quantitative medical probabilities is currently under discussion because of its distortive effect on subjective probability assessment. Previous research, however, always asked participants to imagine a hypothetical scenario. The aim of the present research is to support the existence of the 1-in-X effect in a real setting: when pregnant women have to evaluate their personal risk of Down syndrome according to their maternal age. METHOD: During the first prenatal visit, pregnant women were asked to evaluate their own risk of having a child with Down syndrome according to their age, when such risk was presented either in the 1-in-X or in the N-in-NX format. Then, they were asked to assess their risk of having a child with Down syndrome. RESULTS: Results showed a systematic higher risk assessment when pregnant women were presented with the 1-in-X format (mean = 3.57, standard deviation = 1.4) than with the N-in-NX format (mean = 3.03, standard deviation = 1.4), P = 0.007. Whereas the effect was shown to be not moderated by a differential comprehension of the two numerical formats, women with a low educational level and those who were at their first pregnancy were shown to be significantly more vulnerable. CONCLUSION: The present findings corroborate the existence of the 1-in-X effect in a real-world setting, showing that, in pregnant women, the 1-in-X format actually elicits a higher perceived risk of Down syndrome, compared with the N-in-NX format.

Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis: first case report.

BACKGROUND: Non-invasive prenatal diagnosis has found application in a limited number of genetic diseases due to the difficulty in detecting a few copies of fetal mutated sequences in the presence of a large excess of wild-type maternal alleles, even in the case of single-base mutations. METHODS: We developed conditions for the enrichment of fetal mutated alleles in maternal plasma based on CO-amplification at lower denaturation temperature-PCR (COLD-PCR). In particular, we applied a full COLD-PCR protocol to the identification of a p.A87_G92del mutation in the TWIST1 gene causing craniosynostosis in a couple at risk for the disease. RESULTS: The use of the COLD-PCR protocol coupled with direct sequencing enabled correct identification of the fetal paternally inherited mutated allele, in accordance with the result obtained on DNA extracted from chorionic villi. CONCLUSIONS: COLD-PCR proved to be a simple and powerful tool for the identification of minority mutated alleles even in the case of a moderately large deletion (18 bp) and confirmed to be very suitable for non-invasive prenatal diagnosis of a variety of genetic diseases.

COLD-PCR and innovative microarray substrates for detecting and genotyping MPL exon 10 W515 substitutions.

BACKGROUND: Myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Somatic mutations in exon 10 of the MPL (myeloproliferative leukemia virus oncogene) gene, mainly substitutions encoding W515 variants, have recently been described in a minority of patients with ET or PMF. We optimized analytically sensitive methods for detecting and genotyping MPL variants. METHODS: We used DNA previously isolated from circulating granulocytes of 60 patients with MPN that had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelic-discrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells. RESULTS: Mutations that were present at very low levels in patients who had previously been scored as having an MPL variant by HRM and as wild type by direct sequencing were successfully identified in granulocyte DNA. Notably, the microarray approach displayed analytical sensitivities of 0.1% to 5% mutant allele, depending on the particular mutation. This analytical sensitivity is similar to that obtained with COLD-PCR. The assay requires no enrichment strategy and allows both the characterization of each variant allele and the evaluation of its proportion in every patient. CONCLUSIONS: These procedures, which are transferable to clinical diagnostic laboratories, can be used for detecting very low proportions of minority mutant alleles that cannot be identified by other, conventional methods.

Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment.

BACKGROUND: Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR (coamplification at lower denaturation temperature PCR) is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3 °C. This requirement precludes simultaneous mutation enrichment in sequences of substantially different melting temperature (T(m)) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT) approach (TT-COLD-PCR) that reduces this obstacle. METHODS: We describe thermocycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR. This approach enabled enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation was provided for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and TP53 (tumor protein p53) exons 6-9 by use of dilutions of mutated DNA, clinical cancer samples, and plasma-circulating DNA. RESULTS: A single thermocycling program with a denaturation-temperature window of 2.5-3.0 °C enriches mutations in all DNA amplicons simultaneously, despite their different T(m)s. Mutation enrichments of 6-9-fold were obtained with TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other 2 forms of COLD-PCR, fast-COLD-PCR, and ice-COLD-PCR. CONCLUSIONS: Low-level mutations in diverse amplicons with different T(m)s can be mutation enriched via TT-COLD-PCR provided that their T(m)s fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons and increases significantly the versatility of COLD-PCR.

Study of FTMT and ABCA4 genes in a patient affected by age-related macular degeneration: identification and analysis of new mutations.

BACKGROUND: Age-related macular degeneration (AMD) is a multifactorial disease for which an involvement of alterations in the retinal ABC transporter gene (ABCA4) is still debated. Oxidative stress in retinal pigment epithelial cells has been postulated to contribute to the pathogenesis of the disease. Mitochondrial ferritin (FtMt), an iron-sequestering protein, is expressed in cell types characterized by high metabolic activity and oxygen consumption, including human retina, suggesting a role in protecting mitochondria from iron-dependent oxidative damage. Based on these findings we wanted to investigate whether mutations in this gene could be found in AMD patients. METHODS: Mutational scanning of the FTMTgene was performed in a cohort of 50 patients affected by age-related macular degeneration. The ABCA4 gene was also scanned in one patient carrying an FtMt mutation. In silico analyses were carried out on the identified variants. The recombinant form of FtMt variant was expressed in Escherichia coli and biochemically characterized. RESULTS: One patient was found to be heterozygous for two previously unreported genetic changes: a complex FtMt mutation (c.437_450delinsCT: delAGGACATCAAGAAGinsCT) and a missense p.Leu973Phe (c.2919G>T) mutation in exon 20 of ABCA4. Computational analyses predicted a severe structural impairment for FtMt variant and a mild destabilizing effect for ABCA4. E. coli expression of recombinant FtMt variant yielded a highly insoluble protein that could not be renatured under in vitro conditions suitable for wild-type ferritins. CONCLUSIONS: Our findings suggest that the FtMt mutation may determine a condition similar to haploinsufficiency resulting in a reduced protection from iron-dependent oxidative stress in mitochondria.

Molecular and clinical features of refractory anemia with ringed sideroblasts associated with marked thrombocytosis.

We studied patients with myeloid neoplasm associated with ringed sideroblasts and/or thrombocytosis. The combination of ringed sideroblasts 15% or greater and platelet count of 450 x 10(9)/L or greater was found in 19 subjects fulfilling the diagnostic criteria for refractory anemia with ringed sideroblasts (RARS) associated with marked thrombocytosis (RARS-T), and in 3 patients with primary myelofibrosis. JAK2 and MPL mutations were detected in circulating granulocytes and bone marrow CD34+ cells, but not in T lymphocytes, from 11 of 19 patients with RARS-T. Three patients with RARS, who initially had low to normal platelet counts, progressed to RARS-T, and 2 of them acquired JAK2 (V617F) at this time. In female patients with RARS-T, granulocytes carrying JAK2 (V617F) represented only a fraction of clonal granulocytes as determined by X-chromosome inactivation patterns. RARS and RARS-T patient groups both consistently showed up-regulation of ALAS2 and down-regulation of ABCB7 in CD34+ cells, but several other genes were differentially expressed, including PSIP1 (LEDGF), CXCR4, and CDC2L5. These observations suggest that RARS-T is indeed a myeloid neoplasm with both myelodysplastic and myeloproliferative features at the molecular and clinical levels and that it may develop from RARS through the acquisition of somatic mutations of JAK2, MPL, or other as-yet-unknown genes.

Over-expression of mitochondrial ferritin affects the JAK2/STAT5 pathway in K562 cells and causes mitochondrial iron accumulation.

BACKGROUND: Mitochondrial ferritin is a nuclear encoded iron-storage protein localized in mitochondria. It has anti-oxidant properties related to its ferroxidase activity, and it is able to sequester iron avidly into the organelle. The protein has a tissue-specific pattern of expression and is also highly expressed in sideroblasts of patients affected by hereditary sideroblastic anemia and by refractory anemia with ringed sideroblasts. The present study examined whether mitochondrial ferritin has a role in the pathogenesis of these diseases. DESIGN AND METHODS: We analyzed the effect of mitochondrial ferritin over-expression on the JAK2/STAT5 pathway, on iron metabolism and on heme synthesis in erythroleukemic cell lines. Furthermore its effect on apoptosis was evaluated on human erythroid progenitors. RESULTS: Data revealed that a high level of mitochondrial ferritin reduced reactive oxygen species and Stat5 phosphorylation while promoting mitochondrial iron loading and cytosolic iron starvation. The decline of Stat5 phosphorylation induced a decrease of the level of anti-apoptotic Bcl-xL transcript compared to that in control cells; however, transferrin receptor 1 transcript increased due to the activation of the iron responsive element/iron regulatory protein machinery. Also, high expression of mitochondrial ferritin increased apoptosis, limited heme synthesis and promoted the formation of Perls-positive granules, identified by electron microscopy as iron granules in mitochondria. CONCLUSIONS: Our results provide evidence suggesting that Stat5-dependent transcriptional regulation is displaced by strong cytosolic iron starvation status induced by mitochondrial ferritin. The protein interferes with JAK2/STAT5 pathways and with the mechanism of mitochondrial iron accumulation.

Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms.

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

High resolution melting for the identification of mutations in the iron responsive element of the ferritin light chain gene.

BACKGROUND: Among the causes of hyperferritinemia, hereditary hyperferritinemia cataract syndrome (HHCS) is an autosomal dominant disease characterized by distinctive cataracts and high serum ferritin. It is caused by mutations in the iron responsive element (IRE) of the ferritin light chain gene (FTL). METHODS: To speed up and simplify mutational scanning in this genomic region, we developed a protocol based on high-resolution melting (HRM) analysis. RESULTS: Validation was carried out using 18 wild-type and 14 DNA samples carrying different mutations, each analyzed in replicates of 20. The method allowed for correct identification and genotyping of all mutant samples, and each variant generated a specific profile distinguishable from the wild type. A 5.5% proportion of false positive results were obtained. In addition, in two patients with HHCS, two new mutations were identified by HRM based on an altered melting profile. These mutations were subsequently characterized by direct sequencing (7C>G+40A>G and 49A>C). CONCLUSIONS: The high reliability of HRM in detecting known and new DNA variations indicate that this could be an effective and sensitive method for molecular scanning of mutations in the IRE of the FTL gene in patients presenting with either HHCS or unexplained hyperferritinemia.

Integrated PCR amplification and detection processes on a Lab-on-Chip platform: a new advanced solution for molecular diagnostics.

BACKGROUND: Several microdevices have been developed to perform only a single step of a genotyping process, such as PCR or detection by probe hybridization. Here, we describe a Lab-on-Chip (LoC) platform integrating a PCR amplification microreactor with a customable microarray for the detection of sequence variations on human genomic DNA. METHODS: Preliminary work was focused on developing the single analytical steps including PCR and labeling strategies of the amplified product by conventional reference systems. The optimized protocols included a 1:4 forward:reverse primer ratio for asymmetric PCR, and Cy5-dCTP multiple incorporation for the generation of a labeled PCR product to be hybridized to complementary probes bound to the chip surface. RESULTS: Final conditions were applied to the fully integrated LoC platform for the detection of the IVSI-110 G > A mutation in the human beta-globin (HBB) gene associated with beta-thalassemia, used as a model of genetic application, allowing for correct genotyping of 25 samples that were heterozygous, homozygous or wild-type for this mutation. CONCLUSIONS: The overall results show that the present platform is very promising for rapid identification of DNA sequence variations in an integrated, cost effective and convenient silicon chip format.

Evaluation of a panel of circulating DNA, RNA and protein potential markers for pathologies of pregnancy.

BACKGROUND: Among markers of pregnancy complications, corticotropin-releasing hormone (CRH) mRNA, long pentraxin 3 (PTX3) protein and fetal and total DNA had been reported to be increased in the plasma of women with overt preeclampsia (PE). We developed an optimized protocol to evaluate whether concentrations of CRH mRNA, PTX3 mRNA and protein, fetal and/or total DNA are increased in fetal growth restriction (FGR), and whether they predict complications of pregnancy. METHODS: The protocol included a preamplification step to enrich rare mRNA species. CRH and PTX3 mRNA, DNA and PTX3 protein were measured in the plasma of women with PE or FGR, in women at risk of developing these pathologies and in healthy women matched for gestational age. RESULTS: CRH mRNA, fetal and/or total DNA and PTX3 protein were significantly increased in women with overt PE when compared to controls. Pregnant women who later developed PE or FGR during pregnancy showed total DNA levels that were significantly increased before the onset of both pathologies, while RNA markers were increased only in women who later developed PE. CONCLUSIONS: Our protocol for plasma RNA quantification may allow for the extension of a panel of predictive markers to be investigated in larger patient cohorts.

Application of pyrosequencing to the identification of sequence variations in the cystic fibrosis transmembrane conductance regulator gene.

BACKGROUND: A high number of mutations associated with cystic fibrosis have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, most of which are rare and, therefore, hamper extensive molecular diagnosis. In couples undergoing prenatal diagnosis where no mutation is found in one or both partners, additional analysis of intragenic polymorphisms may allow for the identification of fetal alleles associated with cystic fibrosis. METHODS: We developed novel, rapid and accurate assays for CFTR genotype determination using pyrosequencing technology; a simple, automated and reliable technique with low cost. RESULTS: Assays were optimized for the identification of the seven most frequent CFTR mutations (p.DeltaF508, p.N1303K, p.G542X, c.2183AA>G, c.1717-1G>A, p.W1282X, p.R1162X) in the Italian population and two common intragenic polymorphisms (rs213950 and rs1800136). Blind validation on 15 known control samples, typed for each sequence variation, allowed correct identification of all 135 genotypes. CONCLUSIONS: We demonstrated that this procedure is highly specific for the identification of individual CFTR sequence variations associated with cystic fibrosis, allowing both population screening and prenatal diagnosis.

High-sensitive microarray substrates specifically designed to improve sensitivity for the identification of fetal paternally inherited sequences in maternal plasma.

BACKGROUND: The identification of very low-levels of minority sequences has interesting clinical and diagnostic applications. Among these, non-invasive prenatal diagnosis of genetic diseases on fetal DNA circulating in maternal plasma is an emerging field of application. METHODS: A combined approach based on innovative microarray slides coated with a special copolymer was developed for the identification of three polymorphisms located in the causative gene for cystic fibrosis (CF). This technique was applied to the analysis of fetal DNA in maternal plasma from four couples that carried different allelic variants. RESULTS: The use of highly sensitive slides correctly identified fetal paternally inherited alleles without any enrichment strategy. CONCLUSIONS: Our results confirm that the high fluorescence signal provided by the optimized substrate may be applied to the identification of any fetal paternally inherited sequence. This helps extend the application of non-invasive prenatal diagnosis to genetic diseases caused by predominant mutations or minor rare molecular defects.

Evaluation of human gene variant detection in amplicon pools by the GS-FLX parallel Pyrosequencer.

BACKGROUND: A new priority in genome research is large-scale resequencing of genes to understand the molecular basis of hereditary disease and cancer. We assessed the ability of massively parallel pyrosequencing to identify sequence variants in pools. From a large collection of human PCR samples we selected 343 PCR products belonging to 16 disease genes and including a large spectrum of sequence variations previously identified by Sanger sequencing. The sequence variants included SNPs and small deletions and insertions (up to 44 bp), in homozygous or heterozygous state. RESULTS: The DNA was combined in 4 pools containing from 27 to 164 amplicons and from 8,9 to 50,8 Kb to sequence for a total of 110 Kb. Pyrosequencing generated over 80 million base pairs of data. Blind searching for sequence variations with a specifically designed bioinformatics procedure identified 465 putative sequence variants, including 412 true variants, 53 false positives (in or adjacent to homopolymeric tracts), no false negatives. All known variants in positions covered with at least 30x depth were correctly recognized. CONCLUSION: Massively parallel pyrosequencing may be used to simplify and speed the search for DNA variations in PCR products. Our results encourage further studies to evaluate molecular diagnostics applications.

Genotyping beta-globin gene mutations on copolymer-coated glass slides with the ligation detection reaction.

BACKGROUND: Methods are needed to analyze small amounts of samples for variation in disease-causing genes. One means is to couple the sensitivity and multiplexing capability of the ligation detection reaction (LDR) with the use of simple glass slides specifically functionalized with a novel polymer coating to enhance sensitivity. METHODS: We developed an array-based genotyping assay based on glass slides coated with copolymer (N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate). The assay consists of an LDR with genomic DNA followed by a universal PCR (U-PCR) of genomic DNA-templated LDR product. The LDR occurs in the presence of 3 primers for each sequence variant under investigation: 2 distinguishing primers (allele specific and perfectly complementary to wild-type and mutant alleles) and 1 common locus-specific primer. The 2 allele-specific primers have different capture sequences for binding different complementary probes on a tag array. The LDR product templated from genomic DNA is made fluorescent during the U-PCR via incorporation of a Cy5-labeled universal primer into all LDR products; detection occurs on the coated glass slides. RESULTS: The assay was designed to detect 7 prevalent mutations in the beta-globin gene (HBB, hemoglobin, beta) in a multiplex format, and signals for the different alleles are detected by their fluorescence. The assay was applied to 40 genomic DNA samples from both control individuals and patients with known beta-thalassemia mutations. Results show good correspondence between the patients' genotypes as assessed by DNA sequence analysis and those generated from the LDR assays. CONCLUSIONS: The developed technology allows accurate identification of sequence variants in a simple, cost-effective way and offers good flexibility for scaling to other applications with different numbers of single-nucleotide polymorphisms or mutations to be detected.

Dual-color microchip electrophoresis with single-photon avalanche diodes: application to mutation detection.

A novel microchip electrophoresis instrument based on single-photon avalanche diodes was used for the molecular characterization of mutations in disease genes. The identification of the main mutation causing cystic fibrosis, named DeltaF508, by the Amplification Refractory Mutation System was used to validate the technology. In our implemented protocol the wild-type and mutant allele-specific primers are labeled with Cy5 and Cy5.5, respectively. The protocol enables the amplification of the DNA sample in a single PCR. The genotype was deduced from the fluorescence of the amplicons run in the CE microchip. Validation on 15 DNA samples from either homozygous wild-type or heterozygous and homozygous mutated control subjects proved the complete reliability of the system, thus confirming its high diagnostic potential.