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Stored dried blood spots (DBSs) give significant specimens for retrospective detection of inborn errors of metabolisms (IEM) including for validation studies through newborn screening (NBS) programs. Inborn errors of metabolisms have severe medical outcomes in infants and newborns, and initial identification and errors of metabolism treatment can lessen morbidity and mortality. The study investigated the impacts of different conditions for storage (humidity and temperature) experienced throughout the stored dried blood specimen gathering and transportation of the metabolites tested in the NBS program. Stored dried blood spots collected from healthy newborns and kept at room temperatures (RT, 22–25 °C), 37 °C, and 4 °C were examined for acylcarnitine and amino acid panels for 5 days, 1 month, and 2 months using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The outcomes display the decay patterns of amino acids and acylcarnitines at different temperatures and elevated humidity. The study findings indicate that humidity and temperature during sample transit affect the integrity of stored dried blood within a short period. The packaging of blood spots should be done correctly and sent to a newborn screening laboratory immediately. Including a maintained stored dried blood kept along with earlier specimens utilized in retrospective diagnostic cohort studies would give insights into the sample's condition. [ABSTRACT FROM AUTHOR]

Proficiency testing (PT) is an impartial laboratory performance-evaluating system using an independent body. It is a mandatory accreditation requirement and means for improving the laboratory’s performance. The study aimed to evaluate the performance of PT, with a focus on identifying and discussing determinants that influence PT performance at comprehensive specialized hospitals in northwest Ethiopia. A retrospective cross-sectional study was carried out from 2020 to 2022. Using a convenient sampling technique, laboratory tests with recorded PT results in each hospital laboratory were included. A data collection template and customized checklists were used to collect the data. Epi Data Version 3.1 for data entry and STATA Version 14.1 for cleaning and analysis were used. Binary logistic regression analyses were used. Variables with p < 0.05 in the multivariable logistic regression were considered to be statistically significant. Over nine cycles, 3807 PT challenges were distributed. The total failure rate of the laboratories was 32.4%, with a peak failure rate of 40.3% in 2020, after which the failure rate was decline to 20.6% in 2022. Among the five laboratory sections, molecular biology had the lowest failure rate (22.2%), while microbiology had the highest failure rate (56.5%). Multivariate logistic regression revealed that PT results reported without appropriate unit of measurement (AOR 7.5), lack of corrective action for PT nonconformance (AOR 7.1), and reagent unavailability (AOR 6.1) had significant effects on PT performance (p < 0.001). The results of this study showed that the overall performance of the laboratory was lower. Reporting PT results without appropriate units of measurement and not taking corrective action for PT nonconformance were the major aggravating factors for high failure rates. [ABSTRACT FROM AUTHOR]

Genotype–phenotype correlation in congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency ranges from 45 to 97 %. We performed massively parallel sequencing of CYP21A2 on stored newborn bloodspot samples to catalogue the genotypes present in our patients with CAH and enable genotype–phenotype comparison. Participants ≤15 years old with clinically diagnosed CAH were recruited from The Sydney Children's Hospitals Network. Phenotype was classified from clinical and biochemical details in the medical record as salt wasting (SW), simple virilising (SV), non-classic (NC) or an intermediate phenotype (SW/SV; SV/NC). Amplicon-based sequencing for CYP21A2 was performed on stored newborn bloodspot samples by the New South Wales Newborn Bloodspot Screening Laboratory on MiSeq™Dx (Illumina, California). Available genetic test results were also obtained from the medical records. Samples from 67 participants (43 % female, age 0.3–15 years) were sequenced, including 9 sibships. SW phenotype was present in 33/67 participants (49 %), SV in 9 (13 %) and NC in 16 (24 %). Intermediate phenotypes included SW/SV in seven participants (10 %) and SV/NC in two (3 %). Variants were identified in 90/116 alleles (78 %). A complete genotype was available in 47/67 participants (70 %). The most common genotype was homozygous c.293-13A/C>G (I2G) in 7/47 participants (15 %). Genotype correlated with the most commonly reported phenotype in 36/44 cases (82 %). Correlation was higher in SW and NC phenotypes. This study uses genetic testing of newborn bloodspots to identify and characterise the genotypes present in an ethnically diverse Australian population with CAH. It further strengthens our knowledge of genotype–phenotype correlations in CAH. [ABSTRACT FROM AUTHOR]

Background: Southern white rhinoceroses (Ceratotherium simum simum) are an endangered species in decline due to poaching and negative habitat changes. Conservation of the species has become increasingly important and a focus on better human management has become prevalent. One area of management that impacts southern white rhinoceroses is nutritional health monitoring, which is often conducted through blood analysis. Blood analysis conducted during field research can be difficult due to temperature, distance, and limited technological resources, so new methods of fast, and relatively stable blood collection are being pursued. One method that has been used in humans for many years is beginning to make its way into wildlife studies: the use of dried blood spot (DBS) cards. These cards are used as a tool to store single drops of whole blood on specialized filter paper and, once dried, can be used for nutritional biomarker analysis. An area of interest for southern white rhinoceroses and nutrition is monitoring fatty acid percentages for cardiovascular, immune, and reproductive health. The time and temperature limitations for storing blood fractions or liquid whole blood when analyzing fatty acids have been investigated, but few studies have performed storage studies on DBS cards colder than -20 °C or in non-human species. Methods: In order to better understand the limitations of DBS cards and the impact of temperature on fatty acid DBS samples in long-term storage, triplicate samples from seven adult southern white rhinoceroses at the North Carolina Zoo were collected and subjected to three storage treatments (immediate, room temperature (23 °C), or frozen (-80 °C) for 1 year). Results: Stearidonic (18:4w3) (Δ 0.3%), arachdic (20:0) (Δ 0.1%), eicosatetraenoic (20:4w3) (Δ 0.2%), and erucic acid (22:1w9) (Δ 0.1%) were in higher concentration in frozen than initial. Fatty acids in higher concentrations in the initial samples than frozen were myristic (14:0) (Δ 0.2%), mead (20:3w9) (Δ 0.1%), docosatetraenoic (22:4w6) (Δ 0.2%), nervonic (24:1) (Δ 0.1%), and total highly unsaturated fatty acids (HUFAs) (Δ 0.7%). Stearic (18:0) (Δ 2.2%), stearidonic (18:4w3) (Δ 0.3%), arachdic (20:0) (Δ 0.2%), paullinic (20:1w7) (Δ 0.4%), eicosatetraenoic (20:4w3) (Δ 0.1%), eicosapentaenoic (20:5w3) (Δ 0.1%), docosatetraenoic (22:4w6) (Δ 0.2%), nervonic acid (24:1) (Δ 0.2%), monoenes (Δ 1.9%), and total saturates (Δ 3.6%) had higher concentrations in room temperature than initial. Linoleic (18:2w6) (Δ 4.9%), mead acid (20:3w9) (Δ 0.1%), total polyunsaturated fatty acids (5.3%), and total omega-6 fatty acids (Δ 4.8%) had higher concentrations in initial compared to room temperature. Arachidonic (20:4w6) (Δ 0.4%) and omega-3 docosapentaenoic acid (22:5w3) (Δ 0.1%), had higher concentrations in frozen than in room temperature. Discussion: The frozen samples had the fewest statistical differences compared to room temperature samples and essential omega-3 and -6 fatty acids were stable with freezing up to 1 year. While more research is still warranted, current results suggest that DBS samples are best utilized when immediate analysis or -80 °C storage is available. [ABSTRACT FROM AUTHOR]

The spectrum of somatic genetic variation in colorectal adenomas caused by biallelic pathogenic germline variants in the MSH3 gene, was comprehensively analysed to characterise mutational signatures and identify potential driver genes and pathways of MSH3-related tumourigenesis. Three patients from two families with MSH3-associated polyposis were included. Whole exome sequencing of nine adenomas and matched normal tissue was performed. The amount of somatic variants in the MSH3-deficient adenomas and the pattern of single nucleotide variants (SNVs) was similar to sporadic adenomas, whereas the fraction of small insertions/deletions (indels) (21–42% of all small variants) was significantly higher. Interestingly, pathogenic somatic APC variants were found in all but one adenoma. The vast majority (12/13) of these were di-, tetra-, or penta-base pair (bp) deletions. The fraction of APC indels was significantly higher than that reported in patients with familial adenomatous polyposis (FAP) (p < 0.01) or in sporadic adenomas (p < 0.0001). In MSH3-deficient adenomas, the occurrence of APC indels in a repetitive sequence context was significantly higher than in FAP patients (p < 0.01). In addition, the MSH3-deficient adenomas harboured one to five (recurrent) somatic variants in 13 established or candidate driver genes for early colorectal carcinogenesis, including ACVR2A and ARID genes. Our data suggest that MSH3-related colorectal carcinogenesis seems to follow the classical APC-driven pathway. In line with the specific function of MSH3 in the mismatch repair (MMR) system, we identified a characteristic APC mutational pattern in MSH3-deficient adenomas, and confirmed further driver genes for colorectal tumourigenesis. [ABSTRACT FROM AUTHOR]

Background: Dried blood spots (DBS) are a relatively inexpensive source of nucleic acids and are easy to collect, transport, and store in large-scale field surveys, especially in resource-limited settings. However, their performance in whole-genome sequencing (WGS) relative to that of venous blood DNA has not been analyzed for various downstream applications. Methods: This study compares the WGS performance of DBS paired with venous blood samples collected from 12 subjects. Results: Results of standard quality checks of coverage, base quality, and mapping quality were found to be near identical between DBS and venous blood. Concordance for single-nucleotide variants, insertions and deletions, and copy number variants was high between these two sample types. Additionally, downstream analyses typical of population-based studies were performed, such as mitochondrial heteroplasmy detection, haplotype analysis, mitochondrial copy number changes, and determination of telomere lengths. The absolute mitochondrial copy number values were higher for DBS than for venous blood, though the trend in sample-to-sample variation was similar between DBS and blood. Telomere length estimates in most DBS samples were on par with those from venous blood. Conclusion: DBS samples can serve as a robust and feasible alternative to venous blood for studies requiring WGS analysis. [ABSTRACT FROM AUTHOR]

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM. DNA ligase 1 (LIG1) finalizes eukaryotic nuclear DNA synthesis by sealing Okazaki fragments using DNA end-joining reactions. Here the authors, by studying an engineered low-fidelity LIG1, reveal that LIG1 is a highly accurate DNA ligase in vivo. [ABSTRACT FROM AUTHOR]

Molecular assays for detection of nucleic acids in biologic specimens are valuable diagnostic tools supporting clinical diagnoses and therapeutic decisions. Pre-analytical errors, which occur before or during processing of nucleic acid extraction, contribute a significant role in common errors that take place in molecular laboratories. Certain practices in specimen collection, transportation, and storage can affect the integrity of nucleic acids before analysis. Applying best practices in these steps, helps to minimize those errors and leads to better decisions in patient diagnosis and treatment. Widely acceptable recommendations, which are for optimal molecular assays associated with pre-analytic variables, are limited. In this article, we have reviewed most of the important issues in sample handling from bed to bench before starting molecular tests, which can be used in diagnostic as well as research laboratories. We have addressed the most important pre-analytical points in performing molecular analysis in fixed and unfixed solid tissues, whole blood, serum, plasma, as well as most of the body fluids including urine, fecal and bronchial samples, as well as prenatal diagnosis samples. [ABSTRACT FROM AUTHOR]

The mechanisms that underpin how insertions or deletions (indels) become fixed in DNA have primarily been ascribed to replication-related and/or double-strand break (DSB)-related processes. Here, we introduce a method to evaluate indels, orientating them relative to gene transcription. In so doing, we reveal a number of surprising findings: First, there is a transcriptional strand asymmetry in the distribution of mononucleotide repeat tracts in the reference human genome. Second, there is a strong transcriptional strand asymmetry of indels across 2,575 whole genome sequenced human cancers. We suggest that this is due to the activity of transcription-coupled nucleotide excision repair (TC-NER). Furthermore, TC-NER interacts with mismatch repair (MMR) under physiological conditions to produce strand bias. Finally, we show how insertions and deletions differ in their dependencies on these repair pathways. Our analytical approach reveals insights into the contribution of DNA repair towards indel mutagenesis in human cells. Indels that are commonly found in cancer genomes are characterized by non-random sequence composition and localisation. Here, the authors described a method to investigate transcriptional strand asymmetries and sequence-context specific mechanisms that alter the likelihood of insertions and deletions. [ABSTRACT FROM AUTHOR]

Faithful replication and repair of DNA lesions ensure genome maintenance. During replication in eukaryotic cells, DNA is unwound by the CMG helicase complex, which is composed of three major components: the Cdc45 protein, Mcm2-7, and the GINS complex. The CMG in complex with DNA polymerase epsilon (CMG-E) participates in the establishment and progression of the replisome. Impaired functioning of the CMG-E was shown to induce genomic instability and promote the development of various diseases. Therefore, CMG-E components play important roles as caretakers of the genome. In Saccharomyces cerevisiae, the GINS complex is composed of the Psf1, Psf2, Psf3, and Sld5 essential subunits. The Psf1-1 mutant form fails to interact with Psf3, resulting in impaired replisome assembly and chromosome replication. Here, we show increased instability of repeat tracts (mononucleotide, dinucleotide, trinucleotide and longer) in yeast psf1-1 mutants. To identify the mechanisms underlying this effect, we analyzed repeated sequence instability using derivatives of psf1-1 strains lacking genes involved in translesion synthesis, recombination, or mismatch repair. Among these derivatives, deletion of RAD52, RAD51, MMS2, POL32, or PIF1 significantly decreased DNA repeat instability. These results, together with the observed increased amounts of single-stranded DNA regions and Rfa1 foci suggest that recombinational mechanisms make important contributions to repeat tract instability in psf1-1 cells. We propose that defective functioning of the CMG-E complex in psf1-1 cells impairs the progression of DNA replication what increases the contribution of repair mechanisms such as template switch and break-induced replication. These processes require sequence homology search which in case of a repeated DNA tract may result in misalignment leading to its expansion or contraction. Author summary: Processes that ensure genome stability are crucial for all organisms to avoid mutations and decrease the risk of diseases. The coordinated activity of mechanisms underlying the maintenance of high-fidelity DNA duplication and repair is critical to deal with the malfunction of replication forks or DNA damage. Repeated sequences in DNA are particularly prone to instability; these sequences undergo expansions or contractions, leading in humans to various neurological, neurodegenerative, and neuromuscular disorders. A mutant form of one of the noncatalytic subunits of active DNA helicase complex impairs DNA replication. Here, we show that this form also significantly increases the instability of mononucleotide, dinucleotide, trinucleotide and longer repeat tracts. Our results suggest that in cells that harbor a mutated variant of the helicase complex, continuation of DNA replication is facilitated by recombination processes, and this mechanism can be highly mutagenic during repair synthesis through repetitive regions, especially regions that form secondary structures. Our results indicate that proper functioning of the DNA helicase complex is crucial for maintenance of the stability of repeated DNA sequences, especially in the context of recently described disorders in which mutations or deregulation of the human homologs of genes encoding DNA helicase subunits were observed. [ABSTRACT FROM AUTHOR]

Identifying the predominant microbial species in patients with ethmoidal sinusitis is conducive to its successful treatment. The aim of the present study was to determine the microbial composition and the predominant fungal and bacterial species in patients with ethmoidal sinusitis. A sample was obtained from 3 patients with ethmoidal sinusitis and from the ethmoid sinus of 2 healthy volunteers. Those samples were sequenced using an Illumina/Solexa sequencing platform for mapping to human, fungal, and bacterial genomes. Fungal and bacterial expressions in those samples were analyzed through bioinformatics and statistical methods. The sequencing data revealed that the dominant fungal strains in the ethmoidal sinusitis samples compared with the healthy controls (8_S33 and 10_S9) were Aspergillus oryzae and Aspergillus flavus, and the dominant bacterial strains were Haemophilus influenzae and Haemophilus parainfluenzae. Together, these findings indicate that the development of ethmoidal sinusitis is associated with the presence of fungi and bacteria, which may benefit the successful diagnosis and treatment for patients with ethmoidal sinusitis. [ABSTRACT FROM AUTHOR]

Heterochromatin contains a significant part of nuclear DNA. Little is known about the mechanisms that govern heterochromatic DNA stability. We show here that in the yeast Saccharomyces cerevisiae (i) DNA mismatch repair (MMR) is required for the maintenance of heterochromatic DNA stability, (ii) MutLα (Mlh1-Pms1 heterodimer), MutSα (Msh2-Msh6 heterodimer), MutSβ (Msh2-Msh3 heterodimer), and Exo1 are involved in MMR at heterochromatin, (iii) Exo1-independent MMR at heterochromatin frequently leads to the formation of Pol ζ-dependent mutations, (iv) MMR cooperates with the proofreading activity of Pol ε and the histone acetyltransferase Rtt109 in the maintenance of heterochromatic DNA stability, (v) repair of base-base mismatches at heterochromatin is less efficient than repair of base-base mismatches at euchromatin, and (vi) the efficiency of repair of 1-nt insertion/deletion loops at heterochromatin is similar to the efficiency of repair of 1-nt insertion/deletion loops at euchromatin. [ABSTRACT FROM AUTHOR]

Primary immunodeficiencies (PIDs) are genetic disorders impairing host immunity, leading to life-threatening infections, autoimmunity, and/or malignancies. Genomic technologies have been critical for expediting the discovery of novel genetic defects underlying PIDs, expanding our knowledge of the complex clinical phenotypes associated with PIDs, and in shifting paradigms of PID pathogenesis. Once considered Mendelian, monogenic, and completely penetrant disorders, genomic studies have redefined PIDs as a heterogeneous group of diseases found in the global population that may arise through multigenic defects, non-germline transmission, and with variable penetrance. This review examines the uses of next-generation DNA sequencing (NGS) in the diagnosis of PIDs. While whole genome sequencing identifies variants throughout the genome, whole exome sequencing sequences only the protein-coding regions within a genome, and targeted gene panels sequence only a specific cohort of genes. The advantages and limitations of each sequencing approach are compared. The complexities of variant interpretation and variant validation remain the major challenge in wide-spread implementation of these technologies. Lastly, the roles of NGS in newborn screening and precision therapeutics for individuals with PID are also addressed. [ABSTRACT FROM AUTHOR]

Identifying mutations that cause cystic fibrosis (CF) is important for making an early, unambiguous diagnosis, which, in turn, is linked to better health and a greater life expectancy. In patients of African descent, a molecular diagnosis is often confounded by the fact that the majority of investigations undertaken to identify causative mutations have been conducted on European populations, and CF-causing mutations tend to be population specific. We undertook a survey of published data with the aim of identifying causative CF mutations in patients of African descent in the Americas. We found that 1,584 chromosomes had been tested in only 6 countries, of which 876 alleles (55.3%) still remained unidentified. There were 59 mutations identified. Of those, 41 have been shown to cause CF, 17 have no associated functional studies, and one (R117H) is of varying clinical consequence. The most common mutations identified in the patients of African descent were: ΔF508 (29.4% identified in the United States, Colombia, Brazil, and Venezuela); 3120 + 1G>A (8.4% identified in Brazil, the United States, and Colombia); G85E (3.8% identified in Brazil); 1811 + 1.6kbA>G (3.7% identified in Colombia); and 1342 - 1G>C (3.1% identified in the United States). The majority of the mutations identified (81.4%) have been described in just one country. Our findings indicate that there is a need to fully characterize the spectrum of CF mutations in the diaspora to improve diagnostic accuracy for these patients and facilitate treatment. [ABSTRACT FROM AUTHOR]

Clinical heterogeneity in cystic fibrosis ( CF) often causes diagnostic uncertainty in infants without symptoms and in older patients with milder phenotypes. We performed a cross-sectional evaluation of a comprehensive set of clinical and laboratory descriptors in a physician-defined cohort (N = 376; Children's Hospital of Wisconsin and the American Family Children's Hospital CF centers in Milwaukee and Madison, WI, USA) to determine the robustness of categorizing CF (N = 300), cystic fibrosis transmembrane conductance regulator ( CFTR)-related disorder (N = 19), and CFTR-related ( CRMS) metabolic syndrome (N = 57) according to current consensus guidelines. Outcome measures included patient demographics, clinical measures, sweat chloride levels, CFTR genotype, age at diagnosis, airway microbiology, pancreatic function, infection, and nutritional status. The CF cohort had a significantly higher median sweat chloride level (105 mmol/l) than CFTR-related disorder patients (43 mmol/l) and CFTR-related metabolic syndrome patients (35 mmol/l; p ≤ 0.001). Patient groups significantly differed in pancreatic sufficiency, immunoreactive trypsinogen levels, sweat chloride values, genotype, and positive Pseudomonas aeruginosa cultures (p ≤ 0.001). An automated classification algorithm using recursive partitioning demonstrated concordance between physician diagnoses and consensus guidelines. Our analysis suggests that integrating clinical information with sweat chloride levels, CFTR genotype, and pancreatic sufficiency provides a context for continued longitudinal monitoring of patients for personalized and effective treatment. [ABSTRACT FROM AUTHOR]

ABSTRACT Infants are screened for cystic fibrosis (CF) in New York State (NYS) using an IRT-DNA algorithm. The purpose of this study was to validate and assess clinical validity of the US FDA-cleared Illumina MiSeqDx CF 139-Variant Assay (139-VA) in the diverse NYS CF population. The study included 439 infants with CF identified via newborn screening (NBS) from 2002 to 2012. All had been screened using the Abbott Molecular CF Genotyping Assay or the Hologic InPlex CF Molecular Test. All with CF and zero or one mutation were tested using the 139-VA. DNA extracted from dried blood spots was reliably and accurately genotyped using the 139-VA. Sixty-three additional mutations were identified. Clinical sensitivity of three panels ranged from 76.2% (23 mutations recommended for screening by ACMG/ACOG) to 79.7% (current NYS 39-mutation InPlex panel), up to 86.0% for the 139-VA. For all, sensitivity was highest in Whites and lowest in the Black population. Although the sample size was small, there was a nearly 20% increase in sensitivity for the Black CF population using the 139-VA (68.2%) over the ACMG/ACOG and InPlex panels (both 50.0%). Overall, the 139-VA is more sensitive than other commercially available panels, and could be considered for NBS, clinical, or research laboratories conducting CF screening. [ABSTRACT FROM AUTHOR]

Unlabelled: Newborn screening for cystic fibrosis (CF), a chronic progressive disease affecting mucus viscosity, has been beneficial in both improving life expectancy and the quality of life for individuals with CF. In New York State from 2007 to 2012 screening for CF involved measuring immunoreactive trypsinogen (IRT) levels in dried blood spots from newborns using the IMMUCHEM(™) Blood Spot Trypsin-MW ELISA kit. Any specimen in the top 5% IRT level underwent DNA analysis using the InPlex(®) CF Molecular Test. Of the 1.48 million newborns screened during the 6-year time period, 7631 babies were referred for follow-up. CF was confirmed in 251 cases, and 94 cases were diagnosed with CF transmembrane conductance regulated-related metabolic syndrome or possible CF. Nine reports of false negatives were made to the program. Variation in daily average IRT was observed depending on the season (4-6 ng/ml) and kit lot (<3 ng/ml), supporting the use of a floating cutoff. The screening method had a sensitivity of 96.5%, specificity of 99.6%, positive predictive value of 4.5%, and negative predictive value of 99.5%.Conclusion: Considerations for CF screening algorithms should include IRT variations resulting from age at specimen collection, sex, race/ethnicity, season, and manufacturer kit lots.What Is Known: Measuring IRT level in dried blood spots is the first-tier screen for CF. Current algorithms for CF screening lead to substantial false-positive referral rates.What Is New: IRT values were affected by age of infant when specimen is collected, race/ethnicity and sex of infant, and changes in seasons and manufacturer kit lots The prevalence of CF in NYS is 1 in 4200 with the highest prevalence in White infants (1 in 2600) and the lowest in Black infants (1 in 15,400). [ABSTRACT FROM AUTHOR]

During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Polε using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Polε signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Polε signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Polε and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutSα/β to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles. [ABSTRACT FROM AUTHOR]

Three processes act in series to accurately replicate the eukaryotic nuclear genome. The major replicative DNA polymerases strongly prevent mismatch formation, occasional mismatches that do form are proofread during replication, and rare mismatches that escape proofreading are corrected by mismatch repair (MMR). This review focuses on MMR in light of increasing knowledge about nuclear DNA replication enzymology and the rate and specificity with which mismatches are generated during leading- and lagging-strand replication. We consider differences in MMR efficiency in relation to mismatch recognition, signaling to direct MMR to the nascent strand, mismatch removal, and the timing of MMR. These studies are refining our understanding of relationships between generating and repairing replication errors to achieve accurate replication of both DNA strands of the nuclear genome. [ABSTRACT FROM AUTHOR]

ABSTRACT Cadmium is a cytotoxic, carcinogenic, and mutagenic industrial product or byproduct. The correlation between metal exposure and microsatellite instability (MSI) has been reported by several groups. In the present study, 50 C57BL/6J mice at 6 weeks of age were divided into five groups and intraperitoneally injected with 0, 0.25, 0.5, 1, or 2 mg/kg cadmium chloride quaque die alterna for 4 weeks. Then, the liver, kidney, testis, leukocytes, bone marrow, and small intestine were collected from the treated mice and weighed. Portions of these tissues were fixed for further histological analysis, and the remaining tissues were subjected to genomic DNA extraction for the analysis of a panel of 42 microsatellite markers. The liver and testis weight coefficients were significantly changed in the 1 and 2 mg/kg cadmium chloride-treated groups compared with the control group. Simultaneously, severe histopathologic changes in the liver and kidneys, along with a complete disorganization of testicular structure and obvious severe necrosis in the testes were observed in the cadmium-treated group. The cadmium accumulated in the liver and kidneys of the mice in all cadmium-treated groups; the tissue cadmium concentrations were significantly higher than those in the control group. After STR scanning, MSI was found at three loci (D15Mit5, D10Mit266, and DxMit172) in the kidneys and leukocytes of mice in the lower dose groups (0.25 and 0.5 mg/kg). In summary, we have successfully established a sub-chronic cadmium exposure model and confirmed that cadmium exposure can induce MSI in mice. We also identified two loci that could be regarded as 'hotspots' of microsatellite mutation in mice. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 683-692, 2015. [ABSTRACT FROM AUTHOR]

Background: During an October 2005 algal bloom (i.e., a rapid increase or accumulation in the population of algae) off the coast of Nicaragua, 45 people developed symptoms of paralytic shellfish poisoning (PSP) and one person died. PSP in humans is caused by ingestion of saxitoxin, which is a neurotoxin often associated with shellfish contaminated by algal blooms. To explore the relationship between the algal bloom and human illnesses, we performed a case-control study of residents living in a coastal island. We administered a standardized clinical questionnaire, sampled locally harvested seafood and algae, and obtained urine samples for saxitoxin testing from symptomatic and asymptomatic persons. PSP case-patients were defined as island residents who developed at least one neurological symptom during the November 4-16 intoxication period. Seafood and algal samples were analyzed for saxitoxins using the receptor-binding assay and high-performance liquid chromatography. Two urine samples were analyzed for saxitoxins using a newly developed immunoassay. Findings: Three shellfish and two algal samples tested positive for saxitoxins. Ten (9%) of 107 participants developed neurological symptoms during the specified time period and five required hospitalization. While 6 (67%) of 9 possible case-patients and 21 (21%) of 98 controls had eaten fish (p=0.008), all case-patients and 17 (17%) of controls had eaten clams (P<0.0001). The saxitoxin concentration in the urine of a hospitalized case-patient was 21 ng saxitoxin/g creatinine compared to 0.16 ng saxitoxin/g creatinine in the single control patient's urine. Conclusions: These findings suggest that a bloom of saxitoxin-producing algae resulted in saxitoxin accumulation in local clams and was responsible for the PSP intoxication. [ABSTRACT FROM AUTHOR]

Congenital neuromuscular disorders, such as Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), and Pompe disease (acid maltase deficiency [AMD]), are candidates for universal newborn screening (NBS). In this article, we discuss the future path of NBS for these disorders with particular emphasis on DMD NBS, because of the likely approval of new gene-modifying treatments, the possible benefits of earlier treatment with corticosteroids, and the recently demonstrated feasibility of a 2-tiered approach to NBS with screening by creatine kinase (CK) levels in dried blood spots followed by mutation detection in those with elevated CK. The cystic fibrosis (CF) NBS program is a successful model for NBS. CF outcomes have consistently improved into adulthood following introduction of CF NBS because considerable resources have been devoted to practices that include: attention to improving laboratory screening, consistent confirmatory testing and immediate referral of all newly diagnosed infants to designated CF care centers that follow established practice guidelines, and ongoing evaluation of CF care centers via a centralized clinical database. Like CF, DMD, SMA, and infantile AMD are inexorably debilitating and require lifetime multidisciplinary clinical management. NBS would address the delays in diagnosis that prevent patients from receiving timely treatments. Standardized care following early diagnosis would reduce disparities in clinical care and outcomes. NBS in these neuromuscular disorders should be implemented, utilizing lessons learned from the past 20 years of CF NBS: standardized protocols for all patients identified by DMD NBS, longitudinal follow-up in multidisciplinary clinics, and coordinated oversight of these clinics. Ann Neurol 2015;77:189-197. [ABSTRACT FROM AUTHOR]

Evolvability by means of simple sequence repeat (SSR) instability is a feature under the constant influence of opposing selective pressures to expand and compress the repeat tract and is mechanistically influenced by factors that affect genetic instability. In addition to direct selection for protein expression and structural integrity, other factors that influence tract length evolution were studied. The genetic instability of SSRs that switch the expression of antibiotic resistance ON and OFF was modelled mathematically and monitored in a panel of live meningococcal strains. The mathematical model showed that the SSR length of a theoretical locus in an evolving population may be shaped by direct selection of expression status (ON or OFF), tract length dependent (α) and tract length independent factors (β). According to the model an increase in α drives the evolution towards shorter tracts. An increase in β drives the evolution towards a normal distribution of tract lengths given that an upper and a lower limit are set. Insertion and deletion biases were shown to skew allelic distributions in both directions. The meningococcal SSR model was tested in vivo by monitoring the frequency of spectinomycin resistance OFF→ON switching in a designed locus. The instability of a comprehensive panel of the homopolymeric SSRs, constituted of a range of 5–13 guanine nucleotides, was monitored in wildtype and mismatch repair deficient backgrounds. Both the repeat length itself and mismatch repair deficiency were shown to influence the genetic instability of the homopolymeric tracts. A possible insertion bias was observed in tracts ≤G10. Finally, an inverse correlation between the number of tract-encoded amino acids and growth in the presence of ON-selection illustrated a limitation to SSR expansion in an essential gene associated with the designed model locus and the protein function mediating antibiotic resistance. [ABSTRACT FROM AUTHOR]

The analysis of blood spotted and dried on a matrix (i.e., 'dried blood spot' or DBS) has been used since the 1960s in clinical chemistry; mostly for neonatal screening. Since then, many clinical analytes, including nucleic acids, small molecules and lipids, have been successfully measured using DBS. Although this pre-analytical approach represents an interesting alternative to classical venous blood sampling, its routine use is limited. Here, we review the application of DBS technology in clinical chemistry, and evaluate its future role supported by new analytical methods such as mass spectrometry. [ABSTRACT FROM AUTHOR]

DNA mismatch repair greatly increases genome fidelity by recognizing and removing replication errors. In order to understand how this fidelity is maintained, it is important to uncover the relative specificities of the different components of mismatch repair. There are two major mispair recognition complexes in eukaryotes that are homologues of bacterial MutS proteins, MutSα and MutSβ, with MutSα recognizing base-base mismatches and small loop mispairs and MutSβ recognizing larger loop mispairs. Upon recognition of a mispair, the MutS complexes then interact with homologues of the bacterial MutL protein. Loops formed on the primer strand during replication lead to insertion mutations, whereas loops on the template strand lead to deletions. We show here in yeast, using oligonucleotide transformation, that MutSα has a strong bias toward repair of insertion loops, while MutSβ has an even stronger bias toward repair of deletion loops. Our results suggest that this bias in repair is due to the different interactions of the MutS complexes with the MutL complexes. Two mutants of MutLα, pms1-G882E and pms1-H888R, repair deletion mispairs but not insertion mispairs. Moreover, we find that a different MutL complex, MutLγ, is extremely important, but not sufficient, for deletion repair in the presence of either MutLα mutation. MutSβ is present in many eukaryotic organisms, but not in prokaryotes. We suggest that the biased repair of deletion mispairs may reflect a critical eukaryotic function of MutSβ in mismatch repair. [ABSTRACT FROM AUTHOR]

8-oxoG is one of the most common and mutagenic DNA base lesions caused by oxidative damage. However, it has not been possible to study the replication of a known 8-oxoG base in vivo in order to determine the accuracy of its replication, the influence of various components on that accuracy, and the extent to which an 8-oxoG might present a barrier to replication. We have been able to place a single 8-oxoG into the Saccharomyces cerevisiae chromosome in a defined location using single-strand oligonucleotide transformation and to study its replication in a fully normal chromosome context. During replication, 8-oxoG is recognized as a lesion and triggers a switch to translesion synthesis by Pol η, which replicates 8-oxoG with an accuracy (insertion of a C opposite the 8-oxoG) of approximately 94%. In the absence of Pol η, template switching to the newly synthesized sister chromatid is observed at least one third of the time; replication of the 8-oxoG in the absence of Pol η is less than 40% accurate. The mismatch repair (MMR) system plays an important role in 8-oxoG replication. Template switching is blocked by MMR and replication accuracy even in the absence of Pol η is approximately 95% when MMR is active. These findings indicate that in light of the overlapping mechanisms by which errors in 8-oxoG replication can be avoided in the cell, the mutagenic threat of 8-oxoG is due more to its abundance than the effect of a single lesion. In addition, the methods used here should be applicable to the study of any lesion that can be stably incorporated into synthetic oligonucleotides. [ABSTRACT FROM AUTHOR]

Objective: Endometriosis is a painful disease affecting 10-15% of reproductive-age women. Concentrations of several cytokines and angiogenic factors in peritoneal fluid (PF) have been found to correlate with the severity of the disease. However, levels of some analytes vary across the menstrual cycle, and an ideal biomarker of endometriosis has not yet been identified. We have compared the PF concentrations of different cytokines in proliferative and secretory phases in women with and without the disease using the Bio-Plex platform. Methods: PF was aspirated during laparoscopy ( N = 133) and the PF concentrations of 18 cytokines from Bio-Plex panels I and II determined with the serum protocol. Results: Increased PF concentrations of IL-6, IL-18, eotaxin, and MCP-1 were found in endometriosis with no changes with menstrual cycle. Levels of IL-12(p70), ICAM-1, and GRO- α were higher in the secretory phase, while eotaxin concentrations were lower. Conclusion: Of the 18 cytokines tested, IL-6, IL-18 and MCP-1 were the best PF markers of endometriosis. [ABSTRACT FROM AUTHOR]

Intestinal epithelial cells exist within a complex environment that affects how they interpret and respond to stimuli. We have applied a multi-scale in vivo systems approach to understand how intestinal immune cells communicate with epithelial cells to regulate responses to inflammatory signals. Multivariate modeling analysis of a large dataset composed of phospho-signals, cytokines, and immune cell populations within the intestine revealed an intimate relationship between immune cells and the epithelial response to TNF-α. Ablation of lymphocytes in the intestine prompted a decrease in the expression of MCP-1, which in turn increased the steady state number of intestinal plasmacytoid dendritic cells (pDCs). This change in the immune compartment affected the intestinal cytokine milieu and subsequent epithelial cell signaling network, with cells becoming hypersensitive to TNF-α-induced apoptosis in a way that could be predicted by mathematical modeling. In summary, we have uncovered a novel cellular network that regulates the response of intestinal epithelial cells to inflammatory stimuli in an in vivo setting. [ABSTRACT FROM AUTHOR]

Kluyveromyces marxianus is thermotolerant yeast that is able to utilize a wider range of substrates and has greater thermal tolerance than most other yeast species. K. marxianus can assimilate xylose, but its ability to produce ethanol from xylose in oxygen-limited environments is poor. In the present study, the K. marxianus xylose reductase (KmXR) gene ( Kmxyl1) was cloned and the recombinant enzyme was characterized to clarify the factors that limit xylose fermentation in K. marxianus NBRC1777. KmXR is a key enzyme in the xylose metabolism of K. marxianus, which was verified by disruption of the Kmxyl1 gene. The Km of the recombinant KmXR for NADPH is 65.67 μM and KmXR activity is 1.295 U/mg, which is lower than those of most reported yeast XRs, and the enzyme has no activity with coenzyme NADH. This result demonstrates that the XR from K. marxianus is highly coenzyme specific; combined with the extremely low XDH activity of K. marxianus with NADP, the limitation of xylose fermentation is due to a redox imbalance under anaerobic conditions and low KmXR activity. [ABSTRACT FROM AUTHOR]

The clinical use of pulsed electromagnetic fields (PEMF) in osteoarticular pathology is widely extended, although the mechanisms involved are unknown. The aim of this study was to evaluate the action of a new protocol of treatment with PEMF on liquid medium cultures of fibroblast-like cells derivates of mononuclear peripheral blood cells. Fibroblast-like cells growth was obtained in liquid medium culture from mononuclear cells (MNC) of human peripheral blood. The PEMF irradiation protocol included an intensity of 2.25 mT, a frequency of 50 Hz and an application time of 15 min on days 7, 8 and 9 of cell culture. Immunophenotype was performed with specific heterologous monoclonal antibodies for each cell receptor (Vimentin, Cytokeratin, CD34, CD41, CD61 and CD68). The cytokines' production was determined in the supernatant of the culture medium by means of the Luminex technology. The immunophenotype did not show any statistical difference on comparing treated against non-treated cell cultures on any of the days. In the treatment cell population, the proinflammatory cytokines, IL-1β and TNF-α showed a significant decrease on days 14 and 21 of the culture, whilst IL-10 increased significantly on day 21. It is concluded that PEMF irradiation does not alter the cell immunophenotype of the fibroblast-like cell population, but does provoke a decrease in the production of inflammatory-type cytokines (IL-1β, TNF-α) and an increase in cytokines of lymphocytic origin (IL-10). These facts coincide with the chronology of the clinical effect undergone by patients with osteoarticular pathology after PEMF irradiation. [ABSTRACT FROM AUTHOR]

Background: microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies. Results: Here we have applied with an innovative approach, the Luminex® xMAP™ technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies. Conclusions: We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex® xMAP™ technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies. [ABSTRACT FROM AUTHOR]

Background: Endemic Burkitt's lymphoma (BL) is an extranodal tumor appearing predominantly in the jaw in younger children while abdominal tumors predominate with increasing age. Previous studies have identified elevated levels of antibodies to Plasmodium falciparum schizont extracts and Epstein-Barr virus (EBV) viral capsid antigens (VCA) in endemic BL relative to malaria exposed controls. However, these studies have neither determined if there were any differences based on the site of clinical presentation of the tumor nor examined a broader panel of EBV and P. falciparum antigens. Methods: We used a suspension bead Luminex assay to measure the IgG levels against EBV antigens, VCA, EAd, EBNA-1 and Zta as well as P. falciparum MSP-1, LSA-1, and AMA-1 antigens in children with BL (n = 32) and in population-based age-and sex-matched controls (n = 25) from a malaria endemic region in Western Kenya with high incidence of BL. EBV viral load in plasma was determined by quantitative PCR. Results: Relative to healthy controls, BL patients had significantly increased anti-Zta (p = 0.0017) and VCA IgG levels (p < 0.0001) and plasma EBV viral loads (p < 0.0001). In contrast, comparable IgG levels to all P. falciparum antigens tested were observed in BL patients compared to controls. Interestingly, when we grouped BL patients into those presenting with abdominal tumors or with jaw tumors, we observed significantly higher levels of anti-Zta IgG levels (p < 0.0065) and plasma EBV viral loads (p < 0.033) in patients with abdominal tumors compared to patients with jaw tumors. Conclusion: Elevated antibodies to Zta and elevated plasma EBV viral load could be relevant biomarkers for BL and could also be used to confirm BL presenting in the abdominal region. [ABSTRACT FROM AUTHOR]

This review summarises current experimental adipokine investigations and will focus on some of the procedures and techniques that are currently important in the clinical research laboratory. The complexity of measuring adipokines is discussed and the relative success of the various applications in the transition from the laboratory to clinical diagnosis assessed. In addition, as new adipokines continue to emerge, this review will consider the direction research is taking at the cutting edge of novel adipokine discovery. Finally, how a more comprehensive understanding of the pathobiology related to adipokines may enhance innovative therapeutic strategies designed to attenuate the predicted explosion in obesity related diseases will be discussed. [ABSTRACT FROM AUTHOR]

Abstract??An enrichment microsphere immunoassay (MIA) was developed, based on the Luminex xMAP? technology, for the simultaneous (duplex) detection ofPectobacterium atrosepticum(former nameErwinia carotovorasubsp.atroseptica) (Pca) andDickeya dianthicola(former nameErwinia chrysanthemi) (Dcd) in potato plant extracts. Target bacteria in the extracts were enriched for 48?h in a semi-selective broth containing polypectate under low oxygen conditions. Samples were subsequently incubated with antibody-coated colour-coded microspheres (beads) and with secondary antibodies conjugated with Alexa Fluor? 532, a reporter dye. Samples were analyzed with the Luminex analyzer, in which one laser identified each microsphere and another laser the reporter dye conjugated to the secondary antibodies. The assay required minimal sample preparation, could be completed in 1?h, was performed in 96 wells microtitreplates and required no wash steps. The limit of detection for the duplex enrichment MIA was 100?1000?cfu?ml?1, which was a hundred times lower than of an enrichment-ELISA. Without enrichment, the sensitivity of MIA and ELISA was largely similar and ranged between 106and 107cells ml?1. No difference in sensitivity was found between a MIA in a single or duplex format. In a comparative test with non-infected potato plant extracts and extracts from plants infected with Pca or Dcd, results of the enrichment MIA correlated well with those of the enrichment ELISA and enrichment PCR. These results indicate that MIA can be reliably used for multiplex detection of soft rotEnterbacteriaceaein crude potato plant extracts. The technology is an attractive and cost-effective alternative to other detection methods, including ELISA. [ABSTRACT FROM AUTHOR]

Proof of causality of most neuromental disorders (NMD's) is largely unavailable. Lessons from four-decade investigations of the epidemiology, immunology, pathogenesis, prevention and therapy of perinatal infectious agents, which invade directly the nervous system, have led us to propose a new indirect effect hypothesis: maternal transplacentally-acquired antibodies, to agents with epitope molecular mimicry with the developing nervous system, can cross the fetus/infant's blood–nervous system barriers to cause NMD's, clinically manifest years later.Further rationale is provided by relevant evolutionary/developmental (EVO–DEVO) considerations—applicable also to some vaccines. The hypothesis is being tested in: (a) older pregnancy studies with available maternal and newborn sera, and follow-up of the progeny for NMD's; and (b) NMD registry individuals linked to their stored newborn blood spots. Preliminary results support a possible role for schizophrenia of high-tittered antibodies to some agents (toxoplasma, influenza and herpes simplex type 2 virus).A model that includes likely genetic and postnatal influences is schematized and a list of putative agents and factors, based on varying rationales, is tabulated. In case pilot studies are confirmed, the identified agent(s) and antibodies would need to be tested in new prospectively enrolled pregnant women, so as to establish further risk factors leading to possible preventive modalities. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immunocompromised and immunocompetent people. We developed an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species. [ABSTRACT FROM AUTHOR]