FOXP2 mutations are associated with specific speech and language impairments. In songbirds, experimentally altered FoxP2 expression levels in the striatal song nucleus Area X impair vocal learning and song production. Overall FoxP2 protein levels in Area X are low in adult zebra finches and decrease further with singing. However, some Area X medium spiny neurons (MSNs) express FoxP2 at high levels (FoxP2high MSNs) and singing does not change this. Because Area X receives many new neurons throughout adulthood, we hypothesized that the FoxP2high MSNs are newly recruited neurons, not yet integrated into the local Area X circuitry and thus not active during singing. Contrary to our expectation, FoxP2 protein levels did not predict whether new MSNs were active during singing, assayed via immediate early gene expression. However, new FoxP2high MSNs had more complex dendrites, higher spine density and more mushroom spines than new FoxP2low MSNs. In addition, FoxP2 expression levels correlated positively with nucleus size of new MSNs. Together, our data suggest that dynamic FoxP2 levels in new MSNs shape their morphology during maturation and their incorporation into a neural circuit that enables the maintenance and social modulation of adult birdsong.The transcription factor FOXP2 is crucial for the formation and function of cortico-striatal circuits.  FOXP2 mutations in humans affect both the coordination of fine orofacial movements and language perception3. Because songbirds – like humans – need to learn most of their communicative vocalizations, they offer a unique model to study the role of FoxP2 (for nomenclature FOXP2/FoxP2 see Methods) for vocal learning and for the maintenance of learned vocalizations as adults4. Studying the relationship between FoxP2 and vocal learning in songbirds may inform the neurogenetic mechanism underlying the speech deficits in patients carrying FOXP2 mutations for the following reasons. The FoxP2 protein coding sequence is highly conserved between humans and songbirds as are the brain expression patterns, notably in the cerebellum and striatum7. Moreover, genetic manipulations of FoxP2 expression levels in the striatal song nucleus Area X during the critical phase of song learning lead to inaccurate and incomplete imitation of the tutor’s song and more variable vocal production10. This phenotype bears similarities to the specific speech deficits called developmental verbal dyspraxia, DVD (or childhood apraxia of speech), that patients carrying FOXP2 mutations suffer from. The core-phenotype of DVD consists of altered precision, consistency and sequencing of movements underlying speech in the absence of neuromuscular deficits11. In addition, altered FoxP2 levels in adult Area X affect the dopaminergic modulation of corticostriatal signaling important to song variability and affect song maintenance13, stressing the fact that tight regulation of FoxP2 expression is a prerequisite for correct neural transmission in differentiated neural circuits. Additional effects of Foxp2 and its disruption on the embryonic development and the function of neural circuits have been described in mice23. Further evidence for the biological relevance of tight regulation of FoxP2 expression levels comes from the following studies. FoxP2 expression levels in Area X transiently increase during song learning but are lower in adults24. Singing decreases overall FoxP2 levels in Area X but not in the surrounding striatum and the degree of FoxP2 down regulation correlates with the amount of produced song27. This relationship is missing in deafened birds, pointing to an important role of auditory feedback for singing-driven FoxP2 down regulation27. How does singing affect FoxP2 expression at the cellular level? Medium spiny neurons (MSNs), the most abundant cell type in the avian striatum, predominantly express FoxP2 at low levels (FoxP2low) while a subset expresses FoxP2 at very high levels (FoxP2high). Both subtypes are not equally affected by singing; the density of FoxP2high MSNs is not measurably different after singing, contrary to the decreasing density of FoxP2low MSNs24. The authors hypothesized that the difference might be due to the neuronal age. Adult Area X constantly receives new MSNs that originate at the ventricular zone33. FoxP2high MSNs colocalize more frequently with a marker for new neurons than FoxP2low MSNs24. Recently we showed that new MSNs mature and participate in singing activity – as measured by immediate early gene activation – within a timeframe of six weeks34. Whether FoxP2 influences not only the function but also the integration of new neurons into existing circuits is still an open question. Based on the results of Thompson et al. (ref. 21) we hypothesized that FoxP2high MSNs are newly recruited into Area X and need to become FoxP2low MSNs before they can participate in singing. To test this, we labelled neuronal progenitors in adult zebra finches. At different time points after these cells had migrated into Area X, we quantified their expression levels of FoxP2 and whether they also expressed the immediate early gene expression EGR-1 after singing.The forkhead box P2 transcription factor (FOXP2) is linked to speech and language disorders. Heterozygous 37. Foxp2 expression levels vary in striatal MNSs and these differences may be relevant for the morphology of striatal MSNs. Dopamine receptor 1 (D1) expressing MSNs38 express Foxp2 at higher levels than dopamine receptor 2 (D2) expressing MSNs35. These differences in Foxp2 levels may be linked to anatomical differences between D1 and D2 MSNs39. Furthermore, in mice carrying humanized Foxp2 alleles (Foxp2hum/hum mice), Foxp2high MSNs are more numerous in the dorsal striatum and their MSNs have longer dendrites than wildtype mice37. Based on the latter results we hypothesized that FoxP2 levels of new MSNs in adult songbirds correlate with their neural morphology. To test this, we virally labelled neural progenitors and analyzed their FoxP2 expression, dendrite complexity and spine density after migration into Area X.In rodents, Foxp2 expression is associated with neurite outgrowth, neuronal morphology and synapse formation in cortico-striatal circuitshigh neurons. Neurons within the bottom 30% of the measured pixel intensity distribution were considered as FoxP2low. Because we were interested in the two extremes of the expression levels in this study, we did not analyze the new neurons with intermediate FoxP2 expression levels further . At 21 dpi and at 31 dpi 36.17% ± 6.02 (SEM) and 34.91% ± 2.53 (SEM) percent of all BrdU+ cells were FoxP2high neurons, respectively. At 42 dpi the percentage of FoxP2high cells had significantly decreased to 12.95% ± 2.87 (SEM) on average  and 31 dpi  to 42 dpi . Interestingly there was a significant positive relationship with a low effect size between nucleus size and FoxP2 expression levels in all three experimental groups  and detected BrdU+/FoxP2+ cells after 21, 31 and 42 days post BrdU injection (dpi) in Area X of adult male zebra finches Fig. . We foun26. Undirected singing is also associated with expression of the immediate early gene EGR-1 in Area X, which is therefore often used as a molecular readout of the neuronal activity associated with undirected song44. We hypothesized that FoxP2 levels in new FoxP2high neurons needed to be downregulated before activation by singing could occur, resulting in EGR-1 expression. Consequently, we did not expect to find BrdU+/FoxP2high/EGR-1+ MSNs in Area X. To test this, we analyzed BrdU+/EGR-1 ± /FoxP2+ cells in birds that had sung before sacrifice after 21 dpi, 31 dpi and 42 dpi.In adult zebra finches FoxP2 expression levels in Area X are behaviourally regulated. Undirected singing leads to downregulation of FoxP2 mRNA and proteinhigh/EGR-1+ cells in Area X  and 40.28% ± 13.34 (SEM) of the new neurons that expressed FoxP2high were not activated by singing (BrdU+/FoxP2high/EGR-1-)  nor at 31 dpi . Contrary to our hypothesis we found BrdU+/FoxP2a X Fig.  in all g1-) Fig. . At 42 dhigh and FoxP2low new neurons at 31 dpi  and not mushroom spines  or stubby spines .Because of the relationship of FoxP2 levels and nucleus size we wanted to further characterize the morphology of new neurons expressing different FoxP2 levels. We used a lentiviral approach to express Green Fluorescent Protein (GFP) in progenitor cells at their place of birth in the lateral wall of the lateral ventricle Fig. . After 3dpi Fig.  and founons Fig. . Becausesis Fig. . At 42 d34.In the present study we investigated the dynamics of FoxP2 expression in adult-born MSNs in the striatal song nucleus Area X of adult male zebra finches. We show that the new MSN strongly expressed FoxP2 at their arrival in Area X from the ventricular zone (VZ) where they were born 21 days prior. During this stage and at intermediate maturation stages (31 days) one third of new MSNs expressed FoxP2 at high levels. At the late maturation stage (42 days) most new MSNs expressed FoxP2 at low levels Fig. . Togethe34 here we asked if this was linked to FoxP2 levels. After singing FoxP2 mRNA and protein levels are lower in Area X tissue27 than when birds were silent whereas the expression of the immediate early gene EGR-1 increases linearly the more birds sing undirected song41. Analyzing expression levels in individual neurons revealed that only FoxP2low MSNs, but not Foxp2high MSNs seemed to be subject to singing induced FoxP2 downregulation24. We therefore hypothesized that FoxP2high MSNs were not yet connected into the circuit and therefore not regulated by singing. Our data contradict this hypothesis. We show that the induction of the immediate early gene EGR-1 in MSNs after singing was equally likely in FoxP2low and FoxP2high MSNs, suggesting that both were functionally incorporated into the song circuit.Since our previous work demonstrated that new MSNs participate in singing-associated neural activity in Area X45. In our study, the relationship between FoxP2 downregulation and song quantity was present in new MSNs at 42 dpi but not earlier and we therefore suggest that new MSNs start to receive auditory feedback signals between 31 and 42 dpi.Previous work showed that the degree of FoxP2 downregulation correlates with the quantity of produced song and depends on auditory feedback, which is relayed to Area X via the cortical song nucleus HVCFoxP2 promoter contains EGR-1 binding sites46 and that EGR-1 expression is crucial for functional integration of new neurons in the adult rodent hippocampus47.What might cause the age-dependent FoxP2 downregulation in new MSNs? One possibility is that intrinsic mechanisms, depending more on cell age than on extracellular inputs, downregulate FoxP2 during maturation. Another possibility is that EGR-1 gradually decreases FoxP2 levels during every singing event. This latter scenario is consistent with the findings that the low and FoxP2high MSNs. We now show differences between the morphology of adult generated MSNs that express FoxP2 at high or low levels; high FoxP2 levels were associated with greater dendrite complexity and higher dendritic spine density in comparison to neurons with low FoxP2 levels , a negative regulator of synaptogenesis19. Foxp2 specifically promotes corticostriatal synaptogenesis via the repression of Mef2c. Whether a similar mechanism shapes the integration of new MSNs into the avian corticostriatal network remains to be elucidated by future studies. In zebra finch Area X, two direct FoxP2 targets are associated with neuronal outgrowth; the very-low-density-lipoprotein receptor (VLDLR) and the contactin-associated protein-like 2 (CNTNAP2). Their expression correlates positively with FoxP2 in juveniles and in singing adults54. Thus, in new Area X neurons, VLDLR and CNTNAP2 would be expected to be highly activated during singing in FoxP2high MSN but not in FoxP2low MSNs and may thus generate the diverging MSNs morphology we found.What mechanisms might account for morphological differences between FoxP2high MSNs from former FoxP2low MSNs in later maturation phases. If these two subpopulations persist long-term, they might resemble striato-nigral and striato-pallidal MSNs of the direct and indirect pathway in mammals. These MSNs subtypes are morphologically and neurochemically different. Direct pathway MSNs express the dopamine receptor D1 and their dendrites are more complex than indirect pathway MSNs that express the dopamine receptor D255. High Foxp2 levels in D1 MSNs and low Foxp2 in D2 MSNs have been proposed to be linked to this anatomical dichotomy35. The avian direct and indirect pathway through the basal ganglia however is not characterized by different MSN projections but rather by direct and indirect pallidal-like output neurons that project from Area X to the thalamus56. To date, it is not known if MSN subtypes exclusively synapse on either direct or indirect pallidal-like neurons57. Contrary to mammalian MSNs, more than half of the Area X MSNs express multiple dopamine receptors58 so that they cannot be used as markers for indirect versus direct pathway neurons. Investigating potential avian MSNs subtypes and the developmental role FoxP2 plays in those will be of interest for future studies.We would like to propose some speculations regarding possible functions of two MSNs subpopulations that differ in FoxP2 expression levels. We found that these populations differed in nucleus size, dendritic complexity and spine density during an early time period of their integration into Area X. We do not know if the observed morphological differences persist long-term because of a lack of markers that could distinguish former FoxP234. General MSNs function is feed forward inhibition within a cortico-striatal-thalamo-cortical loop during singing59. We hypothesize that new MSNs in adult zebra finches are entrained to produce a correct firing pattern in a plastic phase during their maturation and thus may counteract song drift, as has been suggested before60. This process might be in influenced by varying FoxP2 expression levels and the resulting morphological differences between putative subpopulations of new MSNs. FoxP2high new neurons with a higher dendrite complexity and more dendritic spines might be more receptive to tuning than FoxP2low new neurons. For further interpretations of our findings it will be crucial to gain additional knowledge on the microcircuitry of Area X and on the role neurons play for its function.What might be the function of new MSNs in Area X and how is it affected by FoxP2 expression levels? Our previous study showed that once matured, new MSNs have similar characteristics as older, resident MSNs and are active during singingIn summary, FoxP2 expression levels vary in adult-born MSN at different maturation times after they have been recruited to Area X. We show that the different FoxP2 expression levels correlate with neuronal morphology and spine density. Varying FoxP2 expression levels during a specific time window might permit different target gene activation important for correct incorporation and function of new MSNs in Area X.61, FOXP refers to the human gene, Foxp refers to the mouse gene and FoxP refers to all other species. FOXP, Foxp2 and FoxP2 correspond to the protein product.We follow the nomenclature proposed byTaeniopygia guttata, age >120 days) were used in the present study, bred and housed at the Department of Animal Behaviour at Freie Universität Berlin. The colony was kept under a 12:12 h light:dark-cycle with food and water ad libitum. All experiments were reviewed and approved by the veterinary department of the Freie Universität Berlin and by the ethics committee of the Regional Office for Health and Social Affairs Berlin and were performed in accordance with relevant guidelines and regulations. The permit numbers are G0116/13 and G0296/15.42 adult male zebra finches  in Area X at three time points, e.g. at 21 days , 31 days  or 42 days  after BrdU injections (dpi). In the second experiment we analyzed FoxP2 expression levels and the expression of the early growth response protein 1 (EGR-1) at 21 dpi , 31 dpi  and at 42 dpi (6 birds 156 neurons). In the third experiment we analyzed FoxP2 expression levels, dendrite morphology and spine density of new neurons that were labelled via lentiviral infection. In total we analyzed 52 neurons of 4 zebra finches  at 31 dpi and 23 neurons of 3 zebra finches at 42 dpi .For the analysis of FoxP2 levels and EGR-1 expression in newborn neurons 35 birds received BrdU (50 µg/g) via intramuscular injections in the mornings for 5 consecutive days. Birds were assigned to three groups with different survival times .62.For FoxP2 expression level analysis after BrdU treatment or lentiviral injections, 17 birds were isolated in sound attenuated chambers for one night before sacrifice. In the following morning, birds were kept from singing by the experimenter sitting nearby for 1.5 h after lights went on. For EGR-1 expression analysis in new neurons after singing, 18 birds were kept in sound attenuated chambers for three nights and were perfused in the morning of the 4th day 1.5 h after the lights went on. During those 1.5 h birds had to sing at least 150 motifs to be included in the subsequent analysis of EGR-1 expression. Vocalizations were continuously monitored via Sound Analysis Pro63 containing a GFP reporter gene was generated as described in Lois et al.63 and stereotactically injected into the ventricular zone of 7 birds under isoflurane anesthesia. Titers ranged from 2 × 106 and 3 × 107 viral particles/µl. Birds were fixed in a stereotaxic head holder, with the beak in a 45° angle from the vertical axis. In each hemisphere, approximately 200 µl of virus containing solution were injected into four sites. Following coordinates relative to the bifurcation of the midsagittal sinus were used: anterior-posterior 3.8–4.1, medial-latera −1.3/+1.3, dorsal-ventral −5.0, injection angle: 10° lateral.To label progenitor cells at the lateral wall of the ventricle, the lentiviral expression vector pFUGWFor immunohistochemical staining birds were overdosed with isoflurane and immediately perfused transcardially with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were dissected, post-fixed in 4% PFA for one night and washed for another night in PBS. Brains were cut sagitally or frontally into 50 µm sections using a vibrating microtome . BrdU antigen retrieval required incubation in 2N HCl for 30 min at 37 °C and neutralization with borate buffer. GFP signal was enhanced via antibody staining. All immunostainings were performed according to standard protocols. The following primary antibodies were used: anti FoxP2 , anti EGR-1 , anti BrdU , anti GFP . Fluorescent secondary antibodies were the following: anti-rat-Alexa-Fluor-488 , anti-rabbit-Alexa-Fluor-568 , anti-goat-Alaxa-Fluor-647 . To visualize nuclei, all sections were counterstained with 4′, 6-Diamidin-2-phenylindol .64. Only neurons with spiny long dendrites were included in the analysis. The Rolling Ball Background Subtraction plugin was used to subtract background. We measured the mean pixel intensity of nuclear FoxP2 expression, by positioning a circle of 4 µm in diameter (12.56 µm2) in the center of the BrdU+ nucleus. In total we analyzed the intensity of the FoxP2 expression dependent fluorescence of 166 BrdU+ cells at 21 dpi (n = 5), 295 BrdU+ cells at 31 dpi (n = 6) and 272 BrdU+ cells at 42 dpi (n = 6). FoxP2high were defined as cells that fell into the top 30% of measured mean pixel intensities in one animal . We decided on the 30% value because it covered the FoxP2high expressing cells in the bimodal distribution of all FoxP2 intensities. We defined the neurons that fell into the bottom 30% of measured mean pixel intensities as FoxP2low. As for the FoxP2high cutoff, the bottom 30% contained the low-intensity peak of the bimodal density distribution. Because we particularly wanted to address the effect of high and low FoxP2 expression levels on neuronal properties, neurons with intermediate FoxP2 expression levels were not considered for further analysis. The Simple Neurite Tracer plugin (Fiji) was used to trace individual neurons and we analyzed their total branch length and number of primary dendrites. The traces were then used by the Sholl analysis plugin in Fiji65. We measured intersections of dendrites with concentric circles that were placed every 10 µm starting from the center of the soma. The maximal number of intersections per neuron was extracted from the Sholl analysis dataset. For dendritic spine analysis images were deconvolved using the Tikhonov-Miller algorithm in the DeconvolutionLab plugin in Fiji66. Prior to deconvolution an individual point spread function was generated for each image using the Born and Wolf 3D optical model in the PSF Generator plugin in Fiji67. Semiautomated dendritic spine counts were performed using the software NeuronStudio68 that uses a spine classification algorithm. For spine classification, the default settings were used to classify spines as mushroom, stubby or thin spines: a head-to-neck ratio threshold of 1.1 µm, a height-to-width ratio threshold of 2.5 µm and a minimum mushroom head size of 0.35 µm. A spine is considered mushroom if the head-to-neck ratio is above the threshold and its head is larger than 0.35 µm. A spine is considered stubby if its head-to-neck ratio and its heights-to-width ratio are below threshold. In all other cases spines were classified as thin. On average, we analyzed spines densities on secondary dendrites along the length of 118 µm ± 1.92  per neuron. In total, we analyzed spines of 52 individual neurons of 4 animals in experimental group 31 dpi, and 23 neurons of 3 animals at 42 dpi. Additionally, we measured the dendrite diameter of 44 new neurons in 4 animals  using the line measuring tool in Fiji64. We took 5 measures on each of 3 secondary dendrites per neuron . The experimenter was blind to FoxP2 levels of individual neurons during the whole quantification process, because cells were selected for quantification based on their BrdU+ fluorescence or their EGR-1 fluorescence and FoxP2 fluorescence in a different channel was quantified last. The datasets generated and analysed during the current study are available from the corresponding author on request.Z-Stacks of BrdU+ or GFP+ cells in Area X were obtained with a SP8 confocal microscope (Leica). For FoxP2 scanning, all microscope settings were kept constant. Scans of BrdU+ nuclei were performed using a 63x lens , an image size of 1024 × 1024 pixels and a z-stack size of 1 µm. Whole neurons (GFP+) were imaged using a 63x lens, an image size of 2042 × 2042 pixel and a z-stack size of 1 µm. Acquired images were processed using the Fiji software package69. Significance level was p < 0.05 for all tests. Plots were generated using the ggplot package in R70. For the analysis of FoxP2high neurons we used a Kruskal-Wallis test followed by a Dunn’s test for pairwise comparison. For the analysis of FoxP2low neurons we used ANOVA followed by a Bonferroni’s multiple comparison test. The relationship of (a) FoxP2 levels and nuclear diameter as well as (b) FoxP2+ neurons and singing were determined using a linear regression analysis. Dendritic spine data  and Sholl data were analyzed using the paired Student’s t-test. Data from the spine type analysis, mushroom head size, total dendrite length, number of primary dendrites and dendrite thickness were analyzed using the Mann Whitney test. Number of maximal intersections was analyzed using the Student’s t-test. Choice of test was based on previous analysis for normality using the Shapiro-Wilk-test and variance analysis using the F-test or Levene’s test.The software R was used to analyze data


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SMN1 and SMN2 copy numbers in spinal muscular atrophy (SMA) samples has not been reported. Described here is a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels. The hydrolysis probe-based multiplex dPCR assay quantifies SMN1, SMN2, and the total SMN (SMN1 + SMN2) while using RPPH1 gene as an internal reference control. The quadruplex assay was evaluated with characterized control DNA samples and validated with 15 blinded clinical samples from a previously published study. SMN1 and SMN2 copy numbers were completely concordant with previous results for both the control and blinded samples. The dPCR-based SMA copy number determination was accomplished in 90 min with a walk-away workflow identical to real-time quantitative PCR (qPCR). In summary, presented here is a simple higher-order multiplexing solution on a novel digital PCR platform to meet the growing demand for SMA genotyping and prognostics.Digital PCR (dPCR) technology has been proven to be highly sensitive and accurate in detecting copy number variations (CNV). However, a higher-order multiplexing dPCR assay for measuring  SMA has an autosomal recessive inheritance pattern with an incidence of 1 in 6000–10,000 births4. SMA results from the loss or mutation of survival motor neuron 1 (SMN1) on the q arm (q13) of chromosome 5 and retention of the paralogous survival motor neuron 2 (SMN2)5. SMN1 and SMN2 are nearly identical save 5 key nucleotide differences at the 3′ ends of the genes. SMN2 is functionally distinguishable from SMN1 by a single nucleotide difference (c.840C>T) in exon 7 that disrupts an exonic splice enhancer7. Even though close to 95% of SMA cases result from the loss of SMN1 and retention of SMN2, there is a wide spectrum of clinical disease severity based on the motor development milestones that are achieved8. Numerous studies using different assays and patient cohorts (reviewed in9) have demonstrated a strong inverse correlation between SMN2 copy number and disease severity. There are currently three USA Food and Drug Administration (FDA) approved therapeutic options; nusinersen , onasemnogene abeparvovec  and risdiplam 14. These agents target SMN2 expression. SMN2 copy number is used to design the therapeutic regimens for SMA patients receiving these approved therapies, with immediate treatment for patients with 2 or 3 copies of SMN216. A simple yet highly accurate and sensitive solution for SMN1 and SMN2 copy number detection is needed to meet the rapidly growing demand for informed SMA treatment.Proximal spinal muscular atrophy (SMA) is an early-onset neurodegenerative disease characterized by the loss of α-motor neurons in the anterior horn of the spinal cord which leads to muscle weakness and atrophySMN1 deletions20. MLPA is a multi-step method requiring post-PCR capillary electrophoresis and a long time-to-result (~ 20 h)19. Alternatively, qPCR requires normalization with standard curves. More importantly, neither MLPA nor qPCR can consistently distinguish unit differences in SMN1 or SMN2 when the copy number is greater than 322. Genomic sequencing (GS) technology has made progress on copy number detection. A recent publication reported SMA diagnosis and carrier identification using whole genome sequencing and advanced copy number analysis software23. However, there are still limitations to GS for diagnosis including the cost, sample processing time, complex data analysis and the need for orthogonal validation.Current genotyping and copy number determination methods for SMA are complex, time consuming or lack resolution with higher copy numbers. Multiplex ligation-dependent probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect 24. With dPCR, the prepared PCR sample is distributed across a large number of physically isolated micro-reaction partitions so that each partition will have single digit counts of template DNA or none at all26. The absolute concentration (and confidence level) of the target gene(s) can be calculated by counting the number of positive partitions (containing at least one target molecule) and the number of negative partitions (containing no target molecules) and using a Poisson-based statistical correction. dPCR can reliably and accurately measure SMN1 and SMN2 copy numbers over a wide range, between 0 and 6 copies27. Due to the optical configuration of first-generation dPCR platforms, most of the currently available multiplex assays are duplex or variations of a duplex assay. A recently published report on multiplex dPCR describes simultaneous identification of SMN1 and SMN2 copy numbers by using different signal amplitudes within the same optical channel28. The Combinati  Absolute Q Digital PCR System  alleviates the limitations previously noted to provide absolute quantification of a target gene within a sample for molecular diagnostics and prognosticsSMN1, SMN2, total SMN (SMN1 + 2), and RPPH1, respectively  hybridizes to nucleotide c.840C while the SMN2 probe (VIC) hybridizes to nucleotide c.840T. The total SMN  targets intron 1—a region that is identical between SMN1 and SMN2. The internal reference control is designed to target the highly conserved RPPH1 gene (TAMRA), which is consistently present as 2 copies30.The hydrolysis probe-based multiplex assay is designed to use FAM, VIC, TYE665, and TAMRA to identify SMN1 and SMN2 copy number assays previously developed on the QuantStudio 3D dPCR System , SMN2 only (NA23255), or both SMN1 and SMN2 (NA03815). Representative images of the dPCR results from the analysis software  were normalized against the reference gene (RPPH1) that contains 2 copies per genome. The scatter plots for SMN1 exon 7 , 3:0 (NA17117), and 1:1 (NA03815). For NA03815, the Absolute Q dPCR data showed copy numbers for SMN1 exon 7, SMN2 exon 7, and total SMN intron 1 as 1.1, 1.1 and 2.2, respectively.The optimized assay was first tested on control DNA samples containing are Fig. A shows sn 7 Fig. B, SMN2 en 7 Fig. C, and ton 1 Fig. D shows rEcoRI-digested genomic DNA. Genomic DNA up to 25 ng was also tested on the Absolute Q. There are no apparent differences in SMN1 and SMN2 copy number quantification completed with 2.5 ng template or with 25 ng genomic DNA (Supplementary Table The Absolute Q dPCR assays require less template genomic DNA (2.5 ng) than the QuantStudio 3D dPCR assays which uses 30–60 ng of SMN1, SMN2 and total SMN in 10 genomic DNA test samples from Coriell Cell Repositories were quantified using the SMA multiplex 4-color assay. All 10 Coriell DNA samples were run in triplicate on the Absolute Q dPCR System for assay verification and repeatability. The intra-assay variability—measured by %CV—between the SMN1  for the SMN2 assay and 0.987 (95% CI 0.963–0.996) for the total SMN assay. The SMN1 and SMN2 copy numbers measured with this multiplex assay are concordant with those provided by Coriell Cell Repositories, including one sample with 5 copies of SMN2. Additionally, the 6 copies of total SMN for NA03814 correctly matched the corresponding sums of SMN1 and SMN2 copy numbers. The total SMN copy numbers in all of the samples except for NA11254 were equal to their corresponding sums of SMN1 and SMN2 copy numbers.Copy numbers of MN1 Fig. A, SMN2  within the cohort. The Absolute Q dPCR identified one of the patient-derived sample (MND10) as containing 1 copy of SMN1 and one copy of SMN2. This sample was from an individual harboring a SMN1(p.A2G) missense mutation27. The Absolute Q dPCR SMN1  with 5 copies of total SMN. Bland Altman analysis of the results from SMN1 , the total SMN copy number at exon 7 (4 copies) was greater than the sum of the SMN1 exon 7 (0 copies) and SMN2 exon 7 (2 copies) copy numbers. Based on this result, this sample was hypothesized to have 2 copies of either SMN1 or SMN2 containing a partial deletion of exon 7. The partial deletion of exon 7 in this sample was confirmed orthogonally by junctional PCR (data not shown) and by GS23.Absolute Q and QuantStudio 3D array dPCR SMA assays were compared on a set of blinded samples derived from patient lines n = 15). This set of samples contained gDNAs from 13 SMA patients and 2 non-SMA controls. Using the MN1 Fig. A, SMN2  and the low DNA input amount (~ 2 to 3 ng per reaction) facilitate single molecule partitioning. Finally, SMN1 and SMN2 genes, each approximately 28 kb in length, are both located in the same region of chromosome 5 but they are separated from each other by about 875 kb of genomic sequence. Mechanical sheering of the genomic DNA during DNA sample isolation may further fragment this region thereby reducing the likelihood of both genes being in the same partition. Eliminating the restriction digestion step in the copy number assay, therefore, simplifies the workflow of the assay. Future studies would assess the effects of specimen source and DNA isolation procedure on the need for either a shearing or digestion step prior to dPCR analysis.Some dPCR copy number variation assays rely on digestion of the template genomic DNA with a restriction endonuclease31. The shortened run-time along with minimal hands-on operation could facilitate platform adoption into diagnostic and prognostic settings.In addition to eliminating the need for a pre-PCR digestion step, the dPCR workflow is simplified by integration of multiple systems into a single instrument. The first-generation of dPCR systems have separate instruments for microfluidic partitioning of the PCR reactions and template DNA, PCR amplification and raw data acquisition. This required significant hands-on-time and training for each unique workflow, which, in turn, increased the probability of operator error or process variability. The Absolute Q dPCR System combines sample partitioning, thermocycling, and raw data acquisition into a single benchtop instrument. As a result, the Absolute Q dPCR System has an average run-time that is 73% faster than first-generation dPCR systemsSMN1 and SMN2 copy numbers, dPCR can also identify partial deletions in SMN1/SMN2 and gene conversion events that generate hybrid SMN genes. By comparing total SMN copy number to the sum of SMN1 exon 7 and SMN2 exon 7 copy numbers, partial deletions of SMN1 or SMN2 can be identified. One of the patient-derived cell lines tested in this study (MND003) contained a partial deletion of exon 7, which was confirmed orthogonally (data not shown). Additionally, one of the control samples (NA11254) possibly contains a partial deletion as the total SMN copy number was found to be 3 at intron 1 while the sum of SMN1 and SMN2 copy numbers at exon 7 was 2. The discrepant results with this sample are not a consequence of a lack of specificity in the intron 1 primers or probe since the in silico specificity scores for these sequences were high, i.e. all had a BLAT score of 22, which exceeds the minimal threshold of 2032. These results need to be confirmed orthogonally by junctional PCR or GS in order to verify that the partial deletion is indeed the reason for the discrepancy. Partial SMN deletions, particularly resulting from the loss of exons 7 and/or 8 (SMN1/2Δ78), have been previously reported in the literature using several approaches including MLPA and GS33. Gene conversion events can create hybrid SMN genes where part of the gene is SMN1 and another part is SMN234. The most commonly identified hybrid gene is SMN1 with c.840T (i.e. SMN2), however there are other SMN1/SMN2 hybrid gene variations. Other SMN1/SMN2 hybrid genes have different exon 7 inclusion efficiencies compared to SMN1 or SMN236. It is important to correctly identify hybrid SMN genes as they can potentially lead to unexpected responses to current therapeutic options. Future work involving the development of a panel of assays targeting the single nucleotide difference between SMN1 and SMN2 at exon 8, as well as the intronic single nucleotide differences, would be useful for the identification of partial deletions in SMN genes as well as hybrid SMN genes.In addition to determining SMN1 are present in cis on the same chromosome. A single nucleotide polymorphism within SMN1  is associated with the cis configuration of SMN137. In addition, this dPCR assay cannot detect intragenic point mutations in SMN1, which account for 3–5% of SMA cases8. Future studies are needed to develop dPCR assays that are able to detect these single nucleotide changes. These assays could potentially be integrated into an expanded multiplexing assay panel alongside the current SMN1/SMN2 differentiating dPCR assays described here.A limitation of the copy number assay described in this study is its inability to detect silent SMA carriers (2 + 0) where both copies of 39. Dried blood spots (DBS) are a common source material for SMA newborn screening. A 3-mm diameter DBS typically yields ~ 140 ng gDNA (in a volume of 50 μL) using column-based DNA extraction40. This study demonstrates that SMN1 and SMN2 copy numbers can be accurately measured with as little as 2–3 ng gDNA, so the Absolute Q and MAP plates could reliably be used for orthogonal validation of SMA carrier status or SMN2 copy number. As the technology is further developed, quadraplex dPCR could potentially become a primary SMA screening tool.Newborn screening of SMA is becoming more available and routine in the United States as well as many other countries41) and other technologies such as mass spectrometry ($3.00 per sample42). These per-sample costs are exclusive of the cost associated with labor, which is reduced with the lower hands-on time of the Absolute Q dPCR System.The acquisition cost for the Absolute Q dPCR System is comparable to high-end qPCR instrumentation, and somewhat lower than other currently available dPCR platforms. In this study, the consumable (SMA reagents and MAP plates) costs were $2.36 per test. This is less than typical per-sample costs for available droplet dPCR systems ($3.80 per sample24). We have demonstrated that this system can identify a rare single nucleotide variant (EGFR(T790M)) associated with non-small cell lung carcinoma as well as fusion genes (BCR-ABL1 and CCDC88C-FLT3) that are associated with different types of cancer29.The Absolute Q dPCR quadruplex assay approach could be extended to identification of copy number variations associated with other diseases, in addition to SMA. dPCR has been used to identify rare gene variants as well as differences in copy number of multiple genes associated with pediatric-onset disorders . NA03814 and NA03815 samples were from known SMA carriers, i.e. 1 copy of SMN1.Control samples of human genomic DNA (n = 10) were obtained from Coriell Cell Repositories  as described previously29. Approval for cell line generation was provided by the Nemours Institutional Review Board and this study is registered on ClinicalTrials.gov (NCT01754441 and NCT02532244). This study was approved by the Nemours Institutional Review Board (#764456). Written informed consent or assent was obtained for each patient-derived cell line. Each cell line was de-identified. For all samples tested, the tester was blinded to the previously measured SMN1 and SMN2 copy numbers. All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or research committees and with the 1964 Helsinki declaration and its later amendments of comparable ethical standards.Patient-derived samples (n = 15) were procured from the Motor Neuron Diseases Research Laboratory . Genomic DNA was isolated from patient-derived fibroblast and lymphoblastoid cell lines and the 29. Each unit is loaded with 10 µL of sample mixture and more than 90% of the loaded sample is partitioned and analyzed in ~ 20,000 pico-scale partitions. The partition volume in MAP plates is defined by the physical dimensions of the microarray chambers and not by a stochastic process such as fluid emulsions, which is important because consistent partition volume is a critical component of the dPCR statistical correction model. The physical array ensures that all samples across all plates yield ~ 20,000 analyzed partitions and minimal sample is lost to dead volume or compromised partitions.The Combinati Absolute Q Digital PCR System consists of Microfluidic Array Partitioning (MAP) consumable plates and a fully integrated instrument that automates partitioning of reagents in the plate, PCR thermocycling and 5-color fluorescence image acquisition. The MAP plate has a standard microplate footprint capable of running up to 16 samples per dPCR runThe Absolute Q digital PCR System has a walk-away workflow identical to traditional qPCR. The MAP plate is loaded via pipette with 10 μL of PCR mix and an overlay of 10 μL Isolation Buffer in each well. The wells are then capped with specialized gaskets. The plate is then placed into the Absolute Q tray and retracted into the system. The Absolute Q uses positive pressure from an on-board compressor to partition the sample within the MAP plate without the need for microfluidic valves, sealing films or other moving parts. The MAP slides are constructed of a cyclo olefin polymer  film that seals the microfluidic features that are molded into a separate piece of thicker material. Four identical slides are bonded to a rigid, microtiter format plate frame that includes the loading wells to complete the plate assembly. The thin film becomes gas permeable when positive pressure is applied to a well containing reagents. As the reagent enters the microfluidic features, air is passed out of the partition through the film. This allows reagent to completely fill dead-ended partitions and prevents any bubbles from forming inside of the microfluidic features. The Isolation Buffer overlay follows the reagent and physically separates the reagent reaction volumes to complete the partitioning. Positive pressure applied to the consumable during the PCR thermocycling prevents any evaporation and ensures that bubbles will not form and disrupt the isolated micro-reactions.Before and after PCR thermocycling, entire arrays are imaged with up to five optical channels configured for the most commonly used dyes, including a ubiquitous quality control dye (ROX) used to verify proper partition filling and finding. The images taken before PCR are subtracted from the after-PCR images to remove background noise. Combinati Analysis software automatically applies optical crosstalk compensation and classifies the partitions using a convoluted neural network algorithm to eliminate false positives/ negatives and ensure robust quantification results. The full dPCR process occurs within the single benchtop instrument without operator’s interaction after setting up the protocol parameters Fig. . The str44 and UCSC In-Silico PCR  using default settings. For Primer-BLAST, each primer was required to have at least 2 total mismatches to unintended targets, including at least 2 mismatches with the last 5 base pairs at the 3′ end. For In-Silico PCR, the minimum match size was 15 and a minimum BLAT32 score of 20, using the GRCh38/hg38 human genome assembly. Primers and probes were also evaluated for primer dimers and cross primer interactions using Multiple Primer Analyzer  with the optimal sensitivity value (3) for dimer detection. All of the oligos were synthesized by Integrated DNA Technologies and Thermo Fisher Scientific. The assay includes a total of 3 pairs of primers and 4 probes with the SMN1 and SMN2 targets sharing a common pair of primers. The FAM-labeled SMN1 and VIC-labeled SMN2 probes were designed utilizing the signature c.840 single nucleotide difference between exon 7 of SMN1 (c.840C) and SMN2 (c.840T). The oligos for total SMN copy number were designed upstream in intron 1 targeting both SMN1 and SMN2 genes, and the probe was labeled with TYE665. The TAMRA-labeled probe for RNase P  targeted the single exon region of RPPH1 gene . All primers were checked for target sequence specificity using NCBI Primer-BLASTSMN1/SMN2 primers, 900 nmol/L RPPH1 primers, 900 nmol/L total SMN primers, and 250 nmol/L of each probe. Each MAP plate well was loaded by hand via pipette with 10 µL of PCR reaction mix then overlaid with 10 µL Isolation Buffer. Gasket caps were placed and the plate was put into the Absolute Q and run with the following conditions: 3 min activation at 95 °C, 40 cycles of 5 s at 95 °C and 30 s at 62 °C. The fully automated Absolute Q Control Software  controls the sample digitization into the MAP partitions, thermal cycling, and imaging.The control DNA samples were diluted to 10 ng/µL working stock. The blinded DNA samples were measured using Qubit 4 fluorometer (Invitrogen) and diluted to 10 ng/µL. The PCR reaction mix contained 1X Combinati MasterMix, 2.5–25 ng human gDNA, 1800 nmol/L SMN1, SMN2 and total SMN are normalized by the reference control RPPH1 using the following equations:1 = number of SMN1 positive partitions, N2 = number of SMN2 positive partitions, N3 = number of total SMN positive partitions and N4 = number of RPPH1 positive partitions.The copy numbers were determined using the Absolute Q Analysis Software (v10.5.4). The software automatically calculates the optimal positive/ negative threshold and in this case the positive signals were five times higher than the negative signals. For each unit, the software displays the total viable partition count and the positive partition count. The software has a setting to identify the sample types as a copy number variation assay and applies the appropriate calculations to automatically display the copy numbers for the samples. The copy numbers of 45 using SigmaPlot v.12.0  was used to measure the agreement between dPCR methods.The coefficient of variability (%CV), defined as the standard deviation divided by the mean value for each set of replicates, was used to assess repeatability, or intra-assay precision. Reliability was also measured by the intraclass correlation coefficient (ICC) using SPSS v.25 . A Bland Altman agreement analysisSupplementary Table S1.


Diabetes is an increasingly important risk factor for ischemic stroke and worsens stroke prognosis. Yet a large proportion of stroke patients who are eventually diabetic are undiagnosed. Therefore, it is important to have sensitive assessment of unrecognized hyperglycaemia in stroke patients.Secondary outcome analysis of a randomized controlled trial focussing on parameters of glucose metabolism and detection of diabetes and prediabetes in patients with acute ischemic stroke (AIS).A total of 130 consecutively admitted patients with AIS without previously known type 2 diabetes mellitus (T2DM) were screened for diabetes or prediabetes as part of secondary outcome analysis of a randomized controlled trial that tested lifestyle intervention to prevent post-stroke cognitive decline. Patients had the oral glucose tolerance test (OGTT) and glycated hemoglobin (HbA1c) measurements in the second week after stroke onset and after 1 year. The detection rates of diabetes and prediabetes based on the OGTT or HbA1c values were compared.By any of the applied tests at the second week after stroke onset 62 of 130 patients (48%) had prediabetes or T2DM. Seventy-five patients had results from both tests available, the OGTT and HbA1c; according to the OGTT 40 (53.3%) patients had normal glucose metabolism, 33 (44%) had prediabetes, two (2.7%) T2DM. In 50 (66.7%) patients the HbA1c results were normal, 24 (32%) in the prediabetic and one (1.3%) in the diabetic range. The detection rate for disorders of glucose metabolism was 10% higher  with the OGTT compared with HbA1c. After 1 year the detection rate for prediabetes or T2DM was 7% higher with the OGTT (26% relative difference).The study intervention led to a more favourable evolution of glycemic status after 1 year.The OGTT is a more sensitive screening tool than HbA1c for the detection of previously unrecognized glycemic disorders in patients with acute stroke with an at least a 25% relative difference in detection rate. Therefore, an OGTT should be performed in all patients with stroke with no history of diabetes. Trial registrationhttp://clinicaltrials.gov. Unique identifier: NCT01109836. Glucose abnormalities, either diabetes type 2 (T2DM) or impaired glucose tolerance (IGT) are common in patients with stroke and impair their prognosis –5. T2DM It is a longstanding paradox that the OGTT is used for diabetes diagnosis whereas HbA1c serves as monitoring parameter for antidiabetic treatment. As the recommended 2-h oral glucose tolerance test (OGTT) may be a time-consuming procedure, an International Expert Committee and the American Diabetes Association (ADA) have recommend the use of HbA1c as a diagnostic test  with ≥ 6The aim of the present study was to find out how well HbA1c results will compare with the determination of FPG and 2-h plasma glucose (2hPG) using an OGTT in screening for T2DM and prediabetes in patients with acute non-disabling ischemic stroke during the early post-acute period and after the 1-year follow-up.In this study we performed a retrospective analysis of the data regarding glucose metabolism that were collected as part of the protocol in the Austrian Polyintervention Study to Prevent Cognitive Decline after Ischemic Stroke (ASPIS) trial. The study details of ASPIS have been described earlier . In shorPatients were tested for diabetes or prediabetes with HbA1c or an OGTT at baseline in the first or second week after the stroke and at 1-year follow-up. Blood samples for FPG and 2hPG were taken in sodium fluoride tubes and analysed immediately with the hexokinase method in a Hitachi Laboratory systems machine. The World Health Organization (WHO) 1999 criteria for diabetes   had already T2DM according to patients’ history, eight patients without previous diagnosis had no results for HbA1c or the OGTT available to allow classification at baseline, leaving 130 patients for further analysis  patients had normal glucose metabolism, 33 (44%) had prediabetes, two (2.7%) T2DM. According to the HbA1c results 50 (66.7%) were normal, 24 (32%) prediabetic and one T2DM (1.3%), see Table After 1 year, 28 patients were either study dropouts or had no further tests for glucose metabolism available, leaving 102 subjects for the follow-up analysis: the results of either tests revealed 56 patients as normal (54.9%), 36 as prediabetes (35.3%) and 10 (9.8%) with T2DM .67 (66%) patients had both tests, OGTT and HBA1c, available for direct comparison: according to OGTT 45 (67.2%) patients had normal glucose metabolism, 19 (28.4%) had prediabetes, and three (4.5%) T2DM. According to HbA1c results 51 (76.1%) were normal and 16 (23.9%) prediabetic. The observed agreement in classification of normal glucose metabolism was 62.7%, of prediabetes only 20.7%, the detection rate for prediabetes or T2DM was 9.9%  higher with OGTT, (see Table Next we looked how glycemic status changed over time in patients who had tests both at baseline and 1-year follow-up: Out of 102 patients 67 (65.7%) remained stable, 16 (15.7%) improved and 19/102 (18.6%) progressed to prediabetes or T2DM. Baseline variables of these three groups are shown in Additional file The main finding of this study was that in patients which subacute minor stroke the OGTT detects up to 25 to 29% more patients with prediabetes or T2DM than HbA1c regardless if performed early after the event or at 1-year follow-up. The odds that prediabetes was detected by the OGTT was 2.6 higher compared to HbA1c measurement alone. As in several previous studies , 12 disoDuring the 1-year observation the classification of glucose disorders in stroke patients in the trial was not invariable, although about two thirds of the patients did not change their original category. A multifactorial lifestyle-oriented intervention resulted in a non-significant statistical trend for a higher percentage of patients who improved in their glucose metabolism in the intervention group than the control group receiving a standard care. With improvements in lifestyle  or treatAmerican Diabetes Association has recommended HbA1c as the tool for diagnosing T2DM and defined a cut-off level 48 mmol/mol to indicate diabetes . Since lThere is also evidence that the parameters of the OGTT correlate better with outcomes of vascular disease than HbA1c. A German study among 1015 patients admitted for coronary angiography found T2DM in 149 patients by an OGTT, but only 42 by HbA1c  and the The strength of our study is that we ascertained consecutively patients admitted that were assessed and reassessed within the settings of a randomized controlled trial. Secondly, we performed a follow-up up investigation after 12 months and therefore our results do not rely only on measurements during the acute stroke period.Weak points of our study are the low number of patients investigated as this was a regionally conducted multi-centre trial and the considerable number of dropouts. Despites this weakness in quantity our results are robust over time from baseline to follow up and confirmatory with other studies. Furthermore, it can be stated that part of the hyperglycaemic states that were classified as diabetes and prediabetes at baseline can be interpreted as acute hyperglycaemic stress responses  especialIn addition, we found a trend for a lower rate of progression of disorders of glucose metabolism among those who underwent multimodal interventions for risk factor control compared with the control group. An improved detection of prediabetes and early T2DM using an OGTT could help to focus and tailor preventive interventions to improve or maintain cardiovascular and metabolic health.In the clinical evaluation of patients with acute stroke the assessment of the glycemic status is important in order to assign adequate preventive management to correct metabolic abnormalities that may influence the prognosis. The OGTT is a more sensitive method than HbA1c to detect and monitor disorders of glucose metabolism in patients with an ischemic stroke. The OGTT should be carried out in all stroke patients who have not been found to be diabetic before the stroke event.Additional file 1: Table S1. Baseline characteristics of patients in different groups of evolution of glucose metabolism (GM) between baseline and 12 months follow up. Both, OGTT and HbA1c were used for diagnosis, patients were classified to PD or T2DM if either the OGTT or HbA1c criteria were fulfilled.


Actinoplanes sp. SE50/110 chromosome via the bacteriophage φC31 integrase and allows cloning of a gene of interest by Golden Gate assembly (BsaI). T4 terminators surround the expression cassette to isolate the transcriptional unit and to prevent antisense transcription. The system can be used in other Actinomycetales by exchanging the promoter.The pSETT4 vector integrates into the  Actinoplanes sp. SE50/110 chromosome via the bacteriophage φC31 integrase and allows cloning of a gene of interest by Golden Gate assembly (BsaI). T4 terminators surround the expression cassette to isolate the transcriptional unit and to prevent antisense transcription. The system can be used in other Actinomycetales by exchanging the promoter.The pSETT4 vector integrates into the  Actinoplanes sp. SE50/110 (strain ATCC 31044) is known as a natural producer of acarbose , assembled by gene splicing by overlap extension (gene SOEing) (For construction of pSETT4 SOEing) , and clo SOEing)  accordingap was digested with NdeI and KpnI and treated with shrimp alkaline phosphatase following the supplier’s instructions . The tipA promoter was amplified from pSETGUS .The complete sequences of pSETT4


The pathogenic BRCA1/2 germline mutations contributed to Hereditary Breast and Ovarian Cancer (HBOC) susceptibility. The features of BRCA1/2 germline mutations in non-small cell lung cancer (NSCLC) have not been systematically studied. Here we performed the first study investigating the characteristics of pathogenic BRCA1/2 germline mutations in Chinese NSCLC patients and compared them with those from Chinese HBOC.Information on BRCA1/2 germline mutations from 9010 Chinese NSCLC patients were collected from available studies and analyzed, and compared with the BRCA1/2 germline mutations from Chinese HBOC BRCA1/2 database .19  pathogenic BRCA1 and 60  pathogenic BRCA2 germline mutations from NSCLC were identified. The carrier frequency of BRCA1/2 in Chinese NSCLC patients (86/9010 = 0.95 %) was significantly lower than that in Chinese breast and ovary cancer patients  (P < 0.001). We found that frameshift and nonsense mutations were the predominant types of BRCA1/2 mutation in NSCLC, with no obvious hot spot mutations. No significant difference in the ratio of frameshift and nonsense mutations was found between BRCA1 and BRCA2 in NSCLC. 5 out of 19 mutations in BRCA1 and 23 out of 60 mutations in BRCA2 were novel mutations found in NSCLC that have never been reported in Chinese HBOC. A trend of higher percentage of BRCA1 nonsense mutations in the carriers was revealed in NSCLC compared with HBOC, while no such difference was found in BRCA2 in all types of mutations.BRCA1/2 germline mutations from NSCLC exhibited distinct characteristics compared with those from HBOC in Chinese population, including lower carrier frequency than HBOC, higher ratio of nonsense mutations and carriers than HBOC, and novel BRCA1/2 germline mutations never found in HBOC. The germline mutations of BRCA1/2 have been comprehensively investigated in Hereditary Breast and Ovarian Cancer (HBOC). The interpretation of pathogenicity of BRCA1/2 germline mutations is based on the American College of Medical Genetics (ACMG) guidelines and provides strong tools for new mutation identification and interpretation [Apart from HBOC, the germline BRCA1/2 mutations have also been found in a variety of other cancers, including pancreatic cancer , lung caThe information on mutations and specific mutation sites were collected from three publications reporting the BRCA1/2 germline mutations in Chinese population –10. MutaThe information on mutations and specific mutation sites was downloaded from the LOVD database following the link below:https://databases.lovd.nl/shared/variants?search_owned_by_=%3D%22Xianqi%20Gao%22.The set of data was reported in a recent publication comprehensively reviewed and summarized the BRCA1/2 germline mutations from HBOC in Chinese population from 1999 to 2017 . Therefohttps://www.r-project.org/). Chi-square test and Fisher test were performed when rate or percentage was compared for significance. P < 0.05 was regarded as significant difference.Statistical analysis was performed, and figures were plotted with GraphPad Prism 5.0 software  or the R software  of lung cancer patients with pathogenic germline mutation, and in 32.35 % (11/34) of patients with likely pathogenic germline mutations . TherefoIn this study, we also identified several novel pathogenic germline mutations in NSCLC that have never been found in Chinese HBOC patients. New germline mutations of BRCA1/2 have been emerging all the time in the past 20 years in HBOC . It appePARP inhibitors (PARPi) have been widely used in the treatment of HBOC, and their effectiveness have been proved by many studies at first-line and multiline levels , 18. MaiAdvances have also been made in the application of PARPi in cancers other than HBOC with BRCA1/2 germline or somatic mutations. For example, for metastatic pancreatic cancer patients with germline BRCA mutations who had not progressed during first-line platinum-based chemotherapy, progression-free survival was longer with maintenance Olaparib than with placebo . There aIn conclusion, our study systematically investigated the characteristics of BRCA1/2 germline mutations in Chinese NSCLC patients, and compared the features of the mutations with those from Chinese HBOC. We found that BRCA1/2 germline mutations in NSCLC had lower carrier frequency than HBOC, potential higher ratio of BRCA1 nonsense mutations and carriers than HBOC, and revealed several novel BRCA1/2 germline mutations that have never been reported in Chinese HBOC.


Based on experiments conducted since this article’s  publicatIn experiments done after the article was published, the authors observed signals for the negative control indicating that there was unspecific binding to the sepharose beads . FurtherGiven these findings, the result and conclusion statements referring to a physical interaction between BAG3 and HSPB7 are not supported. Specifically:The penultimate sentence of the ZBTB17-HSPB7 locus subsection of the Results: “Using GST pull-down experiment and co-immunoprecipitation we also observed that recombinant BAG3 interacts with HSPB7 .”in vitro experiments demonstrate a physical interaction of BAG3 with HSPB7  suggesting functional relationships between the 2 proteins that may be relevant for their genetic implication in DCM pathophysiology.”The fifth sentence of the second paragraph of the Discussion section: “In addition, our The seventh sentence of the second paragraph of the Discussion section: “The interaction signal of BAG3 Arg151 and Cys151 isoforms with HSPB7 was similar (data not shown), suggesting no direct effect of the polymorphism on HSPB7 binding.”PLOS ONE’s Editorial Board confirmed that the main conclusions of the article stand are not substantively affected by this issue.A member of The authors apologize for the errors in the published article.S1 File(PPTX)Click here for additional data file.S2 File(PPTX)Click here for additional data file.


A role for phagocytosis in inducing cell death during thymocyte negative selection. Published 23, December 2019Timd4-/- RIPmOVA thymic slices. We had intended to show CD69 induction on RIPmOVA thymic slices treated with Annexin V in Figure 3b, which are the data described in the results as well as the figure legend, and these data are shown in the corrected version.In the original published version of the article, Figure 3b mistakenly showed the same graph as represented in Figure 4b, which shows CD69 induction on The corrected Figure 3 is shown here:The originally published Figure 3 is also shown for reference:Timd4-/-. The left column should be labelled Timd4+/-. The article has been corrected accordingly.In addition, in Figure 5-figure supplement 1b of the original published version, both columns of the graph are labelled The corrected Figure 5-figure supplement 1 is shown here:The originally published Figure 5-figure supplement 1 is also shown for reference:


Pfhrp2 and Pfhrp 3 gene deletions to false negative malaria RDT results in Ghana.False-negative malaria rapid diagnostic test (RDT) results amongst symptomatic malaria patients are detrimental as they could lead to ineffective malaria case management. This study determined the nationwide contribution of parasites with Plasmodium parasite density and species identity was estimated from the blood smears. Plasmodium falciparum specific 18S rRNA PCR, merozoite surface protein (msp 1) and glutamate rich protein (glurp) gene PCR were used to identify P. falciparum positive samples, which were subjected to Pfhrp 2/3 exon1-2 and exon2 genotyping.This was a cross sectional study where whole blood (~2 ml) was collected from patients presenting with malaria symptoms at 100 health facilities in all the regions in Ghana from May to August 2018. An aliquot of the blood was used to prepare thin and thick blood smears, filter paper blood spots (DBS) and spot a PfHRP 2 RDT kit. The remaining blood was separated into plasma and blood cells and stored at -20°C. P. falciparum positive patients analyzed, 134 (4.69%) had false negative P. falciparum specific RDT results. Samples for PCR analysis was available for 127 of the false negative patients, and the analysis identified 116 (91.3%) as positive for P. falciparum. Only 58.1% (79/116) of the false negative RDT samples tested positive by msp 1 and glurp PCR. Genotyping of exon 1–2 and exon 2 of the Pfhrp 2 gene identified 12.9% (10/79) and 39.5% (31/79) of samples respectively to have deletions. Genotyping exon 1–2 and exon 2 of the Pfhrp 3 gene identified 15.2% (12/79) and 40.5% (32/79) of samples respectively to have deletions. Only 5% (4/79) of the false negative samples had deletions in both exon 1–2 and exon 2 of the Pfhrp 2 gene. Out of the 49 samples that tested positive for aldolase by luminex, 32.6% (16/49) and) had deletions in Pfhrp 2 exon 2 and 2% (1/49) had deletions in both exon 2 and exon 1–2 of the Pfhrp 2 gene.Of the 2,860 microscopically Pfhrp 2 gene the prevalence of parasites with deletions in Pfhrp 2 exon 2 was about a third of the false negative RDT results. The need to ensure rapid, accurate and reliable malaria diagnosis requires continuous surveillance of parasites with Pfhrp 2 gene deletions.The low prevalence of false negative RDT test results provides assurance that PfHRP 2 based malaria RDT kits remain effective in diagnosing symptomatic malaria patients across all the Regions of Ghana. Although there was a low prevalence of parasites with deletions in exon 2 and exon 1–2 of the  Plasmodium falciparum parasites [The diagnosis of symptomatic malaria is typically based on smear microscopy, the gold standard . Howeverarasites , the cauarasites .Pfhrp 2 gene, which codes for the PfHRP 2 antigen, which both cause false negative RDT test results [Pfhrp 2 gene have been found to react positively with PfHRP 2-based malaria RDTs due to cross-reactivity of PfHRP 2 with PfHRP 3 [Pfhrp 2 [Despite the numerous advantages of malaria RDTs over smear microscopy, a few disadvantages exist. Some disadvantages include prolonged PfHRP2 antigen persistence after parasite clearance, which results in false positive test results as well as the prozone effect  and the  results , 10. Par PfHRP 3 . The PfH[Pfhrp 2 .Pfhrp 2 gene that result in false negative malaria RDT results are detrimental and have the potential of missing malaria positive patients in countries like Ghana where testing is largely by PfHRP2 RDTs. The symptomatic malaria patients who test negative may likely not receive appropriate antimalarial treatment [Deletions in the reatment , 14. To reatment .Pfhrp 2 gene can adversely affect the diagnostic accuracy of PfHRP 2 based malaria RDT kits, the WHO has recommended that countries with a Pfhrp 2 gene deletion parasite population at or above 5% should resort to the use of non-PfHRP 2-based RDT kits [Pfhrp 2 gene deletions in symptomatic malaria infections from high endemic countries such as Ghana is complicated by the fact that the infections are predominantly multiclonal [Pfhrp 2 gene deletions can be masked by the presence of co-infecting parasites with intact Pfhrp 2 genes.Since deletions in the RDT kits . Identifticlonal  and the Pfhrp 2 gene was in the Amazon region of Peru [Pfhrp 2 in Ghana, most of which have been conducted on asymptomatic malaria carriers and also without implementing the WHO recommendations [Pfhrp 2 and or Pfhrp 3 gene that result in false negative PfHRP 2-based malaria RDT results.The first reports of the existence of parasites with deletions in the  of Peru  and has  of Peru , includi of Peru . To datendations , 19. DueEthical approval for this study was obtained from the Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana (study # 068/17-18). Written informed consent was obtained from each adult participant (≥18years old) and caregivers of participants less than 18 years old provided parental consent. Written informed assent was also obtained from all participants aged between 12 and 17 years old. Each consenting or assenting participant was informed of the objectives, methods, anticipated benefits and risks of the study as well as their liberty to withdraw without any penalty.This was a cross-sectional survey among all symptomatic individuals seeking care in selected health care facilities in the ten regions of Ghana between May and August 2018.Pfhrp 2/3 gene deletions [As per the WHO protocol on determining the prevalence of eletions , a total® tube. The venous blood was tested on the CareStart® malaria PfHRP 2 RDT kit . Samples were classified as RDT positive if both the control and test lines were positive and RDT negative if only the control line but not the test yielded a positive result.After obtaining written informed consent (for adult participants), parental consent (for children under 18 years old), and assent (for children 12 to 17 years old) the study participants were enrolled into the study. Demographic data was obtained from each participant and entered in KoBoCollect and about 2 ml of venous blood collected into an EDTA vacutainer® No. 3 filter paper. The blood spots were air dried and stored individually in a Ziplock® bag with a desiccant. Packed cells were isolated from the remaining blood samples and stored in a 1.5 ml eppendorf® tube at -20°C until used.Thick and thin blood smears were also prepared using 6 μl and 2 μl of whole blood respectively. Filter paper blood spots were prepared by spotting four 50 μl drops of whole blood on to WhatmanThe blood smears were processed and then stained with 3% Giemsa for 45 min. The stained slides were subsequently viewed under 100X oil immersion microscope . Two ind® and positive by microscopy  were sorted out from the total samples collected. Genomic DNA was extracted from the dry blood spots (DBS) using the Chelex extraction method [Samples that were negative by the routinely used CareStart PfHRP 2 RDTn method  and from18S rRNA gene was adapted from Singh et al. [2, 133 nM each of forward (rPLU6) and reverse (rPLU5) primers (18S rRNA gene from 2 μl of DNA obtained using the Zymo extraction kit or 5 μl of Chelex extracted DNA (~40 ng) in the primary reaction. The nested PCR was performed using similar concentrations of reagents as in the primary reaction mix; however, rFal1 (forward) and rFal2 (reverse) primers were used to amplify 0.5 μl of the primary product in P. falciparum identification. The cycling parameters for the primary and nested reactions are listed in P. falciparum (MRA 102G) was used as a positive control and the no template control served as negative control.The nested PCR amplification of the h et al.  with slih et al. . Briefly primers  and 1 U et al. [msp 1 locus was amplified using nested PCR. The primary reaction contained 200 nM each of M1-0F and M1-0R primers in a reaction containing 2 μl of DNA obtained using the Zymo extraction kit or 5 μl of Chelex extracted DNA (~40 ng) and 0.1 μl One Taq DNA polymerase . In the secondary reaction, 1 μl of the primary PCR product was amplified using 200 nM each of M1-KF+M1-KR , M1- R033R+M1-R033F  and M1-MF+M1-MR  primer sets  of gDNA. Primers G-F4 and GNF (500 nM each) were used in the secondary reaction of amplify 2 μl of the primary PCR product. The cycling conditions for the primary and secondary PCR reactions included an initial denaturation at 95°C for 5 min followed by 35 cycles of a denaturation at 94°C for 30 seconds, primer annealing at 55°C (for primary) or 58°C (for secondary) for 1 minute and an extension at 68°C for 1 minute and a final extension at 68°C for 5 minutes.The Pfhrp 2 and Pfhrp 3 genes were amplified using the protocol described by Abdallah et al.,[Pfhrp2F1 (forward) and Pfhrp2R1 (reverse) primers for Pfhrp2 exon 2 and Pfhrp3F1 (forward) and Pfhrp3R1 (reverse) primers for Pfhrp 3 exon 2 were used to amplify 2 μl of DNA obtained using the Zymo extraction kit or 5 μl of Chelex extracted DNA (~40 ng) using 0.1 μl OneTaq DNA polymerase  in a 15 μl reaction containing 1.8 mM MgCl2 and 0.2 μM dNTP mix. The DNA was initially denatured at 95#x00B0;C for 3 min followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C (Pfhrp 2 gene) or 55°C (Pfhrp 3 gene) for 30 seconds and an extension at 68°C for 1 min. The final extension was performed at 68°C for 5 min and finally held at 4°C. Genomic DNA from Dd2 , HB3  and 3D7 , representing Pfhrp 2 deleted, Pfhrp 3 deleted and wild type parasites respectively were used as controls for the PCR amplifications.Exons 2 and 1–2 of the h et al., with minPfhrp 2 and Pfhrp 3 genes. The primary PCR reactions were carried out in a 15 μl reaction volume containing 2 μl of DNA obtained using the Zymo extraction kit or 5 μl of Chelex extracted DNA (~40 ng), 0.2 μM each of forward and reverse primers 2E12F1 and 2E12R1 for Pfhrp 2 and 3E12F1 and 3E12R1 for Pfhrp 3 respectively), 0.133 μM dNTPmix and 2.5 mM MgCl2 supplemented with 0.1 μl of OneTaq DNA polymerase . The secondary reactions were amplified using similar reaction conditions as the primary reaction, however, the primer sets were replaced with 2E12F2 and 2E12R2 for Pfhrp 2 or 3E12F2 and 3E12R2 for Pfhrp 3. The templates for the secondary reactions were 2 μl of a 1:100 dilution of the respective primary PCR products.A nested PCR protocol was used to amplify exon 1–2 of the PCR products (10 μl) were separated on 2.0% agarose gels containing 0.2 μg/ml ethidium bromide and subsequently viewed under UV illumination using a Bio-Print TX4 gel documentation system . All samples that yielded visible fragments after agarose gel electrophoresis were classified as positive for the particular PCR reaction.3), 50 ml liquid casein, 5 g polyvinyl alcohol, 5 g polyvinylpryrrolidine). Subsequently, 50 μl of PfHRP 2 and pan-Aldolase coated beads (1:500 dilution of each in elution buffer) was aliquoted into each well of the plate. The samples tested included a 50 μl aliquot of the test samples and negative control samples from 86 malaria naïve people. The plates were washed three times with wash buffer. Anti-PfHRP 2 and anti-Aldolase (1:500) detection antibodies (50 μl) were then added to each well of the plate and Incubated for 45 mins with shaking. The plate was subsequently washed three times and then incubated for 30 mins with 50 μl of streptavidin-phycoerythrin (1:200) with shaking. After the final wash, the plates were read on the Luminex 200 (Luminex Corp. USA). The cutoff value for PfHRP 2 and pan-Aldolase antigen positivity was the lognormal mean fluorescence intensity (MFI) of the 86 malaria naïve samples minus the background (bg) MFI signal ± 3 SD.The Multiplex Bead Assay (MBA) was used to determine the PfHRP 2 and pan-Aldolase levels of the samples . BrieflyThe datasets were captured using KoBoCollect, extracted and cleaned in IBM SPSS version 22. IBM SPSS version 22 was used to generate the descriptive statistics including mean, standard error of the mean and the frequency. Kruskal-Wallis test (GraphPad Prism v5) was used to compare the median Regional parasite densities and the age of the study participants. Pearson’s Chi squared test was used to compare the medians of the Regional distribution of male and female participants at a 5% level of significance.The MFI cut off value for positivity for the PfHRP2 and aldolase luminex data was set at 50 and 20 respectively. Spearman’s correlation (GraphPad Prism v5) was used to determine the correlation between parasite density and parasite antigen  concentrations.Plasmodium parasites were identified in 1% (141/13136) of the samples that tested negative by PfHRP 2 RDT kits , leaving 134 false negative (FN) samples. Unfortunately, neither DBS nor whole blood was found for seven of these samples. The age of the patients with FN results ranged between 1 and 94 years and the age distribution were similar across the 10 Regions . Children aged ≤10 years constituted 22.6% and adults 50 years and above constituted 15.8% of the study population. The distribution of males and females across the Regions were similar , with males representing about 30% of the study population.Microscopy identified the presence of malaria parasites in 2890 of the 18618 samples with valid microscopy results. Microscopic densities of RDT kits . Out of The distribution of people carrying parasites that were identified as FN was nationwide, with the highest prevalence found in the Ashanti Region and the lowest prevalence in the Volta Region . The parWhole blood or filter paper samples were available for 127 out of the 134 samples, unfortunately neither whole blood nor DBS could be found for 7 samples.Plasmodium falciparum species PCR identified 116 out of the 127 FN samples as positive  of the 79 false negative samples contained infections with parasite densities of less than 200 p/μl (between 76 and 192 p/μl). All these 7 samples tested negative after 2 exon 2 . The samPfhrp 2 exon 2 gene deletions, only 4 (12.9%) tested negative for Pfhrp 2 exon1-2. Samples with gene deletions in both Pfhrp 2 exon 2 and Pfhrp 2 exon 1–2 were found in only three Regions, the Ashanti, Brong Ahafo and the Central, with the Central Region having the highest prevalence  of samples with deletions in both Pfhrp 2 exon 2 and exon 1–2  of the samples with The PfHRP 2 antigen was absent  in 32.9% (26/79) of the FN samples, whilst the aldolase antigen was absent  in 38% (30/79) of the FN samples. There were 27.8% (22/79) of the FN samples that were negative for both PfHRP 2 antigen and aldolase.About 68% (48/71) of the samples that had parasite densities >200 p/μl tested positive by aldolase luminex. Parasite density exhibited a significant strong positive correlation  with aldolase concentrations and a significant but weak correlation  with PfHRP 2 antigen concentration.Pfhrp 2/3 gene deletions amongst samples that tested positive for aldolase by luminex were predominant in the Ashanti Region and Northern Region  and the South Eastern (Volta and Greater Accra) parts of the country. The low number of false negative RDT results identified in this study is lower than 8.4% and 54% that have been reported in Uganda and Benin respectively . Low parPfhrp 2/3 gene deletions in samples from symptomatic malaria patients. Parasites with Pfhrp 2 exon 2 gene deletions accounted for about a third of the false negative samples. Parasites with Pfhrp 2/3 gene deletions were identified in all but one of the Regions in Ghana, the Eastern Region where no sample was identified to contain parasites with deletions in exon 2 of the Pfhrp 2 gene. All parasites with deletions in Pfhrp 2 exon1-2 also had deletions in Pfhrp 2 exon 2 and were mainly identified in 3 neighboring Regions in the lower mid-section of western Ghana. The pattern of distribution of parasites with deletions in both exon 2 and exon 1–2 of the Pfhrp 3 gene were like that of the Pfhrp 2 gene. Deletions at exon 2 were three times more prevalent than at exon 1–2 for both the Pfhrp 2 and Pfhrp 3 genes. The observed higher prevalence of deletions in exon 2 than exon 1–2 could be a true reflection of parasites with exon 2 gene deletions being more abundant than parasites with exon 1–2 gene deletions as has been reported in other studies such as an earlier report from India where parasites with Pfhrp 2 exon 2 gene deletions were at higher frequencies than parasites with Pfhrp 2 exon 1–2 gene deletions [Pfhrp 2 exon 2 PCR results, some studies have identified samples with higher frequencies of deletions in Pfhrp 2 exon 1–2 than Pfhrp 2 exon 2 [This study identified parasites with eletions . The vareletions , 38. Des2 exon 2 .Pfhrp 3 genes have been found to compensate for the absence of Pfhrp 2 exon 2 in a parasite, especially at high parasite densities and result in a positive PfHRP 2 based malaria RDT test due to the cross-reactivity of PfHRP 3 antigen with the PfHRP 2 antibodies on the RDT kits [Pfhrp 3 exon 2 gene and most likely the corresponding PfHRP 3 antigen in circulation in the false negative samples identified in this study did not result in a positive PfHRP 2 RDT result, even though some of the samples had parasite densities of greater than 1000 parasites per microlitre. The absence of PfHRP 3 antigen compensation has been observed in other studies including one from Kenya [Parasites with intact RDT kits , 40. Howom Kenya . PfHRP 3Pfhrp 2 exon 2 or due to the presence of some parasite isolates producing very low concentrations of aldolase. The prozone effect could have been one of the reasons why samples containing high concentration of PfHRP 2 antigen tested negative by the PfHRP 2 based RDT kit as has been suggested in an earlier report [Despite the high positive correlation between parasite density and aldolase activity, about 38% of the false negative samples had aldolase levels below the cut off for positivity, which suggests that some parasites isolates produced very low aldolase activity. The presence of PfHRP 2 antigen in the false negative samples that had no aldolase activity could have resulted from persisting PfHRP 2 antigen from a recently cleared infection containing parasites with intact r report .Pfhrp 2 exon 2 gene deletions. The confirmation of parasites with Pfhrp 2 gene deletions amongst symptomatic malaria patients with false negative RDT results is detrimental to malaria control in Ghana, especially in communities and facilities where malaria diagnosis is solely based on RDT results. The prevalence of parasites with deletions in the Pfhrp 2 gene circulating in symptomatic malaria patients could be more than identified in this study as only parasites contained in false negative samples were evaluated as opposed to all microscopy positive samples. Overall, the low prevalence of samples classified as false negative resulted in only a small fraction of the samples collected analyzed for Pfhrp 2 gene deletions.There were about a third of the aldolase positive false negative RDT results that were caused by parasites with Pfhrp 2 gene the prevalence of parasites with deletions in Pfhrp 2 exon 2 was about a third of the false negative RDT results. The need to ensure rapid, accurate and reliable malaria diagnosis requires continuous surveillance and monitoring of parasites with Pfhrp 2 gene deletions.The low prevalence but nationwide distribution of samples with false negative RDT test results provides assurance that PfHRP 2 based malaria RDT kits remain effective in diagnosing symptomatic malaria across all the Regions of Ghana. Although there was a low prevalence of parasites with deletions in both exon 2 and exon 1–2 of the S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Plasmodium falciparum infections. Numerous genotypes of haptoglobin have been reported in malaria endemic populations. In this study, the relationship between haptoglobin genotypes and incidence of uncomplicated malaria in a cohort of children living in a malaria-endemic area of Uganda was determined.Haptoglobin (Hp) is an acute phase protein that takes part in systemic regulation of haem during This is an extension of a longitudinal study comprising of 423 children aged between six months and nine years, who were actively followed up for one year. Malaria episodes occurring in the cohort children were detected and the affected children treated with national policy drug regimen. Haptoglobin genotypes were determined by an allele-specific PCR method and their frequencies were calculated. A multivariate negative binomial regression model was used to estimate the impact of haptoglobin genotypes on incidence of uncomplicated malaria in the children’s cohort. In all statistical tests, a P–value of < 0.05 was considered as significant.The prevalence of the Hp 1–1, Hp 2–1 and Hp 2–2 genotypes in the children’s cohort was 41%, 36.2% and 22.9%, respectively. The overall frequency for the Hp 1 allele was 59%, while Hp 2 allele occurred at a frequency of 41%. After adjustment of incidence rates for age, insecticide treated bed net (ITN) use and malaria history, the incidence of uncomplicated malaria for children carrying the Hp 2–2 genotype and those with the Hp 2–1 genotype was statistically similar (P = 0.41). Also, no difference in the incidence of uncomplicated malaria was observed between children carrying the Hp 1–1 genotype and those having the Hp 2–1 genotype (P = 0.84) or between Hp 2–2 Vs Hp 1–1 genotypes (P = 0.50).P. falciparum malaria severity are needed to understand the role of these genotypes in malarial protection.This study showed that the Hp 1–1 and Hp 2–1 genotypes each occur in nearly 4 in 10 children and the Hp 2–2 genotype occurs in 2 of every 10 children. No association with incidence of uncomplicated malaria was found. Additional studies of influence of haptoglobin genotypes on  Plasmodium falciparum malaria infections involves interplay of both parasite and host factors such as host genetic variability . About 88.2% of the participants’ guardians (373 / 423) reported owning and using an insecticide treated bed net (ITN) within their households. Four hundred and three participant’s guardians (95.3%) reported having ever administered an anti-malarial drug to the enrolled child. Throughout the 1-year of longitudinal follow up in this study, malaria episodes were not registered among 217 out of 423 children (51.3%). Among those who experienced malaria episodes  during the 1 year of follow up, the range of annual episodes per child was 1 to 9.Haptoglobin genotyping was performed successfully for 398 samples by determining the presence or absence of 935 bp, 1.2 kb and 1.4 kb DNA fragments corresponding to the Hp2, Hp1S and Hp1F genotypes, respectively. The Hp1-1, Hp2-1 and Hp2-2 genotypes were found in 41%, 36.2% and 22.9% of the cohort children, respectively. The overall allele frequency for the Hp1 allele was 59%, while Hp2 allele occurred at an allele frequency of 41%. The distribution of the Hp genotypes in the study cohort is presented in Table P. falciparum infection, were included in the final multivariate negative binomial regression model to determine the extent to which they were affected by the haptoglobin genotype after adjusting for other independent determinants of malaria incidence identified in an earlier study in the same children’s cohort [This study is an extension of a longitudinal study carried out previously on the same cohort of children . In thiss cohort ; namely There was no statistically significant difference in the distribution of the Hp genotypes among children who did and did not experience uncomplicated malaria throughout the year as shown in Table P. falciparum infection in Dogon, but not in Fulani tribe [Haptoglobin genotypes, Hp1-1, Hp2-1 and Hp2-2 were found in 41%, 36.2% and 22.9% of the cohort children, respectively. The overall allele frequency was 59% for the Hp1 allele and 41% for the Hp2 allele. However, no association with incidence of uncomplicated malaria was found. The present findings differ from results of prospective cohort studies carried out among Kenyan children, in which the Hp 2–2 genotype was associated with lower incidence of clinical malaria , 14. In ni tribe . The finP. falciparum infections [P. falciparum malaria severity are needed to further understand the role of these genotypes in malarial protection.In addition, Hp genotypes have been shown to influence plasma Hp levels , 10 needfections , 25. Thifections , 17. AddThe Hp1 allele was present at an allele frequency of 59%, yet no influence on the incidence of uncomplicated malaria by the Hp1 allele was observed. This is also in line with some earlier studies that found no clear associations between the Hp 1 allele and malaria susceptibility , 16. InsHaptoglobin genotypes, Hp1-1, Hp2-1 and Hp2-2 were found in 41%, 36.2% and 22.9% of the cohort children, respectively. The overall allele frequency was 59% for the Hp1 allele and 41% for the Hp2 allele. Association of Hp genotypes with incidence of uncomplicated malaria was not observed. This lack of detectible association with uncomplicated malaria suggests that these alleles may have been maintained by protection from other infections. Similar studies in different settings are needed to confirm these findings.


After this article  was publpCDNA3.1 and NSP4 panels in Fig 3AMID and C157 panels in Fig 5CThe authors noted that due to figure preparation errors the pCDNA3.1 and MID dot plots were mistakenly duplicated in the NSP4 and C157 panels, respectively. These errors are addressed in the revised Figs PLOS ONE’s Editorial Board advised that the updated figures support the results as reported in the original article.A member of The original data are available upon request from the corresponding author for Figs 1B, The authors apologize for the errors in the published figures.


Genetically engineered murine models involving chromosomal translocation genes have been generated using transgenic approaches or gene knockin.4 However, the closest model is the creation of bona fide chromosomal translocations, which recapitulate the physiologic levels of the fusion genes, the reduced dosage of the wild-type alleles and the contribution of the reciprocal products of the translocations. This has been achieved in vivo using the Cre-loxP system in embryonal stem cells,5 which is labor intensive and time consuming. The CRISPR/Cas9 system is a versatile tool for genomic engineering. Transduction of target cells with vectors encoding Cas9 and sgRNAs targeting two genomic loci causes two DNA double strand breaks (DSBs), which could induce chromosomal rearrangements as a result from non-homologous end joining-based DNA repair.6 This strategy has been used to model oncogenic chromosomal rearrangements in murine10 or human11 cells in vivo. However, given the challenges of delivering CRISPR/Cas9 vectors into hematopoietic cells, the penetrance of leukemogenesis has been shown with low frequency in vivo.11 Here we establish an efficient methodology to generate chromosomal translocations in mice hematopoietic cells based on a vector design enabling convenient lentiviral delivery of multiple sgRNAs to hematopoietic cells from a Cas9 expressing mouse strain.A wide range of chromosomal rearrangements have been identified in human cancers especially in leukemia.KMT2A (MLL) gene constitutes a hotspot for chromosomal translocations in human leukemia, resulting in over 135 different leukemia-associated KMT2A gene fusions.2KMT2A-MLLT1 defines a leukemia subtype with poor outcome. This translocation frequently constitutes the sole genomic lesion to drive the leukemia,12 providing a good model to establish this approach. We designed a strategy to induce Kmt2a-Mllt1 translocations in murine hematopoietic cells using the CRISPR/Cas9 system followed by transplantation into lethally irradiated recipient mice  with special designs: (1) The small size of the backbone with minimum interval sequences between each modules improves the virus titer; (2) The isocaudomer sites flanking the sgRNA cassettes facilitates the combination of multiple single sgRNAs from individual vector into one vector . We firstly cloned several individual sgRNAs targeting the equivalent introns of Kmt2a and Mllt1 that are orthologue regions in the human leukemia translocation . Transduction of this vector coding two sgRNAs in NIH 3T3 cells generated the Kmt2a-Mllt1 translocation efficiently .The 13 we crossbred the Cas9 mice with the inbred C57BL/6 mice and used the F1 progenies (Cas9GFP+/−) as the bone marrow donors. Given the C57BL/6 haploidentical setting, these cells can reconstitute the hematopoietic system in lethally irradiated C57BL/6 recipient mice without inducing graft-versus-host disease. Transduction of these bone marrow cells with the sgRNA vector (sg_ K-M) lead to a detectable genomic rearrangement between the Kmt2a and Mllt1 locus at 4 days after transduction in ex vivo cultures  but not for lymphoid markers  or progenitor markers   in leukemia cells was confirmed by FISH using pan-chromosome probes Fig. E. The KmTCF3-HLF translocation defining a highly aggressive leukemia subtype for which so far no representative mouse model could be engineered.15 Given the frequent PAX5 heterozygous deletions and reduced gene expression identified in this leukemia,16 we tested the hypothesis if the Tcf3-Hlf translocation could induce a mouse leukemia when established in a Pax5 haploinsufficient context . We crossbred the Cas9 mice with the Pax5 heterozygous mice 17 and used the F1 progenies (Cas9GFP+/− Pax5+/+ or Cas9GFP+/− Pax5+/−) as the bone marrow donors. Transduction of the bone marrow cells with validated sgRNAs targeting the equivalent introns of mice Tcf3 and Hlf loci (sg_T-H) lead to detectable Tcf3-Hlf translocation and the expression of the fusion product . The generation of the Tcf3-Hlf translocation was less effective than the Kmt2a-Mllt1 translocation, with ∼0.16% of the cells detected by ddPCR to harbor the translocation .We next attempted to model the Tcf3-Hlf translocation was detected in genomic DNA from peripheral blood and bone marrow at 4 weeks after transplantation . However, no progression to leukemia could be detected even after a follow-up for up to 11 months. Post-mortem examination did not reveal any abnormality of spleen, kidney, liver and bone marrow cells and the Tcf3-Hlf translocation signal was undetectable in bone marrow cells . This suggests a selective disadvantage of cells carrying Tcf3-Hlf in vivo, confirming similar observations made with a knockin model of Tcf3-Hlf.18 Thus, despite the frequency of PAX5 haploinsufficiency in TCF3-HLF positive patients, deletion of Pax5 does not counteract the negative selection pressure by Tcf3-Hlf. Notably, the Kmt2a-Mllt1 myeloid leukemia can be established in the Pax5 +/− background with similar dynamics as in the Pax5 +/+ background confirming that a reduced Pax5 gene dosage does not influence the leukemogenic activity of Kmt2a-Mllt1 .After transplantation of the engineered cells into lethally irradiated C57BL/6 recipients, the Tcf3-Hlf was not sufficient to induce leukemia in this study the model can now be used to evaluate different cells of origin and to screen for required cooperative genetic events. Our approach can be employed to model any pair of genes as long as the orientation to the centromere does not result in dicentric chromosomes. Using the inbred Cas9 mouse strain will further simplify this approach. Besides, our vector design facilitates the combination of multiple sgRNAs to introduce additional oncogenic lesions . Thus, our strategy provides an efficient way to generate precise translocations, enabling functional modeling in mice and constitutes a framework to study the role of cooperative events and the cells of origins in leukemogenesis.Activation of oncogenes by fusion or juxtaposition to ectopic regulatory elements constitutes important cancer initiating events. Here, we established an efficient strategy using the CRISPR/Cas9 system to generate leukemogenic chromosomal translocations in vivo. We generated two types of translocations in bone marrow cells with frequencies of 0.6% and 0.16%. Although the generation of


Journal of Oncology and to the readership for any inconvenience caused. The corrected figure and legend are presented here.In the article titled “UBE2C Induces Cisplatin Resistance via ZEB1/2-Dependent Upregulation of ABCG2 and ERCC1 in NSCLC Cells” [