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1.  Hypomorphic, homozygous mutations in Phosphoglucomutase 3 impair immunity and increase serum IgE levels 
Background
Recurrent bacterial and fungal infections, eczema and elevated serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in STAT3 and DOCK8, involved in signal transduction pathways. However, glycosylation defects have not been described in HIES. One crucial enzyme in the glycosylation pathway is Phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of UDP-GlcNAc which is required for the biosynthesis of N-glycans.
Objective
To elucidate the genetic cause in HIES patients who do not carry mutations in STAT3 or DOCK8.
Methods
After establishing a linkage interval by SNP-chip genotyping and homozygosity mapping in two HIES families from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by Western blotting and glycosylation was profiled by mass spectrometry.
Results
Mutational analysis of candidate genes in a 11.9 Mb linkage region on chromosome 6 shared by two multiplex families identified two homozygous mutations in PGM3 which segregated with the disease status and followed a recessive inheritance trait. The mutations predict amino acid changes in Phosphoglucomutase-3; PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in two other affected families, respectively. These hypomorphic mutations have impact on the biosynthetic reactions involving UDP-GlcNAc. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-/tetra-antennary N-glycans. T cell proliferation and differentiation was impaired in patients. Most patients showed developmental delay and many had psychomotor retardation.
Conclusion
Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity, as biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.
doi:10.1016/j.jaci.2014.02.025
PMCID: PMC4825677  PMID: 24698316
Hyper-IgE syndrome; glycosylation; Staphylococcus aureus; STAT3; DOCK8; PGM3
2.  Next-generation bis-locked nucleic acids with stacking linker and 2′-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes 
Nucleic Acids Research  2016;44(5):2007-2019.
Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson–Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson–Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2′-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.
doi:10.1093/nar/gkw021
PMCID: PMC4797291  PMID: 26857548
3.  Spatial sexual dimorphism of X and Y homolog gene expression in the human central nervous system during early male development 
Background
Renewed attention has been directed to the functions of the Y chromosome in the central nervous system during early human male development, due to the recent proposed involvement in neurodevelopmental diseases. PCDH11Y and NLGN4Y are of special interest because they belong to gene families involved in cell fate determination and formation of dendrites and axon.
Methods
We used RNA sequencing, immunocytochemistry and a padlock probing and rolling circle amplification strategy, to distinguish the expression of X and Y homologs in situ in the human brain for the first time. To minimize influence of androgens on the sex differences in the brain, we focused our investigation to human embryos at 8–11 weeks post-gestation.
Results
We found that the X- and Y-encoded genes are expressed in specific and heterogeneous cellular sub-populations of both glial and neuronal origins. More importantly, we found differential distribution patterns of X and Y homologs in the male developing central nervous system.
Conclusions
This study has visualized the spatial distribution of PCDH11X/Y and NLGN4X/Y in human developing nervous tissue. The observed spatial distribution patterns suggest the existence of an additional layer of complexity in the development of the male CNS.
Electronic supplementary material
The online version of this article (doi:10.1186/s13293-015-0056-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13293-015-0056-4
PMCID: PMC4710049  PMID: 26759715
Sex differences; Human embryo development; Female; Male; Gene expression; Protocadherin; Neuroligin; X chromosome; Y chromosome; Brain; Spinal cord; Cortex; Medulla oblongata; Rolling circle amplification; PCDH11X; PCDH11Y; NLGN4X; NLGN4Y; ISLET1; OLIG2; SOX10; NeuN
4.  Visual Task Demands and the Auditory Mismatch Negativity: An Empirical Study and a Meta-Analysis 
PLoS ONE  2016;11(1):e0146567.
Because the auditory system is particularly useful in monitoring the environment, previous research has examined whether task-irrelevant, auditory distracters are processed even if subjects focus their attention on visual stimuli. This research suggests that attentionally demanding visual tasks decrease the auditory mismatch negativity (MMN) to simultaneously presented auditory distractors. Because a recent behavioral study found that high visual perceptual load decreased detection sensitivity of simultaneous tones, we used a similar task (n = 28) to determine if high visual perceptual load would reduce the auditory MMN. Results suggested that perceptual load did not decrease the MMN. At face value, these nonsignificant findings may suggest that effects of perceptual load on the MMN are smaller than those of other demanding visual tasks. If so, effect sizes should differ systematically between the present and previous studies. We conducted a selective meta-analysis of published studies in which the MMN was derived from the EEG, the visual task demands were continuous and varied between high and low within the same task, and the task-irrelevant tones were presented in a typical oddball paradigm simultaneously with the visual stimuli. Because the meta-analysis suggested that the present (null) findings did not differ systematically from previous findings, the available evidence was combined. Results of this meta-analysis confirmed that demanding visual tasks reduce the MMN to auditory distracters. However, because the meta-analysis was based on small studies and because of the risk for publication biases, future studies should be preregistered with large samples (n > 150) to provide confirmatory evidence for the results of the present meta-analysis. These future studies should also use control conditions that reduce confounding effects of neural adaptation, and use load manipulations that are defined independently from their effects on the MMN.
doi:10.1371/journal.pone.0146567
PMCID: PMC4704804  PMID: 26741815
5.  Travel behaviour change in old age: the role of critical incidents in public transport 
European Journal of Ageing  2015;13:75-83.
Older people’s travel behaviour is affected by negative or positive critical incidents in the public transport environment. With the objective of identifying such incidents during whole trips and examining how travel behaviour had changed, we have conducted in-depth interviews with 30 participants aged 65–91 years in the County of Stockholm, Sweden. Out of 469 incidents identified, 77 were reported to have resulted in travel behaviour change, 67 of them in a negative way. Most critical incidents were encountered in the physical environment on-board vehicles and at stations/stops as well as in pricing/ticketing. The findings show that more personal assistance, better driving behaviour, and swift maintenance of elevators and escalators are key facilitators that would improve predictability in travelling and enhance vulnerable older travellers’ feeling of security. The results demonstrate the benefit of involving different groups of end users in future planning and design, such that transport systems would meet the various needs of its end users.
doi:10.1007/s10433-015-0358-8
PMCID: PMC4769306  PMID: 27034646
Older people; Travel behaviour; Public transport; Critical incidents; Qualitative research
6.  Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia 
Mansouri et al. applied targeted deep sequencing to identify mutations within NF-κB core complex genes in CLL. NFKBIE, the gene encoding the inhibitory IκBε molecule, was most frequently mutated, especially in poor-prognostic subgroups of CLL. The authors show that NFKBIE mutations were associated with significantly reduced IkBε expression and p65 inhibition, ultimately leading to NF-κB activation and a more aggressive disease.
NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.
doi:10.1084/jem.20142009
PMCID: PMC4451125  PMID: 25987724
7.  Susceptibility to infections, without concomitant hyper-IgE, reported in 1976, is caused by hypomorphic mutation in the phosphoglucomutase 3 (PGM3) gene 
Clinical Immunology (Orlando, Fla.)  2015;161(2):366-372.
Phosphoglucomutase 3 (PGM3) is an enzyme converting N-acetyl-glucosamine-6-phosphate to N-acetyl-glucosamine-1-phosphate, a precursor important for glycosylation. Mutations in the PGM3 gene have recently been identified as the cause of novel primary immunodeficiency with a hyper-IgE like syndrome. Here we report the occurrence of a homozygous mutation in the PGM3 gene in a family with immunodeficient children, described already in 1976. DNA from two of the immunodeficient siblings was sequenced and shown to encode the same homozygous missense mutation, causing a destabilized protein with reduced enzymatic capacity. Affected individuals were highly prone to infections, but lack the developmental defects in the nervous and skeletal systems, reported in other families. Moreover, normal IgE levels were found. Thus, belonging to the expanding group of congenital glycosylation defects, PGM3 deficiency is characterized by immunodeficiency, with or without increased IgE levels, and with variable forms of developmental defects affecting other organ systems.
Highlights
•Immunodeficiency reported in 1976, is now identified as caused by a mutation in PGM3.•Replacing Ile322 with Thr reduces protein stability and enzyme activity in PGM3.•Identified mutation in PGM3 induces primary immunodeficiency without hyper-IgE.•A PGM3-PID with increased eosinophils but without skeletal or neurological changes
doi:10.1016/j.clim.2015.10.002
PMCID: PMC4695917  PMID: 26482871
CDG, Congenital defects in glycosylation; EBV, Epstein Barr Virus.; GlcNAc-1-P, N-acetyl-glucosamine-1-phosphate.; GlcNAc-6-P, N-acetyl-glucosamine-6-phosphate; HIES, hyper-IgE syndrome; PGM3, Phosphoglucomutase 3; UDP-GalNac, uridine diphosphate N-acetylglucosamine; Primary immunodeficiency; N-acetylglucosamine-phosphate mutase; hyper-IgE syndrome; Congenital defects of glycosylation; CDG
8.  Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ 
Nucleic Acids Research  2015;43(22):e151.
In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.
doi:10.1093/nar/gkv772
PMCID: PMC4678841  PMID: 26240388
9.  Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio 
Scientific Reports  2015;5:12317.
Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.
doi:10.1038/srep12317
PMCID: PMC4511876  PMID: 26202090
10.  The possibility of lifestyle and biological risk markers to predict morbidity and mortality in a cohort of young men after 26 years follow-up 
BMJ Open  2015;5(5):e006798.
Objectives
To study the association between lifestyle and biological risk markers measured at one occasion, morbidity and mortality from cardiovascular disease (CVD) and cancer, and morbidity from diabetes approximately 26 years later.
Design
A follow-up study of a cohort of men, 33–42 years old at baseline.
Setting
Primary healthcare centre in Sweden.
Participants
All 757 men, living in the community of Habo in Sweden in 1985, and all 652 of these participating in a health examination in 1985–1987.
Interventions
Health profile and a health dialogue with a nurse. A doctor invited the high-risk group to further dialogue and examination. Intervention programmes were carried out in the primary healthcare centre and in cooperation with local associations.
Main outcome measures
CVD and cancer diagnoses from the Swedish National Board of Health and Welfare. Data from pharmacy registers of sold drugs concerning diabetes mellitus.
Results
The participants were divided in three groups based on summarised risk points from lifestyle (smoking, physical activity, alcohol consumption) and biological risk markers (body mass index (BMI), blood pressure, serum cholesterol) selected from the health profile. Comparisons were done between these groups. The group with the lowest summarised total risk points had a significantly lower risk for CVD and cancer compared with the group with the highest summarised risk points. The group with the lowest risk points concerning lifestyle had a significantly lower risk for CVD, and the group with lowest risk points for biological risk markers had a significantly lower risk for both CVD and cancer compared with the groups with the highest risk points. Smoking and serum cholesterol were the most important risk factors. In association to diabetes, BMI and smoking were the most important risk factors.
Conclusions
Risk factors measured on one occasion seemed to be able to predict CVD, cancer and diabetes 26 years later.
doi:10.1136/bmjopen-2014-006798
PMCID: PMC4431125  PMID: 25948404
EPIDEMIOLOGY; PRIMARY CARE; PREVENTIVE MEDICINE
11.  Fourth-generation sequencing in the cell and the clinic 
Genome Medicine  2014;6(4):31.
Nearly 40 years ago, DNA was sequenced for the first time. Since then, DNA sequencing has undergone continuous development, passing through three generations of sequencing technology. We are now entering the beginning of a new phase of genomic analysis in which massively parallel sequencing is performed directly in the cell. Two methods have recently been described for in situ RNA sequencing, one targeted and one untargeted, that rely on ligation chemistry. This fourth generation of sequencing technology opens up prospects for transcriptomic analysis, biomarker validation, diagnosis and patient stratification for cancer treatment.
doi:10.1186/gm548
PMCID: PMC4062057  PMID: 25031621
12.  Diagnostics of Primary Immunodeficiency Diseases: A Sequencing Capture Approach 
PLoS ONE  2014;9(12):e114901.
Primary Immunodeficiencies (PID) are genetically inherited disorders characterized by defects of the immune system, leading to increased susceptibility to infection. Due to the variety of clinical symptoms and the complexity of current diagnostic procedures, accurate diagnosis of PID is often difficult in daily clinical practice. Thanks to the advent of “next generation” sequencing technologies and target enrichment methods, the development of multiplex diagnostic assays is now possible. In this study, we applied a selector-based target enrichment assay to detect disease-causing mutations in 179 known PID genes. The usefulness of this assay for molecular diagnosis of PID was investigated by sequencing DNA from 33 patients, 18 of which had at least one known causal mutation at the onset of the experiment. We were able to identify the disease causing mutations in 60% of the investigated patients, indicating that the majority of PID cases could be resolved using a targeted sequencing approach. Causal mutations identified in the unknown patient samples were located in STAT3, IGLL1, RNF168 and PGM3. Based on our results, we propose a stepwise approach for PID diagnostics, involving targeted resequencing, followed by whole transcriptome and/or whole genome sequencing if causative variants are not found in the targeted exons.
doi:10.1371/journal.pone.0114901
PMCID: PMC4263707  PMID: 25502423
13.  Overall Accessibility to Traveling by Rail for the Elderly with and without Functional Limitations: The Whole-Trip Perspective 
Elderly persons’ perceived accessibility to railway traveling depends on their functional limitations/diseases, their functional abilities and their travel behaviors in interaction with the barriers encountered during whole trips. A survey was conducted on a random sample of 1000 city residents (65–85 years old; 57% response rate). The travels were perceived least accessible by respondents with severely reduced functional ability and by those with more than one functional limitation/disease (e.g., restricted mobility and chronic pain). Those who traveled “often”, perceived the accessibility to be better than those who traveled less frequently. For travelers with high functional ability, the main barriers to more frequent traveling were travel costs and low punctuality. For those with low functional ability, one’s own health was reported to be the main barrier. Our results clarify the links among existing functional limitations/functional abilities, the barriers encountered, the travel behavior, and the overall accessibility to traveling. By operationalizing the whole-trip concept as a chain of events, we deliver practical knowledge on vulnerable groups for decision-making to improve the transport environment for all.
doi:10.3390/ijerph111212938
PMCID: PMC4276655  PMID: 25514149
accessibility; travel behavior; functional limitation; barrier; railway travel; older persons
14.  A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections 
Journal of Clinical Microbiology  2014;53(2):425-432.
To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles of Escherichia coli for two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection.
doi:10.1128/JCM.02434-14
PMCID: PMC4298551  PMID: 25411178
15.  Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification 
PLoS ONE  2014;9(11):e111874.
Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.
doi:10.1371/journal.pone.0111874
PMCID: PMC4219791  PMID: 25369034
16.  Redox State and Mitochondrial Respiratory Chain Function in Skeletal Muscle of LGMD2A Patients 
PLoS ONE  2014;9(7):e102549.
Background
Calpain-3 deficiency causes oxidative and nitrosative stress-induced damage in skeletal muscle of LGMD2A patients, but mitochondrial respiratory chain function and anti-oxidant levels have not been systematically assessed in this clinical population previously.
Methods
We identified 14 patients with phenotypes consistent with LGMD2A and performed CAPN3 gene sequencing, CAPN3 expression/autolysis measurements, and in silico predictions of pathogenicity. Oxidative damage, anti-oxidant capacity, and mitochondrial enzyme activities were determined in a subset of muscle biopsies.
Results
Twenty-one disease-causing variants were detected along the entire CAPN3 gene, five of which were novel (c.338 T>C, c.500 T>C, c.1525-1 G>T, c.2115+4 T>G, c.2366 T>A). Protein- and mRNA-based tests confirmed in silico predictions and the clinical diagnosis in 75% of patients. Reductions in antioxidant defense mechanisms (SOD-1 and NRF-2, but not SOD-2), coupled with increased lipid peroxidation and protein ubiquitination, were observed in calpain-3 deficient muscle, indicating a redox imbalance primarily affecting non-mitochondrial compartments. Although ATP synthase levels were significantly lower in LGMD2A patients, citrate synthase, cytochrome c oxidase, and complex I+III activities were not different from controls.
Conclusions
Despite significant oxidative damage and redox imbalance in cytosolic/myofibrillar compartments, mitochondrial respiratory chain function is largely maintained in skeletal muscle of LGMD2A patients.
doi:10.1371/journal.pone.0102549
PMCID: PMC4117472  PMID: 25079074
17.  Gold Nanowire Based Electrical DNA Detection Using Rolling Circle Amplification 
ACS Nano  2014;8(2):1147-1153.
We present an electrical sensor that uses rolling circle amplification (RCA) of DNA to stretch across the gap between two electrodes, interact with metal nanoparticle seeds to generate an electrically conductive nanowire, and produce electrical signals upon detection of specific target DNA sequences. RCA is a highly specific molecular detection mechanism based on DNA probe circularization. With this technique, long single-stranded DNA with simple repetitive sequences are produced. Here we show that stretched RCA products can be metalized using silver or gold solutions to form metal wires. Upon metallization, the resistance drops from TΩ to kΩ for silver and to Ω for gold. Metallization is seeded by gold nanoparticles aligned along the single-stranded DNA product through hybridization of functionalized oligonucleotides. We show that combining RCA with electrical DNA detection produces results in readout with very high signal-to-noise ratio, an essential feature for sensitive and specific detection assays. Finally, we demonstrate detection of 10 ng of Escherichia coli genomic DNA using the sensor concept.
doi:10.1021/nn4058825
PMCID: PMC3936482  PMID: 24433087
gold nanoparticles; rolling circle amplification; nanoelectronics; gold conjugation; metal enhancement
18.  DNA repair genes are selectively mutated in diffuse large B cell lymphomas 
The Journal of Experimental Medicine  2013;210(9):1729-1742.
Mutations in DNA damage response and repair genes correlate with genomic instability in diffuse large B cell lymphomas.
DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis.
doi:10.1084/jem.20122842
PMCID: PMC3754869  PMID: 23960188
19.  In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics 
Oncotarget  2013;4(12):2407-2418.
Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.
PMCID: PMC3926836  PMID: 24280411
Padlock probes; RCA; in situ; KRAS; cancer diagnostics
20.  The Effects on Absorbed Dose Distribution in Intraoral X-ray Imaging When Using Tube Voltages of 60 and 70 kV for Bitewing Imaging 
ABSTRACT
Objectives
Efforts are made in radiographic examinations to obtain the best image quality with the lowest possible absorbed dose to the patient. In dental radiography, the absorbed dose to patients is very low, but exposures are relatively frequent. It has been suggested that frequent low-dose exposures can pose a risk for development of future cancer. It has previously been reported that there was no significant difference in the diagnostic accuracy of approximal carious lesions in radiographs obtained using tube voltages of 60 and 70 kV. The aim of this study was, therefore, to evaluate the patient dose resulting from exposures at these tube voltages to obtain intraoral bitewing radiographs.
Material and Methods
The absorbed dose distributions resulting from two bitewing exposures were measured at tube voltages of 60 and 70 kV using Gafchromic® film and an anatomical head phantom. The dose was measured in the occlusal plane, and ± 50 mm cranially and caudally to evaluate the amount of scattered radiation. The same entrance dose to the phantom was used. The absorbed dose was expressed as the ratio of the maximal doses, the mean doses and the integral doses at tube voltages of 70 and 60 kV.
Results
The patient receives approximately 40 - 50% higher (mean and integral) absorbed dose when a tube voltage of 70 kV is used.
Conclusions
The results of this study clearly indicate that 60 kV should be used for dental intraoral radiographic examinations for approximal caries detection.
doi:10.5037/jomr.2013.4302
PMCID: PMC3887573  PMID: 24422035
dental radiography; dental digital radiography; bitewing radiography; radiation dosage; radiographic image enhancement.
21.  Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection 
PLoS ONE  2013;8(7):e69813.
Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.
doi:10.1371/journal.pone.0069813
PMCID: PMC3713053  PMID: 23874999
22.  National surveys: a way to manage treatment strategies in Parkinson’s disease? Pharmaceutical prescribing patterns and patient experiences of symptom control and their impact on disease 
Background
The purpose of this study was to draw conclusions from patient-reported experiences in two national surveys from Scandinavia with the intention of comparing treatment strategies and increasing our knowledge of factors that affect the experiences of patients with Parkinson’s disease (PD).
Methods
A total of 2000 individuals in Sweden and 1300 in Norway were invited to complete postal surveys covering PD-related issues. Patient experiences of diagnostic procedures, symptom control, and follow-up in PD and the effects on symptom-related quality of life were collected. Pharmaceutical prescription data on anti-PD drugs and administrative data were collected from national registries.
Results
The surveys were completed by 1553 (78%) of the Swedish cohort and 1244 (96%) of the Norwegian cohort. Only small differences were seen in disease duration and age distribution. Statistically as well as clinically significant differences in symptom control, diagnostic, and follow-up procedures, as well as in pharmacological treatment and impact on quality of life, were found between the national cohorts independent of disease duration.
Conclusion
Information from separate national surveys has the potential to increase our knowledge of patient experiences in PD and can be used to compare, evaluate, educate, and guide health care staff and administrators in optimizing health care for patients with the disease.
doi:10.2147/JMDH.S44451
PMCID: PMC3704404  PMID: 23847426
parkinson’s disease; diagnosis; follow-up; pharmaceutical prescription; quality of life; survey
23.  Human Enterovirus Species B in Ileocecal Crohn's Disease 
OBJECTIVES:
Advanced ileocecal Crohn's disease (ICD) is characterized by strictures, inflammation in the enteric nervous system (myenteric plexitis), and a high frequency of NOD2 mutations. Recent findings implicate a role of NOD2 and another CD susceptibility gene, ATG16L1, in the host response against single-stranded RNA (ssRNA) viruses. However, the role of viruses in CD is unknown. We hypothesized that human enterovirus species B (HEV-B), which are ssRNA viruses with dual tropism both for the intestinal epithelium and the nervous system, could play a role in ICD.
METHODS:
We used immunohistochemistry and in situ hybridization to study the general presence of HEV-B and the presence of the two HEV-B subspecies, Coxsackie B virus (CBV) and Echovirus, in ileocecal resections from 9 children with advanced, stricturing ICD and 6 patients with volvulus, and in intestinal biopsies from 15 CD patients at the time of diagnosis.
RESULTS:
All patients with ICD had disease-associated polymorphisms in NOD2 or ATG16L1. Positive staining for HEV-B was detected both in the mucosa and in myenteric nerve ganglia in all ICD patients, but in none of the volvulus patients. Expression of the cellular receptor for CBV, CAR, was detected in nerve cell ganglia.
CONCLUSIONS:
The common presence of HEV-B in the mucosa and enteric nervous system of ICD patients in this small cohort is a novel finding that warrants further investigation to analyze whether HEV-B has a role in disease onset or progress. The presence of CAR in myenteric nerve cell ganglia provides a possible route of entry for CBV into the enteric nervous system.
doi:10.1038/ctg.2013.7
PMCID: PMC3696939  PMID: 23804031
24.  Correction: The Influence of Sulphate Deposition on the Seasonal Variation of Peat Pore Water Methyl Hg in a Boreal Mire 
PLoS ONE  2013;8(5):10.1371/annotation/c81e29f5-0042-4155-bcba-19839ed573b3.
doi:10.1371/annotation/c81e29f5-0042-4155-bcba-19839ed573b3
PMCID: PMC3654722
25.  Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead-Based Readout 
PLoS ONE  2013;8(4):e62015.
Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.
doi:10.1371/journal.pone.0062015
PMCID: PMC3632517  PMID: 23630621

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