ABSTRACT
Transmembrane protein 184B (TMEM184B) is an endosomal 7-pass transmembrane protein with evolutionarily conserved roles in synaptic structure and axon degeneration. We report six pediatric patients who have de novo heterozygous variants in TMEM184B. All individuals harbor rare missense or mRNA splicing changes and have neurodevelopmental deficits including intellectual disability, corpus callosum hypoplasia, seizures, and/or microcephaly. TMEM184B is predicted to contain a pore domain, wherein many human disease-associated variants cluster. Structural modeling suggests that all missense variants alter TMEM184B protein stability. To understand the contribution of TMEM184B to neural development in vivo, we suppressed the TMEM184B ortholog in zebrafish and observed microcephaly and reduced anterior commissural neurons, aligning with patient symptoms. Ectopic TMEM184B expression resulted in dominant effects for K184E and G162R. However, in vivo complementation studies demonstrate that all other variants tested result in diminished protein function and indicate a haploinsufficiency basis for disease. Expression of K184E and other variants increased apoptosis in cell lines and altered nuclear localization of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, suggesting disrupted nutrient signaling pathways. Together, our data indicate that TMEM184B variants cause cellular metabolic disruption likely through divergent molecular effects that all result in abnormal neural development.
Subject(s)
Genetic Testing , Preimplantation Diagnosis , Humans , Female , Pregnancy , Blastocyst , Risk Factors , Delivery of Health CareABSTRACT
OBJECTIVE: Genomics Quality Assessment has provided external quality assessments (EQAs) for preimplantation genetic testing (PGT) for 12 years for eight monogenic diseases to identify sub-optimal PGT strategies, testing and reporting of results, which can be shared with the genomics community to aid optimised standards of PGT services for couples. METHOD: The EQAs were provided in two stages to mimic end-to-end protocols. Stage 1 involved DNA feasibility testing of a couple undergoing PGT and affected proband. Participants were required to report genotyping results and outline their embryo testing strategy. Lymphoblasts were distributed for mock embryo testing for stage 2. Submitted clinical reports and haplotyping results were assessed against peer-ratified criteria. Performance was monitored to identify poor performance. RESULTS: The most common testing methodology was short tandem repeat linkage analysis (59%); however, the adoption of single nucleotide polymorphism-based platforms was observed and a move from blastomere to trophectoderm testing. There was a variation in testing strategies, assigning marker informativity and understanding test limitations, some clinically unsafe. Critical errors were reported for genotyping and interpretation. CONCLUSION: EQA provides an overview of the standard of preimplantation genetic testing-M clinical testing and identifies areas of improvement for accurate detection of high-risk embryos.
Subject(s)
Preimplantation Diagnosis , Pregnancy , Female , Humans , Preimplantation Diagnosis/methods , Genetic Testing/methods , Blastocyst , AneuploidyABSTRACT
We describe a novel deletion causing heterozygous εγδß-thalassemia (εγδß-thal) across three generations of a Greek family. The Greek deletion is about 72 kb in length, spanning from the hypersensitive site 4 (HS4) in the locus control region (LCR) to the 3' end of the ß-globin gene, thus encompassing the entire ß-globin gene cluster. The deletion caused severe but transient neonatal anemia and a non transfusion-dependent chronic hemolytic anemia state later in life, resembling mild ß-thalassemia intermedia (ß-TI) rather than ß-thalassemia (ß-thal) trait, as had been previously reported. Apart from the presentation of clinical and laboratory characteristics, the challenges involving clinical management are also discussed.
Subject(s)
Thalassemia , beta-Thalassemia , Greece , Humans , Phenotype , Thalassemia/genetics , beta-Globins/genetics , beta-Thalassemia/diagnosis , beta-Thalassemia/geneticsABSTRACT
BACKGROUND: Deletions removing 100s-1000s kb of DNA, and variable numbers of poorly characterised genes, are often found in patients with a wide range of developmental abnormalities. In such cases, understanding the contribution of the deletion to an individual's clinical phenotype is challenging. METHODS: Here, as an example of this common phenomenon, we analysed 41 patients with simple deletions of ~177 to ~2000 kb affecting one allele of the well-characterised, gene dense, distal region of chromosome 16 (16p13.3), referred to as ATR-16 syndrome. We characterised deletion extents and screened for genetic background effects, telomere position effect and compensatory upregulation of hemizygous genes. RESULTS: We find the risk of developmental and neurological abnormalities arises from much smaller distal chromosome 16 deletions (~400 kb) than previously reported. Beyond this, the severity of ATR-16 syndrome increases with deletion size, but there is no evidence that critical regions determine the developmental abnormalities associated with this disorder. Surprisingly, we find no evidence of telomere position effect or compensatory upregulation of hemizygous genes; however, genetic background effects substantially modify phenotypic abnormalities. CONCLUSIONS: Using ATR-16 as a general model of disorders caused by CNVs, we show the degree to which individuals with contiguous gene syndromes are affected is not simply related to the number of genes deleted but depends on their genetic background. We also show there is no critical region defining the degree of phenotypic abnormalities in ATR-16 syndrome and this has important implications for genetic counselling.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/genetics , DNA Copy Number Variations/genetics , Intellectual Disability/genetics , Monosomy/genetics , alpha-Thalassemia/genetics , Chromosome Deletion , Chromosomes, Human, Pair 16/genetics , Female , Gene Deletion , Humans , Intellectual Disability/diagnosis , Intellectual Disability/pathology , Male , Monosomy/diagnosis , Monosomy/pathology , Phenotype , alpha-Thalassemia/diagnosis , alpha-Thalassemia/pathologyABSTRACT
Next-generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single-gene profession is now developing into gene panel sequencing and whole-exome and whole-genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.
Subject(s)
Genome, Human/genetics , Exome/genetics , High-Throughput Nucleotide Sequencing , Humans , Methyl-CpG-Binding Protein 2/genetics , Polymorphism, Single Nucleotide/geneticsABSTRACT
BACKGROUND: Screening for "non-deletion" α-chain haemoglobin variants resulting from point mutations or short deletions/insertions has attracted an increased interest during recent years, especially in areas where α-thalassaemia is prevalent. We describe a method utilising high resolution melting analysis for detecting the 13 most common "non-deletion" α-thalassaemia mutations in populations around the Mediterranean and Middle East. METHODS: The method comprises: (1) amplification of a 1087 bp fragment for each of the duplicated α-globin genes (HBA1 and HBA2) flanking all 13 mutations using a common forward primer and different reverse primers specific for HBA1 and HBA2, respectively; (2) nested amplification of three fragments in HBA2 flanking 10 mutations and two fragments in HBA1 flanking 5 mutations; (3) High resolution melting analysis of the amplicons using a LightScanner Instrument and LC Green. RESULTS: All 13 "non-deletion" α-chain haemoglobin variants were successfully detected by high resolution melting analysis. All heterozygote samples and eight out of 10 available homozygotes were clearly differentiated from each other and from wild type in the same amplicon. Although not all homozygote samples were distinguishable from wild type samples, this should not present a problem in a clinical setting since all DNA results should be evaluated alongside the haematological and (if relevant) clinical findings in each case. CONCLUSIONS: The 13 "non-deletion" α-chain haemoglobin variants were successfully genotyped by high resolution melting analysis using LightScanner instrument and LCGreen Plus saturating dye. High resolution melting analysis is an accurate mutation scanning tool, advantageous as a closed-tube method, involving no post-PCR manipulations and requiring only around 5 min post-PCR analysis.
Subject(s)
DNA Mutational Analysis/methods , Glycated Hemoglobin/genetics , Hemoglobin A2/genetics , Mutation , Nucleic Acid Denaturation , alpha-Thalassemia/genetics , DNA/blood , DNA/genetics , Genotype , Humans , Polymerase Chain ReactionABSTRACT
BACKGROUND: Alpha-thalassaemia is an autosomal recessive disorder characterized by defective production of the alpha chain of haemoglobin. It is caused mainly by deletions of one or both of the duplicated alpha-globin genes on chromosome 16, and/or by nucleotide variations, known as "nondeletion" mutations. Definition of the alpha globin genotype in carriers supports genetic counselling, and in patients with Hb H disease is useful to predict prognosis and management options. Here, we report a method that facilitates direct detection by naked eye of the 13 most common "nondeletion" alpha-globin gene mutations in populations around the Mediterranean and Middle East. METHODS AND RESULTS: The method comprises (i) PCR amplification of a single 1087 bp fragment for each HBA1 and HBA2 gene (separately); (ii) multiplex primer extension reaction of just 10 cycles, using unpurified amplification product as a template, to incorporate biotin into those allele-specific primers that extend and, finally, (iii) visual detection of the reaction products within minutes by the dipstick biosensor. The method was evaluated by analysing 105 samples of known genotypes and the results were found fully concordant with those obtained by the reference methods. CONCLUSIONS: The proposed assay is particularly suited for small molecular-diagnostic laboratories with a limited budget and a low-to-medium sample volume. In addition this platform represents a very simple and useful genotyping tool to support gene scanning methods whenever nucleotide variations have to be specified.
Subject(s)
Biosensing Techniques/methods , Glycated Hemoglobin/genetics , Hemoglobin A2/genetics , Multiplex Polymerase Chain Reaction/methods , alpha-Globins/genetics , alpha-Thalassemia/diagnosis , Biosensing Techniques/economics , DNA Primers/chemical synthesis , DNA Primers/genetics , Gene Expression , Genetic Testing , Genotype , Humans , Mediterranean Region , Sensitivity and Specificity , alpha-Globins/analysis , alpha-Thalassemia/geneticsABSTRACT
We report the case of a 5-year-old child with clinical and hematological findings consistent with the diagnosis of α-thalassemia intermedia (α-TI). Molecular analysis disclosed the common 3.7 kb deletion in the α-globin gene cluster in trans to a novel in-frame 6 bp deletion in the HBA2 gene. It removes the sequence CCTGGG (or GCCTGG) that normally encodes for alanine (codon 13) and tryptophan (codon 14). Even though several hemoglobin (Hb) variants with mutations affecting codons 13 or 14 have been described, Hb Souli (HBA2: c.[41-46delCCTGGG]) is, to the best of our knowledge, the first variant to be reported where both amino acid residues, α13Ala and α14Trp, are deleted, leading to unstable and rapidly degraded α-globin chains.
Subject(s)
Hemoglobin A2/genetics , Hemoglobins, Abnormal/genetics , alpha-Globins/genetics , alpha-Thalassemia/genetics , Adult , Base Sequence , Child, Preschool , Female , Humans , Male , Sequence Deletion , alpha-Thalassemia/bloodABSTRACT
BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is characterized by complex genetics linked to DNA rearrangements in a polymorphic genomic region of tandemly repeated D4Z4 segments. A panel of FSHD biomarkers including contracted D4Z4 array repeat combined with the 4qA(159/161/168)PAS haplotype has been proposed as molecular signature for defining alleles causally related to FSHD. The aim of the present study was to develop a simple approach for FSHD molecular testing in order to extend studies related to the applicability of FSHD molecular signature in Greek population. METHODS AND RESULTS: The method comprises: (i) visual genotyping of the common 4qA and 10qA subtelomeric haplotypes by a multiplex assay in a dipstick format. (ii) Detection of 4qA161 haplotype in D4Z4 contracted alleles by tri-primer PCR. (iii) Detection of PAS SNP in PLAM region and G>C SNP in the first proximal D4Z4 unit by tri-primer PCR. The method was evaluated by analysing DNA from monoallelic sources representing common 4q and 10q haplotypes, samples from 3 FSHD families, 36 unrelated probands and 38 control individuals of Greek origin. CONCLUSIONS: The proposed method could be a very useful tool for FSHD testing making it more accessible to clinical diagnostic laboratories and the wider FSHD community.
Subject(s)
Molecular Diagnostic Techniques/methods , Muscular Dystrophy, Facioscapulohumeral/diagnosis , Muscular Dystrophy, Facioscapulohumeral/genetics , Alleles , Base Sequence , Chromosomes, Human, Pair 10/genetics , Chromosomes, Human, Pair 4/genetics , Electrophoresis , Genome, Human/genetics , Haplotypes , Humans , Polymerase Chain Reaction , Polymorphism, Genetic , Polymorphism, Single Nucleotide , Regulatory Sequences, Ribonucleic Acid/geneticsABSTRACT
Hemoglobinopathies constitute the most common monogenic disorders worldwide, caused by mutations in the globin genes that synthesize the globin chains of hemoglobin. Synthesis may be reduced (thalassemia) or underlie abnormal hemoglobins. Mutation interactions produce a wide range of disorders. For neonatal and antenatal screening, identification of affected newborns or carriers is achieved by hematological tests. DNA analysis supports definitive diagnosis, and additionally facilitates prenatal diagnosis procedures. Most methods used today have been developed over several decades, with few recent advances in hematology methods. However, DNA methods evolve continuously. With global migration and multiethnic societies the trend is from targeted, population-specific methods towards generic methods, such as Sanger sequencing (point mutations) and multiplex ligation probe amplification (deletions). DNA microarrays constitute an advanced DNA method for some mutation categories. The newest DNA technology is next-generation sequencing. Although not completely ready for routine use currently, next-generation sequencing may soon become a reality for some hemoglobin diagnostic laboratories.
Subject(s)
DNA/analysis , Hemoglobinopathies/diagnosis , Oligonucleotide Array Sequence Analysis , Chromatography, High Pressure Liquid , Electrophoresis , Hemoglobinopathies/genetics , Hemoglobins/genetics , Humans , Mass Spectrometry , Mutation , Sequence Analysis, DNA , Thalassemia/diagnosis , Thalassemia/geneticsABSTRACT
Human leukocyte antigen (HLA) typing of in vitro fertilization (IVF) embryos, aims to establish a pregnancy that is HLA compatible with an affected sibling who requires hematopoietic stem cell transplantation (HSCT). It can be performed with or without preimplantation genetic diagnosis (PGD) for exclusion of a single-gene disorder (SGD) and it is a multistep, technically challenging procedure at every stage. Our purpose was to address the difficulties of genetic analysis by developing a fast, reliable and accurate PGD-HLA protocol, to simplify patient work-up and PGD application, while providing high flexibility for combination with any SGD. Requests included PGD-HLA for ß-thalassemia (ß-thal)/sickle cell disease (most common request), Diamond-Blackfan anemia (DBA), chronic granulomatous disease (CGD) and preimplantation-HLA typing only. For HLA haplotyping, we selected a panel of 26 short tandem repeats (STRs) distributed across the entire HLA locus, following PGD guidelines. When required, mutation detection was performed by both a direct and indirect approach. To support concurrent SGD exclusion and HLA typing, a one-step, single-tube, multiplex fluorescent touchdown-polymerase chain reaction (PCR) was optimized. The described touchdown-PCR was successfully applied for all PGD-HLA protocols. Eight clinical cycles were performed with a diagnosis achieved for 94.7% of amplified biopsied blastomeres. Embryo transfer took place in six cycles, with two pregnancies achieved and two healthy female infants (from a twin pregnancy) born so far. Our protocol enables HLA typing in a single PCR, reducing the risk of contamination and the cost, and providing faster results. It requires minimum optimization before clinical application, irrespective of the SGD involved, decreasing the waiting time from referral to treatment for all PGD-HLA cases.
Subject(s)
Fertilization in Vitro , Genetic Diseases, Inborn/diagnosis , Genetic Diseases, Inborn/genetics , Histocompatibility Testing , Preimplantation Diagnosis , Female , Genetic Testing , Humans , Molecular Typing , Pregnancy , beta-Thalassemia/diagnosis , beta-Thalassemia/geneticsABSTRACT
ß-thalassaemia is one of the commonest autosomal recessive single-gene disorders worldwide. Prenatal tests use invasive methods, posing a risk for the pregnancy itself. Development of a noninvasive prenatal diagnostic method is, therefore, of paramount importance. The aim of the present study is to identify high-heterozygote informative single-nucleotide polymorphisms (SNPs), suitable for the development of noninvasive prenatal diagnosis (NIPD) of ß-thalassaemia. SNP genotyping analysis was performed on 75 random samples from the Cypriot population for 140 SNPs across the ß-globin cluster. Shortlisted, highly heterozygous SNPs were then examined in 101 carrier families for their applicability in the noninvasive detection of paternally inherited alleles. Forty-nine SNPs displayed more than 6% heterozygosity and were selected for NIPD analysis, revealing 72.28% of the carrier families eligible for qualitative SNP-based NIPD, and 92% for quantitative detection. Moreover, inference of haplotypes showed predominant haplotypes and many subhaplotypes with sufficient prevalence for diagnostic exploitation. SNP-based analyses are sensitive and specific for the detection of the paternally inherited allele in maternal plasma. This study provides proof of concept for this approach, highlighting its superiority to NIPD based on single markers and thus providing a blueprint for the general development of noninvasive prenatal diagnostic assays for ß-thalassaemia.
Subject(s)
Polymorphism, Single Nucleotide , Prenatal Diagnosis/methods , beta-Globins/genetics , beta-Thalassemia/diagnosis , Female , Haplotypes , Heterozygote , Humans , Pregnancy , beta-Thalassemia/geneticsABSTRACT
For definitive diagnosis of thalassemia carriers and patients, as well as for prenatal diagnosis, genotype analysis is of fundamental importance. We report a dry-reagent, lateral flow dipstick test that enables visual genotyping (detection by naked eye) of 15 mutations common in Mediterranean populations in the beta-globin gene (HBB). The method comprises 3 simple steps: (i) PCR amplification of a single 1896 bp segment of the beta globin gene flanking all 15 mutations; (ii) a multiplex (10-plex and/or 30-plex) primer extension reaction of the unpurified amplification product using allele-specific primers. Biotin is incorporated in the extended product; (iii) a dry-reagent multi-allele (10-plex) dipstick assay for visual detection of the primer extension reaction products within minutes. The total time required for PCR, primer extension reaction and the dipstick assay is ~2 h. The method was evaluated by genotyping 45 DNA samples of known genotypes and 54 blind samples. The results were fully concordant with reference methods. The method is simple, rapid, and cost-effective. Detection by the dipstick assay does not require specialized instrumentation or highly qualified personnel. The proposed method could be a particularly useful tool in laboratories with limited resources and a basis for point-of-care diagnostics especially in combination with PCR amplification from whole blood.
Subject(s)
DNA Mutational Analysis/instrumentation , DNA Mutational Analysis/methods , Globins/genetics , Mutation/genetics , Genotype , Humans , Point-of-Care Systems , Polymerase Chain Reaction , beta-Thalassemia/diagnosis , beta-Thalassemia/geneticsABSTRACT
Hemoglobinopathies, especially ß-thalassemia (ß-thal), represent an important health burden in Mediterranean countries like Turkey. Some couples prefer the option of preimplantation genetic diagnosis (PGD). However, clinical application of PGD, especially for the monogenic disorders is technically demanding. To ensure reliable results, protocols need to be robust and well standardized. Ideally PGD-PCR (polymerase chain reaction) protocols should be based on multiplex and fluorescent PCR for analysis of the disease-causing mutation(s) along with linked markers across the disease-associated locus. In this study, we aimed to constitute a protocol in single cells involving first round multiplex PCR with primers to amplify the region of the ß-globin gene containing the most common mutations. Two microsatellites linked to the ß-globin gene cluster (D11S4891, D11S2362) and two unlinked (D13S314, GABRB3) microsatellite markers, were used to rule out allele dropout (ADO) and contamination; followed by nested real-time PCR for genotyping the ß-globin mutations. We also investigated the allele frequencies and heterozygote rates of these microsatellites in the Turkish population that have not been reported to date. This protocol was tested in 100 single lymphocytes from heterozygotes with known ß-globin mutations. Amplification failure was detected in one lymphocyte (1%) and ADO was observed in two lymphocytes (2%). No contamination was detected. All results were concordant with the genotypes of the patients. Overall, this protocol was demonstrated to be sensitive, accurate, reliable and rapid for the detection of ß-globin mutations in single cells and shows potential for the clinical application of PGD for hemoglobinopathies in the Turkish population.
Subject(s)
Genotyping Techniques/methods , Lymphocytes/metabolism , Mutation , beta-Globins/genetics , Adolescent , Adult , Alleles , Child , DNA Mutational Analysis/methods , Female , Gene Frequency , Genotype , Humans , Infant , Lymphocytes/cytology , Male , Microsatellite Repeats , Polymerase Chain Reaction/methods , Pregnancy , Pregnancy Complications, Hematologic/diagnosis , Pregnancy Complications, Hematologic/genetics , Preimplantation Diagnosis/methods , Reproducibility of Results , Sensitivity and Specificity , Single-Cell Analysis/methods , Turkey , beta-Thalassemia/diagnosis , beta-Thalassemia/geneticsABSTRACT
In this study we report the development of a generic protocol for preimplantation genetic diagnosis (PGD) of severe α-thalassemia (α-thal) syndromes in α-thal carrier couples of Mediterranean origin. The in silico identification and design of primers for multiplex analysis of short tandem repeats (STRs), was followed by the optimization of polymerase chain reaction (PCR) conditions for multiplexed STR analysis within the α-globin gene cluster (16p3.3) and subsequent optimization and validation of a single-cell multiplex reaction including the selected STRs. Three simple dinucleotide repeats were selected based on their rate of heterozygosity, multiplex PCR efficiency and product size, and location within the α-globin gene cluster. The multiplex PCR was optimized in single lymphocytes with PCR efficiency ranging from 92.5 to 98% and an allele drop-out (ADO) rate of 0 to 9.0% for the three loci. The optimized method was applied in two clinical PGD cycles and genotypes were achieved in 17 out of 18 blastomeres (94%). Transfer of unaffected embryos led to a singleton pregnancy in one of the two couples. The triplex PCR validated for Greek and Cypriot populations is a robust generic method for α-thal PGD.
Subject(s)
Microsatellite Repeats/genetics , Multigene Family , Preimplantation Diagnosis/methods , alpha-Globins/genetics , alpha-Thalassemia/diagnosis , alpha-Thalassemia/genetics , Cyprus , Female , Greece , Humans , Male , Multiplex Polymerase Chain Reaction/methods , Pregnancy , Pregnancy Complications, Hematologic/diagnosis , Pregnancy Complications, Hematologic/genetics , Reproducibility of Results , Sensitivity and Specificity , SyndromeABSTRACT
OBJECTIVES: To determine the molecular basis in a Greek child suspected of having HbH disease and ß-thalassemia trait. METHODS: Standard hematology, Hb electrophoresis, and HPLC. Multiplex ligation-dependent probe amplification (MLPA), direct sequencing, and breakpoint characterization by NimbleGen fine-tiling array analysis. RESULTS: The index patient showed a moderate microcytic hypochromic anemia with normal ZPP and elevated HbA(2) , indicative for ß-thalassemia trait. However, the moderate microcytic hypochromic anemia along with the observation of HbH inclusions in occasional red blood cells suggested a coexisting α-thalassemia. Molecular analysis indicated that the propositus inherited the ß(+) -thalassemia mutation IVS2-745 (c>g) and a novel α(0) -thalassemia deletion from the mother, and the common non-deletion α-thalassemia allele α(2) (-5nt)α from the father. The α(0) -thalassemia deletion, named -â-(BGS) , is approximately 131.6 kb in length. It removes the major regulatory elements along with the functional α-globin genes but leaves the theta-gene intact. CONCLUSIONS: The compound interaction of a ß-thalassemia defect along with a single functional α-globin gene is quite rare. Although patients with HbH/ß-thal and simple HbH disease have comparable levels of Hb, the absence of free ß-globin chains and thus detectable non-functional HbH means that in HbH/ß-thal, the levels of functional Hb are higher, resulting in a better compensated functional anemia. Rare large deletions as the one described here remain undetected by gap-PCR in routine molecular screening. The introduction of MLPA as a diagnostic screening tool may improve laboratory diagnostics for these defects. The use of NimbleGen fine-tiling arrays may give additional information about the precise location of breakpoints.
Subject(s)
alpha-Globins/genetics , alpha-Thalassemia/genetics , beta-Globins/genetics , beta-Thalassemia/genetics , Alleles , Child, Preschool , Chromatography, High Pressure Liquid/methods , Comparative Genomic Hybridization , Family Health , Female , Gene Deletion , Greece , Humans , Male , Sequence Analysis, DNAABSTRACT
BACKGROUND: Toll-like receptor-4 (TLR4) is a central regulators of innate immune response as it interacts with bacterial lipopolysaccharide (LPS) and also with endogenous molecules, such as heat-shock proteins and fibrinogen. Two common single nucleotide polymorphisms, A896G (Asp299Gly) and C1196T (Thr399Ile), have been found in the exon 3 of human TLR4 gene, which lead to structure alteration of the extracellular domain of TLR4 thereby influencing the receptor ability for recognition and ligand binding. METHODS: We propose a simple, rapid and reliable method for the simultaneous detection of the two SNPs in TLR4 gene that involves: (a) exponential amplification of the genomic region that spans the two SNPs, (b) quadruple primer extension (PEXT) reaction using two allele-specific primers per SNP, and (c) a simple-to-perform dipstick test that allows visual and simultaneous detection of the four alleles within minutes without the need for specialized instrumentation. RESULTS: The method was applied to the simultaneous detection of the two SNPs in 90 samples of general Greek population and the results showed 100% concordance with those obtained by direct sequencing. The entire assay, starting from genomic DNA, can be run in less than 1.5h. CONCLUSIONS: The dipstick test eliminates multiple incubation and washing steps that are common in microtiter well-based assays and does not require highly trained personnel. Because of these advantages, it is suitable for the routine clinical laboratory or even for point-of-care testing.
Subject(s)
G-Quadruplexes , Polymerase Chain Reaction , Polymorphism, Single Nucleotide/genetics , Toll-Like Receptor 4/genetics , Alleles , Genotype , HumansABSTRACT
In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.