Direct DNA Analysis Research Articles

Label-free plasmonic spectral profiling of serum DNA

Genetic and epigenetic modifications are linked to the activation of oncogenes and inactivation of tumor suppressor genes. Likewise, the associated molecular alternations can best inform precision medicine for personalized tumor treatment. Therefore, performing characterization of genetic and epigenetic alternations at the molecular level represents a crucial step in early diagnosis and/or therapeutics of cancer. However, the prevailing methods for DNA analysis involve a series of tedious and complicated steps, in which important genetic and epigenetic information could be lost or altered. To provide a potential approach for non-invasive, direct, and efficient DNA analysis, herein, we present a promising strategy for label-free molecular profiling of serum DNA in its pristine form by fusing surface-enhanced Raman spectroscopy with machine learning on a superior plasmonic nanostructured platform. Using DNA methylation and single-point mutation as two case studies, the presented strategy allows a well-balanced sensitive and specific detection of epigenetic and genetic changes at the single-nucleotide level in serum. We envision the presented label-free strategy could serve as a versatile tool for direct molecular profiling in pristine forms of a wide range of biological markers and aid biomedical diagnostics as well as therapeutics.

Comparison of Three DNA Extraction Kits for Assessment of Bacterial Diversity in Activated Sludge, Biofilm, and Anaerobic Digestate

Direct DNA analysis is the most widely used approach for microorganism characterization in natural and built environments; therefore, reliable and effective methods of nucleic acid extraction for samples from particular types of environments are needed. In this study, we compared three commercial kits for metagenomic DNA extraction from three types of biomass: activated sludge, biofilm, and anaerobic digestate. The yield, purity, and quality of DNA were measured, and the effect that the DNA kit had on the subsequent microbial community analysis was assessed with amplified ribosomal intergenic spacer analysis (ARISA). Amplicons were analyzed automatically utilizing capillary electrophoresis. For the activated sludge and digestate, the suggested kit is FastDNA™ Spin Kit for Soil (MP Biomedicals). This kit allowed the highest DNA yield to be obtained and provided the highest biodiversity. For biofilm with a high content of extracellular polymeric substances, the FavorPrep™ Soil DNA Isolation Mini Kit (FAVORGEN) is recommended. This kit allowed to obtain the highest biodiversity and provided the most reliable results of genetic distance assessment in this type of biomass.

Genetic and clinical characterization of 73 Pigmentary Mosaicism patients: revealing the genetic basis of clinical manifestations

BackgroundPigmentary mosaicism constitutes a heterogeneous group of skin pigmentation alterations associated with multisystem involvement. The aim of this study was to establish a complete cytogenetic and molecular characterization of PM patients, emphasizing on searching for possible low chromosomal mosaicism and on establishing an accurate genotype-phenotype correlation.ResultsA total of 73 patients were included (3 months to 18 years of age), 52% male and 48% female. Observed in 69 (95%) patients, the most frequent pattern of pigmentation was fine and whorled BL, which was associated with disseminated skin extent in 41 (59%) patients. Central nervous system (84%) alterations were the most frequent observed in the group of patients, followed by the musculoskeletal (53%) and ophthalmologic (27%) alterations. Considering the pattern of pigmentation, no significant differences in association with skin extent or extracutaneous manifestations were detected. Following a strict cytogenetic analysis strategy, screening metaphases from three different tissues (peripheral blood, hyperpigmented and hypopigmented skin) we found that 23/73 patients had chromosomal abnormalities classified as follows: 1) Mosaic with 2 or more different cell lines with structural alterations n = 19; 2) Polyploidy (mosaic) n = 1 and 3) Alterations in all cells in three different tissues n = 3. SNP array, array CGH and FISH were useful for the complete characterization of the chromosomal aberrations, for the detection of microdeletions in patients with normal karyotype but with strong clinical suspicious of chromosomal alteration, and for a better establishment of genotype-phenotype correlation. In 2 patients we found genes associated with some of the extracutaneous manifestations (SHH, MNX1, PPP2R2C).ConclusionsThis group of 73 patients finely described is the largest series of patients with pigmentary mosaicism reported worldwide. As we showed in this study, the followed analysis strategy allowed the detection of cytogenetic and molecular abnormalities, and made possible the establishment of genotype-phenotype associations in some patients. An important limitation of our study was the analysis of fibroblasts cultures instead of melanocytes and keratinocytes. In some cases the direct molecular DNA analysis of skin biopsy could be another choice.

Evaluation of simulation models to mimic the distortions introduced into squiggles by nanopore sequencers and segmentation algorithms.

Nucleotides ratcheted through the biomolecular pores of nanopore sequencers generate raw picoamperage currents, which are segmented into step-current level signals representing the nucleotide sequence. These ‘squiggles’ are a noisy, distorted representation of the underlying true stepped current levels due to experimental and algorithmic factors. We were interested in developing a simulation model to support a white-box approach to identify common distortions, rather than relying on commonly used black box neural network techniques for basecalling nanopore signals. Dynamic time warped-space averaging (DTWA) techniques can generate a consensus from multiple noisy signals without introducing key feature distortions that occur with standard averaging. As a preprocessing tool, DTWA could provide cleaner and more accurate current signals for direct RNA or DNA analysis tools. However, DTWA approaches need modification to take advantage of the a-priori knowledge regarding a common, underlying gold-standard RNA / DNA sequence. Using experimental data, we derive a simulation model to provide known squiggle distortion signals to assist in validating the performance of analysis tools such as DTWA. Simulation models were evaluated by comparing mocked and experimental squiggle characteristics from one Enolase mRNA squiggle group produced by an Oxford MinION nanopore sequencer, and cross-validated using other Enolase, Sequin R1_71_1 and Sequin R2_55_3 mRNA studies. New techniques identified high inserted but low deleted base rates, generating consistent x1.7 squiggle event to base called ratios. Similar probability density and cumulative distribution functions, PDF and CDF, were found across all studies. Experimental PDFs were not the normal distributions expected if squiggle distortion arose from segmentation algorithm artefacts, or through individual nucleotides randomly interacting with individual nanopores. Matching experimental and mocked CDFs required the assumption that there are unique features associated with individual raw-current data streams. Z-normalized signal-to-noise ratios suggest intrinsic sensor limitations being responsible for half the gold standard and noisy squiggle DTW differences.

Fast-Track Strategy for the Prevention of Hb Bart’s Hydrops Fetalis Syndrome

We propose a fast-track strategy [direct blood DNA analysis using a quantitative real-time polymerase chain reaction (PCR) technique] for the early risk detection and prenatal diagnosis of α(0)-thalassemia (SEA and Thai deletion). Blood DNA samples were obtained from a volunteer group of 1235 ANC couples. They were assessed using quantitative real-time PCR to detect carriers of α(0)-thalassemia (SEA and Thai deletion). At-risk couples were identified, and further prenatal diagnosis by amniocentesis was implemented. Fetal DNA was isolated from the amniotic cells and characterized by quantitative real-time PCR to detect the α(0)-thalassemia mutation, which was reconfirmed using the droplet digital PCR method. Fifteen at-risk couples were identified. The timing of prenatal diagnosis was appropriate for all couples and four of the fetuses were diagnosed with Bart’s hydrops fetalis. The results were compatible with those calculated using the Hardy-Weinberg equation for a recessively inherited single gene disorder. The conclusion was that the fast-track strategy could shorten screening policy timelines, promoting early risk detection for couples and early prenatal diagnosis. The fast-track strategy might be beneficial for the prevention of hemoglobin Bart’s hydrops fetalis syndrome.

1993-2014: two decades of predictive testing for Huntington's disease at the Medical Genetics Unit of the University of Genoa.

BackgroundPredictive testing for Huntington's disease has been available at the Medical Genetics Unit of the University of Genoa from 1987. In 1989, an integrated counseling protocol (geneticist, psychologist, and neurologist) was developed following International Guidelines.MethodsThis is a retrospective analysis of the clinical charts and motivation questionnaires of persons seeking predictive testing through direct DNA analysis from 1993 until 2014, with the aim to evaluate their individual characteristics, motivations, and the outcomes of the counseling protocol.ResultsA total of 299 persons (164 women, 135 men) applied for predictive testing. Most applicants’ features and motivations were similar to those previously described, but surprisingly the percentage of completed protocols was higher among men, 68.5% versus 53.5% (P = 0.011). Likewise, persons over 25 years of age were more likely to take the test than younger applicants (18–25 years): 63.4% versus 48.1% (P = 0.043). In addition, relationship status, having children, and the gender of the affected parent showed different effects on the decision about testing in males and females. No catastrophic reactions were reported during the study period.ConclusionsWe observed that factors influencing the decision‐making process might differ between males and females, and that predictive testing appears a safe procedure if framed within an integrated counseling protocol.

Direct DNA Analysis with Paper-Based Ion Concentration Polarization.

DNA analysis is essential for diagnosis and monitoring of many diseases. Conventional DNA testing is generally limited to the laboratory. Increasing access to relevant technologies can improve patient care and outcomes in both developed and developing regions. Here, we demonstrate direct DNA analysis in paper-based devices, uniquely enabled by ion concentration polarization at the interface of patterned nanoporous membranes in paper (paper-based ICP). Hepatitis B virus DNA targets in human serum are simultaneously preconcentrated, separated, and detected in a single 10 min operation. A limit of detection of 150 copies/mL is achieved without prior viral load amplification, sufficient for early diagnosis of hepatitis B. We clinically assess the DNA integrity of sperm cells in raw human semen samples. The percent DNA fragmentation results from the paper-based ICP devices strongly correlate (R(2) = 0.98) with the sperm chromatin structure assay. In all cases, agreement was 100% with respect to the clinical decision. Paper-based ICP can provide inexpensive and accessible advanced molecular diagnostics.

High heterozygosity frequency of three exonic SNPs of factor V gene (F5): implications for genetic diagnosis

High heterozygosity frequency of three exonic SNPs of factor V gene (F5): implications for genetic diagnosis

Immunohistochemical detection of mutations in the epidermal growth factor receptor gene in lung adenocarcinomas using mutation-specific antibodies.

BackgroundThe recent development of antibodies specific for the major hotspot mutations in the epidermal growth factor receptor (EGFR), L858R and E746_A750del, may provide an opportunity to use immunohistochemistry (IHC) as a screening test for EGFR gene mutations. This study was designed to optimize the IHC protocol and the criteria for interpretation of the results using DNA sequencing as the gold-standard.MethodsTumor sections from fifty lung adenocarcinoma specimens from Chinese patients were immunostained using L858R and E746_A750del-specific antibodies using three different antigen retrieval solutions, and the results were evaluated using three different sets of criteria. The same specimens were used for DNA purification and analysis of EGFR gene mutations.ResultsIn this study the optimal buffer for antigen retrieval was EDTA (pH 8.0), and the optimal scoring method was to call positive results when there was moderate to strong staining of membrane and/or cytoplasm in >10% of the tumor cells. Using the optimized protocol, L858R-specific IHC showed a sensitivity of 81% and a specificity of 97%, and E746_A750del-specific IHC showed a sensitivity of 59% and a specificity of 100%, both compared with direct DNA analysis. Additionally, the mutant proteins as assessed by IHC showed a more homogeneous than heterogeneous pattern of expression.ConclusionsOur data demonstrate that mutation-specific IHC, using optimized procedures, is a reliable prescreening test for detecting EGFR mutations in lung adenocarcinoma.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2059012601872392

Fabry's Disease: A Prospective Multicenter Cohort Study in Young Adults with Cryptogenic Stroke

Stroke in young adults is etiologically diverse and may represent a diagnostic challenge remaining cryptogenic in one-fourth of cases. Limited information is available on the prevalence of Fabry's disease, a treatable multisystem inherited lysosomal storage disorder, and disability in young patients with cryptogenic stroke. The Canadian Fabry Stroke Screening Initiative (CFSSI) is a prospective multicenter cohort study of young adults (age 18-55) presenting with an ischemic stroke, transient ischemic attack, or intracerebral haemorrhage of unknown etiology to stroke centres across Canada. Diagnosis of Fabry's disease is made by direct DNA analysis of blood samples for α-galactosidase gene mutations or polymorphisms. Demographics, clinical information, and investigations including brain Magnetic Resonance Imaging (MRI) are collected. Functional neurological assessment includes neurological examination, the National Institutes of Health (NIH) stroke scale, modified Rankin scale, and the Barthel index. A follow-up interview is conducted by telephone or in person approximately six-months after the index stroke/transient ischemic attack/intracerebral haemorrhage to determine patient outcomes, quality of life, and patient use of medications. Prevalence of positive DNA mutation or single nucleotide polymorphism screens for Fabry's disease as a proportion of total cryptogenic stroke. Secondary outcomes include incident risk of new or recurrent vascular event at six-months, discharge disposition, disability at six-months as measured by the modified Rankin scale, mean time from symptoms onset to the definite etiological diagnosis, and length of hospital stay. This study constitutes the first initiative to determine the prevalence of a positive screen for Fabry's disease in young adults with stroke in Canada. Moreover, the Canadian Fabry Stroke Screening Initiative will provide information on recurrent vascular events, disability at six-months (modified Rankin scale), and disposition in this understudied population.

Componentes de variância genética e ambiental nos padrões de atividade física de gêmeos. Exploração das possibilidades da entropia aproximada DOI: 10.5007/1980-0037.2010v12n4p246

The main objective of this study was to estimate the contribution of genetic factors to physical activity patterns (PAPs) in twins using approximate entropy (ApEn) statistics. The sample consisted of 162 monozygotic and dizygotic twins from Portugal aged 6 to 18 years. Physical activity was measured with a Tritrac-RT3 triaxial accelerometer over 5 days of a usual week. PAPs were described by ApEn using the Cine Wizard software. Zygosity was assessed by direct DNA analysis. Data were analyzed using the SYSTAT 10, STATA 10 and Twinan92 softwares. PAPs were estimated for 5, 3 and 2 days. In addition, structural equation modeling was used to compute different sources of variance genetic, common environmental and unique environmental variance. The level of significance was set at 5%. Sibling aggregation was identified by ApEn analysis, with monozygotic twins showing greater homogeneity. In conclusion, genetic factors accounted for 44 to 89% of the total variation in PAP.

Study of mutations in p53 tumour suppressor gene in human sporadic breast cancers.

Using Polymerase Chain Reaction followed by Single Strand Conformation Polymorphism analysis, we studied thirty seven cases of primary human breast cancer for the presence of mutations in exons 5 and 7 of p53 tumour suppressor gene. Only one of the thirty seven tumours tested (2.7%) showed the electrophoretic mobility shift indicative of a mutation in exon 5 of the p53gene, while no such mobility shift was noted in exon 7. Our finding is in contrast to the findings in previous studies, wherein the mutation frequency has been reported to be 13-30% by direct DNA analysis. This may be related to the ethnicity and local population prevalence rather than technique.

HLA-A allele associations with viral MER9-LTR nucleotide sequences at two distinct loci within the MHC alpha block

The study of the association of the Human Leukocyte Antigen (HLA) alleles and polymorphic retrotransposons such as Alu, HERV, and LTR at various loci within the Major Histocompatibility Complex allows for a better identification and stratification of disease associations and the origins of HLA haplotypes in different populations. This paper provides sequence and association data on two structurally polymorphic MER9-LTR retrotransposons that are located 54 kb apart and in close proximity to the multiallelic HLA-A gene involved in the regulation of the human immune system. Direct DNA sequencing and analysis of the PCR products identified DNA nucleotide variations between the MER9-LTR sequences at the two loci and their associations with HLA-A alleles as potential haplotype and evolutionary markers. All MER9-LTR sequences were haplotypic when associated with common HLA-A alleles. The number of SNP loci was 2.5 times greater for the solo LTR at the AK locus, which is located closer to the HLA-A gene than the solo or 3' LTR at the HG locus. Our study shows that the nucleotide variations of the MER9-LTR DNA sequences are additional informative markers in fine mapping HLA-A genomic haplotypes for future population, evolutionary, and disease studies.

Identical genotypes of an ericoid mycorrhiza-forming fungus occur in roots of Epacris pulchella (Ericaceae) and Leptospermum polygalifolium (Myrtaceae) in an Australian sclerophyll forest

Assemblages of fungi associated with roots of cooccurring Epacris pulchella (Ericaceae) and Leptospermum polygalifolium (Myrtaceae) seedlings at a sclerophyll forest site in New South Wales, Australia, were investigated by direct DNA extraction and analysis of rRNA gene internal transcribed spacer (ITS) products by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analyses. While ordination of the DGGE data suggested that the assemblages did not differ significantly between the two plant taxa, T-RFLP data provided marginal statistical support for the presence of different assemblages. Fungi isolated from roots of both plants were identified by ITS sequence comparisons largely as ascomycetes, several of which had close sequence identity to Helotiales ericoid mycorrhizal (ERM) fungi. One isolate morphotype from E. pulchella had close sequence similarity to ectomycorrhizal fungi in the Cenococcum geophilum complex, and neighbour-joining analysis grouped this strongly with other Australian C. geophilum-like sequences. Distribution of genotypes of an ERM Helotiales ascomycete in root systems of the two plant taxa was also investigated using inter-simple sequence repeat (ISSR)-PCR. Nineteen ISSR genotypes were identified, two of which were present in roots of both plant taxa. The results are discussed in the context of potential mycelial connections between Ericaceae and non-Ericaceae plants.

The genetic basis of hereditary medullary thyroid cancer: clinical implications for the surgeon, with a particular emphasis on the role of prophylactic thyroidectomy

Medullary thyroid cancer (MTC) may occur either sporadically or on a hereditary basis. Hereditary MTC may be observed with either multiple endocrine neoplasia syndromes (MEN 2A and MEN 2B) or as familial MTC (FMTC). Despite the rarity of these syndromes, early diagnosis is especially important, since MTC is a lethal disease if not promptly and appropriately treated. Recently, the development of genetic testing and direct DNA analysis allows the identification of asymptomatic patients. Surgical prophylaxis should be considered in these cases, ideally to prevent the development of MTC. During the recent decade, the concept of 'codon-directed' timing of prophylactic surgery emerged as a reasonable strategy in the management of these patients. Currently, genetic analysis offers the possibility to define genotype-phenotype correlations and to adjust the time of prophylactic surgery. Hereditary MTC is a model of genetically determined cancer in which both diagnostic and therapeutic strategies rely on the identification of specific mutations.

Electroactive intercalators for DNA analysis on microchip electrophoresis

Miniaturized analytical systems, especially microchip CE (MCE), are becoming a promising tool for analytical purposes including DNA analysis. These microdevices require a sensitive and miniaturizable detection system such as electrochemical detection (ED). Several electroactive DNA intercalators, including the organic dye methylene blue (MB), anthraquinone derivatives, and the metal complexes Fe(phen)3 2+ and Ru(phen)3 2+, have been tested for using in combination with thermoplastic olefin polymer of amorphous structure (Topas) CE‐microchips and ED. Two end‐channel approaches for integration of gold wire electrodes in CE‐ED microchip were used. A 250 μm diameter gold wire was manually aligned at the outlet of the separation channel. A new approach based on a guide channel for integration of 100 and 50 μm diameter gold wire has been also developed in order to reduce the background current and the baseline noise level. Modification of gold wire electrodes has been also tested to improve the detector performance. Application of MCE‐ED for ssDNA detection has been studied and demonstrated for the first time using the electroactive dye MB. Electrostatic interaction between cationic MB and anionic ssDNA was used for monitoring the DNA on microchips. Thus, reproducible calibration curves for ssDNA were obtained. This study advances the feasibility of direct DNA analysis using CE‐microchip with ED.

Assessment of Liquid Microbead Arrays for the Screening of Newborns for Spinal Muscular Atrophy

Spinal muscular atrophy is a common neurodegenerative disorder that has recently been considered for inclusion in the next generation of newborn screening regimens. We sought to validate liquid microbead arrays for the identification of affected individuals by direct DNA analysis. Assays were created to detect the homozygous deletions in exon 7 of the SMN1 gene found in approximately 95% of affected individuals by use of 2 different microbead chemistries on the Luminex 200: MultiCode-PLx and Tag-It. A series of 367 blood spots including 164 from affected individuals, 46 from known carriers, and 157 from unaffected individuals were then analyzed with each assay. The MultiCode-PLx assay required 4.2 h to perform and provided correct identification of all 164 samples from affected individuals. Correct exclusion was also made for all 46 carrier and 157 unaffected individual samples. The Tag-It assay required 6.8 h, detected all samples from affected individuals, and excluded all but 1 (99.5%) of the samples from carriers and unaffected individuals. Neither method was sensitive to increasing copy numbers of the SMN2 gene. Both methods showed high sensitivity and specificity for the detection of patients with spinal muscular atrophy. For both methods, ample DNA was extracted from all blood spots for analysis, and SMN2 copy numbers did not interfere. Liquid bead arrays represent a robust method for DNA analysis in newborn screening laboratories.

Evaluating the clinical utility of a molecular genetic test for polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is estimated to affect 1/600–1/1000 individuals worldwide. The disease is characterized by age dependent renal cyst formation that results in kidney failure during adulthood. Although ultrasound imaging may be an adequate diagnostic tool in at risk individuals older than 30, this modality may not be sufficiently sensitive in younger individuals or for those from PKD2 families who have milder disease. DNA based assays may be indicated in certain clinical situations where imaging cannot provide a definitive clinical diagnosis. The goal of this study was to evaluate the utility of direct DNA analysis in a test sample of 82 individuals who were judged to have polycystic kidney disease by standard clinical criteria. The samples were analyzed using a commercially available assay that employs sequencing of both genes responsible for the disorder. Definite disease causing mutations were identified in 34 (∼42%) study participants. An additional 30 (∼37%) subjects had either in frame insertions/deletions, non-canonical splice site alterations or a combination of missense changes that were also judged likely to be pathogenic. We noted striking sequence variability in the PKD1 gene, with a mean of 13.1 variants per participant (range 0–60). Our results and analysis highlight the complexity of assessing the pathogenicity of missense variants particularly when individuals have multiple amino acid substitutions. We conclude that a significant fraction of ADPKD mutations are caused by amino acid substitutions that need to be interpreted carefully when utilized in clinical decision-making.

Celocentesis for In Utero Stem Cell Therapy: Where We Now Stand and Future Directions

As the number of inborn errors of metabolism that can be diagnosed prenatally by direct DNA analysis increases, so will the number of patients interested in having prenatal diagnosis for these conditions. The phenotypic expression of these inherited inborn errors of metabolism in theory could be prevented by the in utero transplantation of donor stem cells. It is the authors' opinion that ultrasound-guided celocentesis performed in the timed-pregnant baboon animal model could have an important role for determining if stem cell transplantation in the nonfunctional immunocompetent embryo has any clinical potential.

A common mutation in the COG7 gene with a consistent phenotype including microcephaly, adducted thumbs, growth retardation, VSD and episodes of hyperthermia

We describe the clinical and biochemical characteristics in three patients from two different families diagnosed with Congenital Disorder of Glycosylation type IIe owing to a defect in Conserved Oligomeric Golgi complex (COG)7; one of the eight subunits of the COG. The siblings and an unrelated single child of consanguineous parents presented with growth retardation, progressive, severe microcephaly, hypotonia, adducted thumbs, feeding problems by gastrointestinal pseudo-obstruction, failure to thrive, cardiac anomalies, wrinkled skin and episodes of extreme hyperthermia. A combined disorder in the biosynthesis of N- and O-linked glycosylation with hyposialylation was detected. Western blot analysis showed a severe reduction in the COG5 and 7 subunits of the COG. A homozygous, intronic splice site mutation (c.169+4A>C) of the COG7 gene was identified in all patients. The phenotype is similar to that previously described in two patients of North African ethnicity with the same mutation, except for the lack of skeletal anomalies and only a mild liver involvement in our patients. We suggest performing protein glycosylation studies and Western blot for the different COG subunits in patients with progressive microcephaly, growth retardation, hypotonia, adducted thumbs and cardiac defects, especially in association with skin anomalies or episodes of hyperthermia. The presence of the characteristic phenotype might warrant direct DNA analysis.