Prunus species) breeding is currently difficult to achieve due to the polygenic nature of the most relevant agronomic traits linked to fruit quality. Genotyping by sequencing (GBS), however, provides a large quantity of useful data suitable for fine mapping using Single Nucleotide Polymorphisms (SNPs) from a reference genome. In this study, GBS was used to genotype 272 seedlings of three F1 Japanese plum  progenies derived from crossing “98–99”  with “Angeleno,” “September King,” and “September Queen” as male parents. Raw sequences were aligned to the Peach genome v1, and 42,909 filtered SNPs were obtained after sequence alignment. In addition, 153 seedlings from the “98–99” × “Angeleno” cross were used to develop a genetic map for each parent. A total of 981 SNPs were mapped (479 for “98–99” and 502 for “Angeleno”), covering a genetic distance of 688.8 and 647.03 cM, respectively. Fifty five seedlings from this progeny were phenotyped for different fruit quality traits including ripening time, fruit weight, fruit shape, chlorophyll index, skin color, flesh color, over color, firmness, and soluble solids content in the years 2015 and 2016. Linkage-based QTL analysis allowed the identification of genomic regions significantly associated with ripening time (LG4 of both parents and both phenotyping years), fruit skin color (LG3 and LG4 of both parents and both years), chlorophyll degradation index (LG3 of both parents in 2015) and fruit weight (LG7 of both parents in 2016). These results represent a promising situation for GBS in the identification of SNP variants associated to fruit quality traits, potentially applicable in breeding programs through MAS, in a highly heterozygous crop species such as Japanese plum.Marker-assisted selection (MAS) in stone fruit ( Prunus salicina Lindl.) is a crop tree species native to China. It has a diploid genome (2n = 2x = 16), unlike the European plum (P. domestica L.), which has a hexaploid genome (6n = 2x = 48), although both species belong to the Rosaceae family, subfamily Prunoideae, genus Prunus, and subgenus Prunophora .Japanese plum  Batsch] (2n = 2x = 16); sweet cherry (P. avium L.) (2n = 2x = 16); and apricot (P. armeniaca L.) (2n = 2x = 16).Marker-assisted selection (MAS) is an important biotechnological tool used in Meloidogine incognita Kofoid et White and Meloidogine arenaria  Chitwood] resistance using plum progenies and interspecific plum × almond [P. amygdalus (Batsch) syn. P. dulcis (Miller) Webb] (2n = 2x = 16) × peach progenies  technology makes it possible to generate more saturated genetic maps, thus facilitating more accurate QTL identification. This next generation technology makes it possible to build libraries of thousands of DNA fragments (tags), as has been done in maize (IBM) and barley (Oregon Wolfe Barley), for which 200,000 and 25,000 sequence tags, respectively, have been mapped. A single GBS experiment can provide 25,000 SNPs, which can be used for germplasm characterization, plant breeding progeny studies and QTL mapping  were generated in the Rinconada de Maipu Experimental Station  in 2011 by bumble bee pollination under a fine plastic mesh cage using a common female parent genotype “98–99” and three different pollen donors, including “September King,” “September Queen,” and “Angeleno.” The “98–99” genotype is a mid-early maturing selection with red skin and yellow flesh and with high organoleptic quality. “Angeleno” is a mid-late maturing cultivar with purple skin color and excellent postharvest performance, while “September King” and “September Queen” are both very late maturing genotypes.AD), firmness (FIRM), and the soluble solids concentration (SSC). RT was calculated in Julian days and FW in grams. SHP and fruit color  were visually determined and classified in numerical categories. SHP was defined according to the following scale: elongated (1), hearted (2), spherical (3), or oblate (4), while SKC was classified as green (1), yellow (2), red (3), purple (4), or black (5). FLSC was classified as white (1), green (2), yellow (3), or red (4). In addition, OVC was determined on a scale of 1–4, determined as percentages of color covering the fruit: 25% (1), 50% (2), 75% (3), and 100% (4). On the other hand, FIRM was quantified in Newton (N) by a texture analyzer  using a 7.9 mm diameter plunger, and SSC was determined using an ATAGO® hand-held refractometer calibrated as the percentage of sucrose at 20°C. IAD chlorophyll content-related maturity index was measured by DA-meter  and 1 week after harvest .Fifty-five seedlings from the “98–99” × “Angeleno” progeny were phenotyped in the years 2015 and 2016. Nine agronomic traits linked to fruit quality were evaluated including ripening time (RT), fruit weight (FW), shape (SHP), skin color (SKC), flesh color (FLSC), over color (OVC), chlorophyll index  using Restricted Maximum Likehood (REML) was calculated for determining the genetic effect of each seedling by Best Linear Unbiased Predictors (BLUPs), considering the genotype factor as a random effect. All analyses and histograms were calculated and edited using INFOSTAT v16 software with the exception of the GLMM analysis, which was calculated using an interface between INFOSTAT and R.Young leaves for each individual were collected and lyophilized for DNA extraction, which was carried out according to the CTAB procedure described by Doyle and Doyle . A totalp = 0.05 using SNPs from each parent was applied in order to discern the parentage of the 272 seedlings. A total of 161 individuals were obtained from “Angeleno,” 62 from “September King” and 15 from “September Queen,” and the genotype “98–99” was the common female. One hundred fifty-three seedlings from the “98–99” × “Angeleno” progeny were used for genetic mapping. Genotyping errors, under-calling homozygotes and heterozygotes and SNPs with over 10% missing data were eliminated prior to genetic mapping. A total of 14,697 SNPs were obtained, including common SNPs  and uncommon SNPs for “98–99”  and “Angeleno” . Genetic linkage maps for each parent were constructed by JoinMap v.3.0 software  using numeric data joined to genotype data and principal component analysis (PCA) was used for generating Manhattan plots for each trait and year. In addition, a Mixed Lineal Model (MLM) was applied using kinship data to define the relationship between individuals, because MLM sometimes has higher statistical power than GLM and may detect more true associations. The width of the QTL intervals was calculated by interval-mapping (a parametric test) and the Kruskal–Wallis test (a non-parametric test) using MAPQTL v4. LOD significance thresholds were determined for each trait and year with a test of 1,000 permutations between phenotypic and genotypic data using the “Permutation Test” function. The strongest marker cofactors of each QTL were identified by “Automatic Cofactor Selection” using SNP markers that exceeded the LOD significance threshold as the most significant marker. Linkage maps and QTL intervals were drawn using MapChart 2.3 software  between years, suggesting that these traits might be mostly influenced by genotype, rather than the year effect , all of which were significant with 05 Table . Other p05 Table  and apri05 Table  show esp05 Table .Prunus species, with an average number of reads per genotype of 1,843,261 in cherry  were generated, and over 220 million reads were read per lane, resulting in over 16 million tags for each plate or lane. This made it possible to get 2,272,102 good barcoded reads per each individual Table . SimilarFigure 3). This implies a mean genetic distance between markers of below 2 cM. As expected, the LG1 was the longest chromosome with 156.3 cM in “98–99” and 153.1 cM in “Angeleno.” LG5 was the shortest chromosome, with 61.5 cM in “98–99” and 79.8 cM in “Angeleno.” This supposes close to 45 Mbp for LG1 and below 20 Mbp for LG5  were identified by IM and KW for 2015 and 2016. The majority of these QTLs were localized in LG3, including IG6 Table , S6. S4_Regarding the SKC QTL, LG3, and LG4 were the LGs most linked to this trait for both parents and years Tables , S6. TheAD at different maturity states (IAD_1-2). For this QTL, S3_4166803 and S3_8549572 were identified as the cofactors of “98–99” and “Angeleno,” respectively. The explained phenotypic variance was over 30% for both parents  as a candidate gene encoding transcription factors of the NAC family  as transcription factors (TFs) involved in the expression of both traits , cherry (PavMYB10) and apple (MdMYB1/MdMYBA/MdMYB10). In apple, several authors claim that anthocyanin biosynthesis is regulated by MYB transcription factors that are positively correlated with red skin color . This strong effect was confirmed by the finding QTLs segregating in both parents, on two harvest seasons and located in a region that spans between positions 10,446,158 and 14,698,248 of chromosome 3. Marker showing the most significant effect in trait variation was S3_13359114, segregating only in “Angeleno” (Expl% = 41.9–85.5). Other important component of this trait corresponded to the QTLs mostly detected in “98–99” parent, and co-localizing with ripening time QTLs. Several QTLs for fruit skin color have been reported for peach in different LGs: in LG2 ; INVERTASE INHIBITOR ; and SUSY (Prupe.1G131700).FW QTL located in the LG7 means a promising QTL because usually fruit weight or fruit size are strongly influenced by climatic conditions, with a very polygenic nature, as reported in t et al.  placed dt et al.  located t et al.  located t et al.  identifit et al.  located t et al.  identifiAD_1-2), skin color (SKC), over color (OVC), soluble solids content (SSC), weight (FW), and shape (SHP). QTLs with a significant and consistent effect through years were found for RT, IAD_1-2, and SKC. Due to the complexity of these polygenic traits, it will be necessary to carry out a more detailed study in order to validate the reported QTLs, considering the phenotyping of more seedlings, in order to include more recombinants, and thus obtain QTLs covering narrower genomic regions for fine mapping of the traits; moreover, more phenotyping years must be included to have a more precise estimation of genetic and seasonal effects on these traits. Moreover, new molecular markers specifically designed for each QTL region must be included in order to assist fine mapping of genes controlling these traits. Such further studies would make it possible to use MAS as a breeding tool for Japanese plum. Finally, from a postharvest point of view, non-destructive control of the firmness of the fruit during the critical maturation period , chlorophyll degradation index (IJS: Writer and analyst of results. IP: Analyst of results and reviewer. PS: Management and technical support. PZ and CS: Laboratory technician. MA and DV: Adviser and rewiewer. DR and PM: Writer and reviewer. RI: Corresponding author and reviewer.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There are few studies comparing diagnostic accuracy of different lung function parameters evaluating dose–response characteristics of methacholine (MCH) challenge tests (MCT) as quantitative outcome of airway hyperreactivity (AHR) in asthmatic patients. The aim of this retrospectively analysis of our database  was, to assess diagnostic accuracy of several lung function parameters quantitating AHR by dose–response characteristics.eff) as estimate of the degree of bronchial obstruction were compared with concomitantly measured forced expiratory volume in 1 s (FEV1) and forced expiratory flows at 50% forced vital capacity (FEF50). According to the GINA Guidelines the patients (n = 484) were classified into asthmatic patients (n = 337) and non-asthmatic subjects (n = 147). Whole-body plethysmography  was performed using ATS-ERS criteria, and for the MCTs a standardised computer controlled protocol with 3 consecutive cumulative provocation doses  was used. Break off criterion for the MCTs were when a decrease in FEV1 of 20% was reached or respiratory symptoms occurred.Changes in effective specific airway conductance  3.7 times, at the 2nd level step (1 mg MCH) 2.4 times, and at the 3rd level step (2.2 mg MCH) 2.0 times more pronounced for sGeff, compared to FEV1. A much better diagnostic odds ratio of sGeff (7.855) over FEV1 (6.893) and FEF50 (4.001) could be found. Moreover, the so-called dysanapsis, and changes of end-expiratory lung volume were found to be important determinants of AHR.In the assessment of AHR, whole-body plethysmography offers in addition to spirometry indices of airways conductance and thoracic lung volumes, which are incorporated in the parameter sGApplying plethysmographic tidal breathing analysis in addition to spirometry in MCTs provides relevant advantages. The absence of deep and maximal inhalations and forced expiratory manoeuvres improve the subject’s cooperation and coordination, and provide sensitive and differentiated test results, improving diagnostic accuracy. Moreover, by the combined assessment, pulmonary hyperinflation and dysanapsis can be respected in the differentiation between “asthmatics” and “non-asthmatics”.The online version of this article (doi:10.1186/s12931-016-0470-0) contains supplementary material, which is available to authorized users. Airway hyperreactivity (AHR) is a characteristic feature of bronchial asthma, and methacholine challenge testing (MCT) is well established to quantitate AHR in patients with unexplained symptoms such as cough, chest tightness and/or dyspnea, when the diagnosis of asthma is uncertain –10. Comp1), measured with spirometry, decreases at least 20% compared to baseline (PD−20FEV1), is currently the most commonly used outcome of assessing AHR by detection of flow limitation in consequence of airway narrowing [aw) or an increase of specific airway resistance (sRaw) of 100% [eff) has never been evaluated as a target parameter.Following MCT, the cumulative provocation dose (PD) at which the forced expiratory volume in 1 s , in relation to sGeff differentiating different diagnostic and functional groups and to encounter possible differences of AHR in relation to pulmonary hyperinflation and/or the phenomenon of dysanapsis [Therefore, the purpose of the present study was to compare diagnostic value of the response characteristics to MCH assessed by FEVsanapsis –17.n = 337; 69.8%), and a group of non-asthmatic subjects . Bronchial asthma was diagnosed when subjects presented with a medical history of atopy , and documented with a positive MCT (PD−20FEV1). The groups of non-asthmatic subjects was composed of 4 subgroups according to the potential origin of pulmonary symptoms differentiating either between (i) an “upper airway syndrome” (UACS), previously also termed as post-nasal-drip-syndrome [n = 55; 37.4%), (ii) a gastroesophageal reflux disease , (iii) “persistent chronic cough” lasting more than 8 weeks [n = 30; 20.4%), and patients with (iv) a “symptom complex” such as unexplained dyspnoea, chest tightness, hyperventilation, or somatisation , which could not be attributed to a clear diagnosis. There were no patients with chronic obstructive pulmonary disease (COPD), bronchiectasis, cystic fibrosis, obstructive sleep apnoea syndrome (OSAS), or interstitial lung disease. Short-acting ß2-agonists were withdrawn for 8 h, long-acting beta-agonists for 48 h, and leukotriene receptor antagonists for 24 h prior to the lung function testing. Inhaled corticosteroids were withdrawn 7 days before MCT.The study was conducted in the Division of Pulmonary Medicine, Clinic Barmelweid, CH-5017 Barmelweid, Switzerland, recruiting patients referred to the pulmonary function laboratory for baseline pulmonary function and MCT in patients with symptoms suggestive for asthma, such as cough, shortness of breath, wheezing or chest tightness. According to the classical definition of the GINA dissemination committee report , and bassyndrome   [http://www.atsjournals.org/doi/suppl/10.1164/rccm.200603-423OC/suppl_file/onlinesup200603-423ocr2.pdf) [http://www.biomedcentral.com/content/supplementary/1465- 9921-10-106-S1.doc) [Spirometry and plethysmographic measurements were performed using standard techniques according to ATS-ERS recommendations , 22 prevods.pdf) ; http://cr2.pdf) ; http://-S1.doc) .eff, and its reciprocal value the sGeff resp., were assessed while the subject breathed tidally, in an upright position, through filter and measuring head in the body plethysmograph [Using a Jaeger MasterLab whole-body plethysmograph , measurements of airway mechanics, slow spirometry and hence assessment of static lung volumes were carried out first and only afterwards forced flow-volume loops were performed according to the ATS-ERS recommendations . The sRsmograph , without1 and FEF50 together with sGeff. sGeff is computed as the ratio between the integral of the area of the tidal flow-volume loop as numerator (∮V′dVT) and the integral of the area enclosed by the specific resistive work of breathing (sWOB = ∮ΔVboxdVT) [amb is the barometric pressure, PH2O the saturated vapour water pressure at body temperature. The mathematical background to obtain sGeff as the reciprocal value of the effective specific resistance (sReff), has been given previously [eff were measured in a first phase of testing. In a second phase of testing functional residual capacity (FRCpleth) was assessed by normal resting breathing against the closed shutter (no panting). In a third phase a slow maximal vital capacity manoeuvre, carefully linked with the shutter-measurement was performed to get the static lung volumes, functional residual capacity (FRCpleth), residual volume (RV) and total lung capacity (TLC). However, only in a forth phase of testing 3 consecutive forced flow-volume-loops were recorded. The median of at least 5 consecutive single measurements of sGeff and 3 measurements of FRCpleth were computed, and from the triplet of forced expiratory manoeuvres the best effort was analysed. The pulmonary function test data were expressed as a percentage of predicted normal values, and z-transformed accordingly [In order to obtain a parallel synoptical presentation of the MCT, we routinely present the reaction of FEVoxdVT)  accordineviously –25, 28, ordingly , 29, 30.eff, which is computed real-time by an integral, multi-dimensional approach. Details are given in the Additional file i) better diagnostic accuracy, (ii) leading by independency of deep inspirations and hence changes in the volume-history [iii) by independency from the subject’s cooperation (need of forced breathing manoeuvres) circumvention of inadvertent change of the airway responsiveness during test procedure [The assessment of the dose–response characteristics to MCH, observed not only by forced spirometry, but additionally by whole-body plethysmography, was chosen because a certain paradigm shift at least in the technical approach of the assessment of airway mechanics is established since several years, albeit the combined assessment of AHR by MCH by spirometry and whole-body plethysmography is not yet well validated and established in its clinical use. However, improved reliability of test results can be expected by assessing the bronchial reaction to sG-history , 31–36 trocedure , 37, 38.MCT was performed according to a protocol routinely used in several pulmonary function laboratories in Europe, applying a modified dose oriented, single concentration sequence  with the1: 0.2 mg; CD2: 1.0 mg; CD3: 2.2 mg. The measurements of whole-body plethysmography and spirometry were performed two minutes after each MCH-inhalation. On the basis of the inhalation time and the nebulizer output provided by the APS and the known concentration of the MCH, the applied doses of MCH was individually computed at each provocation level. Response to MCH was assessed by linear regression against the logarithmic doses of MCH giving the individual PD causing either a 20% decrease of FEV1 (PD−20FEV1), a 40%, 45% or 50% decrease of sGeff , or a 20% decrease of FEF50 (PD−20FEF50). A synopsis of the assessment of AHR by this technique is presented in the Additional file 1 > 20% was reached or symptoms occurred. After having defined PD−40sGeffas the most discriminating factor and in order to distinguish the degree of AHR to MCH, patients were classified into 4 functional severity groups: (1) non-reactive if PD−40sGeff ≥ 2.0 mg, (2) low-reactive if PD−40sGeff ≥ 1.0 mg but < 2.0 mg, (3) medium-reactive if PD−40sGeff ≥ 0.2 mg but < 1.0 mg, or (4) severe AHR, if PD−40sGeff < 0.2 mg. Each provocation test was terminated with 2 puffs of salbutamol inhaled from spacer device.After baseline measurements, MCH (5%) was administered in 3 steps of increasing dose. However, in contrast to a previously proposed 4-step procedure , a one-ceff, FEV1 and FEF50) using Chi-squared tests. Statistical analysis was performed with the IBM SPSS version 24.0 . The limit of significance was a p-value of 0.05.The discriminative power of each lung function parameter was evaluated by measures of diagnostic accuracy such as sensitivity, specificity, positive predictive values (PPV), negative predicting values (NPV), likelihood ratios , the area under receiver operating curves (ROC), the Youden’s index (J) and diagnostic odds ratios (DOR). Statistical comparisons were performed applying McNemar testing. Using these different statistical procedures several aspects of diagnostic accuracy, such as predictive ability and/or discriminative property of the MCT, could be evaluated . The Addn = 337; 69.6%) and non-asthmatic subjects  previously diagnosed. Table pleth > +1.645 SD), without or in combination with bronchial obstruction (sGeff < −1.645 SD). Regarding spirometry, normal flow-volume curve indices were found in 90.5% of asthmatic patients and 93.9% of non-asthmatic subjects. Only a very minor percentage of asthmatic patients (7.7%) presented with flow limitation (FEV1 < −1.645 SD) at baseline.A total of 484 patients  were eligible for inclusion in the study stratifying patients into a group of asthmatic patients (eff was much quicker and much more pronounced than the response assessed by FEV1 or FEF50. The cumulative percent-responses at each cumulative dose- (CD)-level demonstrate completely different response characteristics regarding the 3 target parameters. In asthmatic patients the cumulative percent-response of sGeff was at the 1st PD-level (0.2 mg MCH) 3.7 times more pronounced than with FEV1, 1.6 times than with FEF50 resp. At the 2nd CD-level (1 mg MCH) the percent-response of sGeff was 2.4 times more pronounced than with FEV1, 1.4 times more than with FEF50 resp., and at the 3rd CD-level (2.2 mg MCH) the percent-response of sGeff was 2.0 times more pronounced than with FEV1, 1.3 times more than with FEF50 resp. By nature the percent-response in non-asthmatic subjects was 1.5 to 2.9 times lower than in asthmatic patients, but the differences in percent-responses between the lung function parameters were even more pronounced.In Fig. eff is not yet clearly defined, we first compared the 3 potentially valuable thresholds PD−40sGeff, PD−45sGeff, and PD−50sGeff. Optimal determination as represented by the highest sensitivity, highest negative predictive values, highest diagnostic odds ratios (DOR) [−40sGeff, indicating that PD−40sGeff is the most appropriate threshold for evaluating AHR by sGeff. Whereas the PD−40sGeff level was reached in all subjects by sGeff, the PD−20FEV1 was not reached by FEV1 in 9.1% of subjects and in 6.4% PD−20FEF50 by FEF50 respectively. Therefore, PD−40sGeff measures AHR in comparison with PD−20FEV1 and PD−20FEF50 with better properties of diagnostic accuracy.All indices contributing to the assessment of diagnostic accuracy  , lowest −40sGeff compared to PD−20FEV1, or PD−20FEF50 . Consequently, the provocation doses to which subjects are exposed to MCH were significantly lower for PD−40sGeff compared to PD−20FEV1, or PD−20FEF50 .We found it of clinical relevance to compare the test duration and MCH-doses to achieve PD-levels by the different target lung function parameters. Table 1. In 63 of 337 asthmatic patients (18.7%) pulmonary hyperinflation was observed prior to testing, or was developed during MCT. In Fig. −40sGeff: 7.5%, n.s.; PD−20FEV1: 5.8% p < 0.001; PD−20FEF50: 7.6%, n.s.). Noteworthy to realise that pulmonary hyperinflation influenced AHR measured by PD−20FEV1 significantly. Moreover interestingly, differences were not significant for PD−40sGeff. This may be a formal proof, that the assessment of AHR using the integral method of sGeff, evaluates changes of airway mechanics concomitantly with changes of EELV during MCT.Changes of end-expiratory lung volume (EELV) during MCT cannot be assessed by spirometry, especially not a shift of the flow-volume curve towards total lung capacity in consequence of dynamic hyperinflation, which results in an elevated residual volume and a decrease of inspiratory capacity and vital capacity as well. However, a shift of EELV toward total lung capacity may have an influence on the magnitude of FEVeff yielded the greatest value for PD−40sGeff  compared to PD−45sGeff and PD−50sGeff on one hand, and compared to PD−20FEV1  and PD−20FEF50  5% CI: .680-.778) on the other hand. Considerable differences are also found regarding the Youden J-index and the likelihood ratios, both estimates of diagnostic test performance.Receiver operating curves (ROC) describing the relationship between sensitivity and specificity of percent changes during MCT of the 3 lung function parameters are given in Fig. 25–75/FVC thought to be a surrogate measure of airway size relative to lung size significantly associated with AHR [eff, and its reciprocal value sGeff respectively, as the most useful target parameters in the detection of AHR. Due to our observations of much higher percent-response to MCH by PD−40sGeff than by PD−20FEV1, we suggest, that some false negative PD−20FEV1 tests resulted in an underestimation of the severity of AHR, and hence potentially missed the diagnosis of asthma. In the following discussion we would like to focus on some important aspects of MCT performed by whole-body plethysmography, if combined with the spirometric assessment. Furthermore, we evaluate the physiological aspects of the so-called dysanapsis [The principal goal of our study was to demonstrate that the performance of MCTs using the plethysmographic technique in addition to the spirometric approach offers some fundamental benefits, such as independence from deep inspiration, and hence, modulation of the airway calibre , 31–36, with AHR . The prewith AHR  reinforcsanapsis , 14, whisanapsis –17, 45.−20FEV1-results they found that both challenge tests were equivalent in diagnosing asthma. In the present study, however, we first look at the accuracy of different target lung function parameters, which could be discriminative between “asthma” and “non-asthma”.There is an increasing interest to define proximity of measurement results comparing different test procedures. Recently Porpodis et al.  comparedeff with a provocation level of PD−40presented the highest diagnostic accuracy  for the diagnosis of asthma. In comparison with PD−20FEV1 and PD−20FEF50 the response-pattern to MCH evaluated by PD−40sGeff was consistently different. Identical agreement regarding severity of AHR was only found in 159 (32.9%) cases . In comparison to PD−40sGeff, AHR-responses of PD−20FEV1 were observed in 83 (24.9%) cases only one PD-level later, in another 73 (21.9%) cases only two PD-levels later, and in 20 (6.0%) cases even only three PD-levels later.The most remarkable finding of the present study is that the sG−40sGeff, the level of PD−20FEV1was not reached in 65 cases (13.5%). It follows that in 67.1% of asthmatic patients, a disagreement regarding MCH-response between PD−40sGeff and PD−20FEV1 was found. Since the diagnosis of asthma was primarily based on the results of AHR evaluated by PD−20FEV1 the question remains open whether or not some asthmatics are hidden in the collective of patients diagnosed as chronic cough disease. This question, however, can only be answered by a properly planned prospective study.Furthermore, in contrast to PDIt has well been recognised that deep inspirations play a major role in modulating airway calibre and airway responsiveness. In healthy subjects, deep inspirations reduce the level of pharmacologically induced airway obstruction by bronchodilation , whereasFor many years it has been recognised that one factor determining the presence of AHR to broncho-provocative agents such as MCH is airway size. It varies from one individual to another, and some of this variability cannot be explained by differences in lung size between individuals , 16. The25–75) is considered as the most effort-independent part of the flow volume curve. This fraction is very sensitive regarding flow-limitation in peripheral airways, when chronic airflow obstruction is present [25–75 and forced vital capacity (FVC) are measured routinely during forced breathing manoeuvres, the ratio between FEF25–75 and FVC has been taken as a surrogate measure of dysanapsis [25–75/FVC ratio, subjects with lowest ratio also had the lowest PD−20FEV1, supporting the notion that subjects who are more sensitive to MCH have smaller airway calibres in relation to their lung size. They concluded that baseline FEF25–75/FVC ratio could be a determinant of AHR to MCH. Moreover, Mirsadraee et al. [−20FEF25–75/FVC and PD−20FEF50 were similar to PD−35sGaw and superior to PD−20FEV1.Forced expiratory flow between 25 and 75% of vital capacity  regarding dysanapsis expressing the FEF25–75/FVC ratio as age- and gender-specific quartiles showed significant association to the severity of AHR assessed by PD−40sGeff, PD−20FEV1, and PD−20FEF50. However, within these quartiles of dysanapsis the response-pattern of the 3 lung function parameters were completely different. The differences between severe and medium AHR can be much better presented by PD−40sGeff  than by PD−20FEV1 , or by PD−20FEF50 , if related to the phenomenon of “airway narrowing” by FEF25–75/FVC ratios < 25%ile, 25 to 50%ile. These associations existed for both male and female asthmatics, indicating that as opposed to Gaw and sGaw, AHR assessed by sGeff represents a much wider range of central and peripheral airways within the bronchial tree. In line with Parker et al. [−40sGeff is in strong association with dysanapsis, and may well provide a new conceptual approach, and hence better understanding of mechanisms that predispose an individual to asthma. Within the group of non-asthmatic subjects (n = 148) 14 (9.5%)  presented with a PD−40sGeff to be labelled as “severe”, within the subgroup of dysanapsis with a ratio of FEF25–75/FVC < 25%ile, and the question remains open whether in these patients the diagnosis “asthma” was missed, because the diagnosis “asthma” was only expressed when PD−20FEV1 demonstrated AHR.The analysis in a subgroup of our asthmatic patients , as well as the phenomenon of dysanapsis to be included into the differentiation between “asthmatic” and “non-asthmatics”.Important findings in regard to the performance of MCT as a hallmark in the diagnosis of bronchial asthma could be discovered, if the spirometric approach is combined with the assessment by whole-body plethysmography. Where diagnostic accuracy is required, there are striking differences regarding the choice of target parameters. Although the spirometric assessment based on changes in FEV


We performed a 1-year longitudinal study of Six Minute Walk Test (6MWT), North Star Ambulatory Assessment (NSAA), and timed function tests in Becker muscular dystrophy (BMD). Skeletal muscle dystrophin was quantified by immunoblot. We grouped deletions ending on exon 45  or 51 ; isolated exon 48 deletion ; and other mutations (n = 21). Only patients in the “del 45-x” or “other” groups became non-ambulatory  or unable to run . All measures correlated positively with dystrophin quantity and negatively with age, and were significantly more impaired in the “del 45-x” and “other” groups. After one year, NSAA score decreased significantly ; in the “del 45-x” group, both NSAA  and 6MWT  decreased. We conclude that patients with “del x-51” or “del 48” mutations have mild or asymptomatic BMD, while “del 45-x” mutations cause comparatively severe weakness, and functional deterioration in 1 year. Furthermore, exon 51 skipping could be more effective than exon 45 skipping in Duchenne muscular dystrophy. DMD mutations, consisting approximately of 70% large deletions, 15% duplications, and 15% small mutations, leading to altered, but detectable dystrophin expression in muscle fibers1Becker muscular dystrophy (BMD) is an X-linked disorder caused by non-truncating 2. However, BMD also includes patients with calf hypertrophy and/or elevated creatine kinase, but virtually no muscle weaknessThe “typical” presentation of BMD features a juvenile onset of muscle wasting and weakness, predominant at the thigh extensors and pelvic girdle, calf hypertrophy, gradual progression leading to loss of motor function over years or decades, and frequent dilated cardiomyopathy, not proportional in severity to muscle involvementDifferent BMD deletions affect the properties of the resulting dystrophin protein: the loss of functionally critical N- or C-terminal domains may result in DMD-like phenotypes458111239131416DMD gene mutations  and skeletal muscle dystrophin content (when available). We aimed to explore if these measures are feasible and clinically meaningful in BMD as they are in DMD, and to refine the description of the natural history of relevant BMD mutational subgroups.The longitudinal description of validated, clinically meaningful outcome measures is needed for the design of BMD clinical trials. Unlike DMD181920212425All evaluations involving patients and experiments involving muscle tissue samples were performed in accordance with relevant guidelines and regulations, and were approved by the Padova Ethics Committee for Clinical Experimentation. All patients, or their legal guardians, provided their written informed consent to study procedures.DMD mutation; or (b) muscle immunoblot or immunofluorescence showing non-absent dystrophin, and any DMD mutation.We selected male BMD patients with (a) an in-frame Protein samples from diagnostic biopsies were separated by SDS-PAGE on 3–8% gradient Tris-glycine gel, and transferred for 5 hours onto a nitrocellulose membrane. We used a primary monoclonal antibody against the dystrophin C-terminus. Visualization on X-ray films was performed by ECL-chemiluminescence (Amersham). Adult male control samples were loaded in the same gel to determine relative abundance. Dystrophin quantity was determined by densitometry of dystrophin bands (ImageJ software), normalized to myosin bands in the post-transfer Coomassie blue stained gels, with subtraction of background.DMD exons and flanking regions. Single-exon deletions identified by MLPA were confirmed by PCR, using primers which did not overlap MLPA probe binding regions.Molecular analyses were performed in the context of diagnostic testing and included multiplex ligation-dependent probe amplification (MLPA) and genomic sequence of 6MWT, NSAA, and TFTs  were performed as described212425DMD deletions predicted to result at transcript level from skipping of exons 45 and 51: respectively, del 45–47, 45–48, 45–49, and 45–55 (“del 45-x”), and del 34–51, 45–51, 48–51, and 50–51 (“del x-51”). There was a double rationale to this grouping criterion: first, “del 45-x” BMD mutations have been described in the literature to be associated with a more severe phenotype than “del x-51”891011We grouped 2), including patients with previous symptoms/signs of heart failure that in most severe cases needed cardiac transplantation. We also gathered data about LVEF and LVEDV in a time window of one year before or after baseline evaluations, when available, to assess the impact of cardiac dysfunction on motor outcome measures, and especially the 6MWT, which is most influenced by fatigability and exercise tolerance, and thus most likely modified by cardiac impairment.While an in-depth discussion of cardiomyopathy is beyond the purposes of this paper, in order to better describe the clinical characteristics of the studied cohort, we assessed the frequency of dilated cardiomyopathy (DCM) in the overall cohort and in defined mutation groups. We defined DCM echocardiographically as left ventricular ejection fraction (LVEF) <50%, and/or increased left ventricular diastolic volume  and cardiac dysfunction  on 6MWT distance. Significance of functional changes was tested by Friedman rank sum test. Dystrophin quantity was treated as a continuous variable for correlations, and categorized  for descriptive statistics. Statistical significance was set at p < 0.05. Analyses were performed with R (v3.2.1), and power calculations with PS v3.0.4328 (paired t-test).We recruited 69 BMD patients, aged 6 to 69 years, from 61 unrelated families. Familial cases included one sibship of three and four sibships of two brothers, one uncle-nephew pair, and one grandfather-grandson pair .in silico to disrupt an exon splicing enhancer (ESE)Fifty-six/69 (81%) patients harboured large deletions. Two (3%) harboured duplications. In-frame microdeletions were identified in one patient  and three brothers  . Two broAverage dystrophin quantity was 59.5 ± 31.3% of control. Dystrophin was more abundant in the “del x-51” than “del 45-x” group (p = 0.03). Deletions 45–55, 10–25, 10–29, and 11–30 showed dystrophin quantities similar to control .LoA. At baseline, 3/69 patients were full time wheelchair users: one 60-year-old ; one 47-year-old ; and one 18-year-old . Two patients lost ambulation during follow-up: one 22-year-old (out-of-frame microdeletion); and one 38-year-old (missense mutation). LoA only occurred in the “del 45-x” or “other” groups  or “other”  groups stopped running. Nine could not date LoR precisely, and were excluded from time-to-event analyses. Median age at LoR was 31 overall, 95% confidence interval (CI) 26 - undetermined, and 26 in the “del 45-x” group, 95% CI 17–27. Age at LoR differed significantly between mutations  . All tesNSAA was scored in 68/69 patients (one missing score because of recent trauma) and averaged 25.3 ± 10.8, with a range of 2–34 and a right-skewed distribution (median 32.5) . Twenty-One patient refused to perform the 10 m walk/run because of recent trauma, and one could not perform TFTs because of a logistic issue. Due to disease progression, 4/67 patients (6%) were unable to walk 10 m, 18/68 (26%) were unable to rise from the floor, and 9/68 (13%) were unable to climb 4 standard steps (“zero” velocities) .All functional measures showed a moderate positive correlation with dystrophin quantity, a moderate negative correlation with age, and strong positive inter-correlations. Due to NSAA “ceiling effect”, correlations between NSAA and other parameters appeared more linear in more severe groups , although still significant in the overall BMD cohort .All functional measures differed significantly between the four mutation groups at baseline  . CompareAs all functional measures were negatively correlated with age, and patients in the “del 48” and “del x-51” mutation groups, who presented milder or no functional impairment, were on average younger than those in the “del 45-x” group, we asked if functional differences between these two group could be wholly attributed to age, or if there was an independent effect of mutations. We performed analyses of covariance (ANCOVA) with baseline NSAA and 6MWT as dependent variables, and mutation group and age as independent variables. This showed that mutation group and age have significant, independent effects on NSAA (p < 0.001 for both mutation and age) and 6MWT (p = 0.004 for mutation and p < 0.001 for age). The different slope in NSAA and 6MWT values, plotted against age, in different mutation groups, is evident in One-year 6MWT was stable overall (+3 ± 66 m). Conversely, NSAA decreased significantly  . TFTs weWe asked how many BMD patients would be needed to demonstrate effectiveness of a 1-year treatment in slowing NSAA decrease against placebo. As NSAA decrease was significant in the “del 45-x” group, we used data from this group, which we deem to be representative of a “typical” BMD population (excluding asymptomatic and DMD-like patients). Assuming α = 0.05 and power = 0.8, identifying the effect of an intervention able to arrest disease progression (1.3-point difference in NSAA change) would take approximately 15 patients per study arm, or larger numbers for smaller effects .2, with no significant differences between groups. Frequencies of cardiomyopathy in the whole cohort and in defined mutation groups, as well as average LVEF and LVEDV in the same groups, are detailed in per se, or to association of DCM to more severe muscle weakness, or both. In order to test these hypotheses, we devised an ANCOVA model with 6MWT distance as dependent variable, and two independent predictors: the presence of DCM, and NSAA score . While the NSAA score remained strongly correlated with 6MWT outcome (p < 0.0001), the presence of DCM showed no significant effect on 6MWT distance in this model, suggesting that cardiomyopathy did not hinder 6MWT performance in this population. This is in keeping with our clinical observation that no patients complained of dyspnea, dizziness, excessive fatigue, or other cardiac symptoms during the 6MWT.The frequency of DCM was 61% (35/69 patients) overall, higher in the “del 45-x”  and “other”  than “del x-51”  and “del 48”  mutation groups. Dystrophin quantity assessed by immunoblot in 30 patients who developed DCM was lower, compared to 22 patients who did not  . MeasureOur study population recapitulates typical genetic and clinical features of BMD: mutations consisting mostly (81%) of single- or multi-exon deletion, and strikingly variable phenotypes2The “del 45-x” group, conversely, was confirmed to display “typical” BMD1112Therefore, as previously proposed by others, we expect skipping of exon 51 to be more effective than skipping of exon 45 in DMD9in vitro proof-of-principle studies are warranted.Furthermore, the observation that exon 45–51 and 45–55 deletions have mild or asymptomatic phenotypes suggests that “typically” severe BMD patients with 45–47, 45–48, and 45–49 deletions might also benefit from multi-exon skipping up to exon 51 or 55. On the other hand, inefficient multi-exon skipping could lead to out-of-frame transcripts which would be degraded through nonsense-mediated decay, with the risk of diminishing overall dystrophin levels. Therefore, such an approach remains highly speculative and Some individual patients offered interesting insights into genoype-phenotype correlations. A 13-year-old patient with an exon 3–9 deletion, ablating most of the N-terminal actin binding domain, nevertheless presented a mild phenotype (NSAA 34/34), suggesting functional rescue through secondary actin-binding sitesThe novel missense mutation p.Thr160Pro (exon 6) was associated to “typical” BMD (LoA at 38 years), the mutant proline probably disrupting secondary structures within the actin-binding domain.A synonym nucleotide substitution , likely disrupting an ESE in exon 31One patient carrying the nonsense mutation c.4980G > A, p.Trp1660* (exon 35) presented relatively mild BMD  with 29% dystrophin, while another, carrying the mutation c.3843G > A, p.Trp1281* (exon 28), displayed substantial weakness  with 17% dystrophin. A BMD phenotype is sometimes observed with nonsense mutations within in-frame exons with low splice acceptor site strength and ESE density, leading to endogenous exon skippingTwo brothers had an out-of-frame microdeletion in exon 74 , and were included because of reduced IHC signal with the anti-rod DYS-1 antibody. One brother lost ambulation at the age of 22, and one is ambulatory at 17. The distal situation of this frameshifting mutation may determine a partial escape of the transcript from nonsense-mediated decay. Unfortunately, no muscle tissue was available for immunoblot.Two patients with duplications (dup 13–42 and 19–41) and abnormally large dystrophin (estimated around 550 kDa), presented symptomatic BMD. The larger duplication (13–42) was previously described as causing a mild phenotypeDMD transcripts and/or dystrophin proteins. We did not observe a clear correlation between dystrophin quantity and phenotype within the most frequent mutation groups  always walked more than 400 m, a threshold suggesting low probability of short-term changes in DMDThe NSAA and TFTs, on the one hand, showed obvious “ceiling effects” in the “del 48” and “del x-51” mutation groups. On the other hand, in “typical” BMD (e.g. “del 45-x”) these measures showed a symmetric distribution, a tight inverse correlation with age (similar to muscle strength scores in the Dutch cohort)The 6MWT distance was stable overall after one year. Breakdown of 6MWT changes by mutation revealed apparent “improvements” in a few mildly affected patients, probably due to a “learning effect”. However, a decreasing trend (−12 m) was observed in typical BMD, hinting at a possible detection of significant changes with longer observation, and supporting the clinical meaningfulness of longitudinal 6MWT in BMD.NSAA changes, although not informative for mild patients , detected a statistically significant progression (−0.9), driven by a decrease in the “del 45-x” group (−1.3), in itself statistically significant and clinically meaningful, as this decrease relates directly to loss of a motor ability or need for compensation to perform it independently.These observations have several implications. First, we conclude that functional inclusion criteria (for instance NSAA <32) would be useful when one seeks to measure disease progression in a one-year trial. Second, we provided a preliminary power calculation for a hypothetical BMD trial, which represents a step towards clinical trial readiness. Third, in-depth analyses of loss of function patterns are warranted to improve our understanding of BMD natural history and possibly adapt the DMD-tailoredFor the sake of clarity, we purposely left an in-depth study of DCM onset, outcomes, and genotype-phenotype correlations out of this manuscript, which focuses on skeletal muscle functional measures. Nevertheless, a brief description of DCM frequencies within mutation groups, and a simple comparison of skeletal muscles dystrophin quantity between patients with and without DCM, seem to suggest that more severe skeletal muscle phenotypes are associated with a somewhat higher probability of developing DCM. None of the patients in our cohort showed overt symptoms of heart failure at the time of evaluations. Among outcome measure explored in this study, the 6MWT, which has been originally devised for patient with cardiac and respiratory diseasesWe acknowledge several limitations to this study: lack of baseline test/retest, unavailability of immunoblot in all patients, and some loss to longitudinal follow-up. We plan to continue data collection and expand the cohort. Furthermore, the “del 48” and “del x-51” patients were younger, which might bias comparisons to some extent, although older individuals in these groups never showed functional deterioration.In conclusion, we characterized mutation-specific BMD subphenotypes with longitudinal measures, and predicted better outcomes for exon 51 than 45 skipping in DMD. We showed that only BMD patients with measurable muscle weakness at baseline incur functional deterioration after one year. NSAA and 6MWT hold promise as clinically meaningful outcome measures for BMD trials.How to cite this article: Bello, L. et al. Functional changes in Becker muscular dystrophy: implications for clinical trials in dystrophinopathies. Sci. Rep.6, 32439; doi: 10.1038/srep32439 (2016).


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We assessed if patients with known hypoglycaemia present on other occasions with non-specific symptoms associated with (but not diagnosed as) hypoglycaemia, potentially representing missed hypoglycaemia.335 primary care records (5/2/12-4/2/13) from patients aged >65  were assessed for hypoglycaemia episodes and consultations with non-specific symptoms, “hypo clues”.27/79(34%) insulin-treated patients had >1 documented hypoglycaemia episode, compared to 4/85(5%) sulphonylurea-treated patients, 2/121(2%) metformin-only treated patients, and none without diabetes, p < 0.001.“Hypo clue” consultations were common: 1.37 consultations/patient/year in insulin-treated patients, 0.98/patient/year in sulphonylurea-treated, 0.97/patient/year in metformin only-treated, and 0.78/patient/year in non-diabetic patients, p = 0.34. In insulin-treated patients with documented hypoglycaemia, 20/27(74%) attended on another occasion with a “hypo clue” symptom, compared to 21/52(40%) of those without hypoglycaemia, p = 0.008. No significant difference in the other treatment groups.Nausea, falls and unsteadiness were the most discriminatory symptoms: 7/33(21%) with hypoglycaemia attended on another occasion with nausea compared to 14/302(5%) without hypoglycaemia, p = 0.002; 10/33(30%) vs 36/302(12%) with falls, p = 0.007; and 5/33(15%) vs 13/302(4%) with unsteadiness, p = 0.023.Non-specific symptoms are common in those >65 years. In insulin-treated patients at high hypoglycaemia risk, nausea, falls and unsteadiness should prompt consideration of hypoglycaemia. The practice’s Egton Medical Information Systems (EMIS) database was used to identify all patients aged 65 or over who were treated with insulin (n = 79), sulphonylureas (but not insulin) (n = 85), or metformin only (n = 121), and 50 age-matched non-diabetic patients.One author (SH: a geriatrician) systematically reviewed patients’ consultation notes over a one year period (5/2/12-4/2/13), to identify any episodes of hypoglycaemia (defined below), or any “hypo clue consultations” – consultations with non-specific symptoms known to be associated with hypoglycaemia (see below), where no other obvious explanation or subsequent diagnosis was recorded. The records were reviewed sequentially using the practice’s internal computer number for each patient . Review of the consultation records was performed independently of patient characteristics which were collected on a separate occasion: age, diabetes details, treatment, and glycated haemoglobin (HbA1c) blood test results.2.1Hypoglycaemia episodes were defined as episodes having been directly confirmed by a doctor or nurse, paramedic or hospital .2.2A “hypo clue consultation” was defined as one or more of the following symptoms recorded in the primary care records, without an obvious explanation or subsequent diagnosis documented – or documentation that hypoglycaemia had been considered. The symptoms (or synonyms) included were based on the work by Jaap et al. 2.32/Fisher’s exact tests used for comparing frequencies across groups and for the binary analyses of “at least one” hypoglycaemia episode or “hypo clue” consultation over the year by treatment group.The majority of the data was non-parametric; thus median results and interquartile ranges are presented, and chi2/Fisher’s exact tests.Frequency of presentation with individual “hypo clue” symptoms was assessed. Individual symptom frequencies were compared in patients who had, and those who had not had, a recognised episode of hypoglycaemia on another occasion, using chiThe median HbA1c of those with/without at least one hypoglycaemia or “hypo clue” consultation per treatment group was compared using the Mann Whitney test.2.4The research project was based on an initial audit within the practice, which did not require ethical permission.33.1At least one episode of hypoglycaemia was recorded for 27/79 (34%) insulin-treated patients, compared to 4/85 (5%) sulphonylurea-treated patients, 2/121 (2%) metformin-only treated patients, and none in patients without diabetes. The total frequency was significantly higher in insulin-treated patients: 51 episodes (0.65 episodes/patient/year), compared to 5 episodes in the 85 patients with sulphonylureas (0.06 episodes/patient/year), and 3 in the 121 (0.02 episodes/patient/year) for the metformin-only treated patients, p < 0.001, 3.2Even patients without diabetes had frequent consultations with at least one non-specific symptom without other obvious documented explanation (feasibly due to hypoglycaemia in an at-risk patient), 0.78 consultations/patient/year (39 consultations in 50 patients). Rates of “hypo clue” consultations were similar for all patients with diabetes, regardless of treatment , 3.3The most commonly reported non-specific symptoms overall in this study, in decreasing order of frequency, were lethargy/tiredness , falls , feeling unwell , dizziness/light-headedness , depression symptoms , nausea , and unsteadiness .3.4In those patients who were insulin-treated and had at least one documented episode of documented hypoglycaemia over the year, 20/27 (74%) had presented on at least one other occasion with a “hypo clue” symptom, 3.5When the rates were compared overall in those with/without at least one episode of documented hypoglycaemia, the symptoms that were significantly more common were nausea , falls , unsteadiness , and depression symptoms .The majority of patients  with at least one documented episode of hypoglycaemia were insulin-treated. Of these, 9/27 (33.3%) had presented on another occasion with a fall, compared to 4/52 (7.7%) insulin-treated patients without a documented episode of hypoglycaemia, p = 0.008; and a higher proportion had presented with unsteadiness , p = 0.043. Presentation with nausea was also more frequent in those insulin-treated patients with a recognised/reported episode of hypoglycaemia over the year: 6/27 (22.2%) vs 1/52 (1.9%), p = 0.006.3.6Hypoglycaemia was unrelated to HbA1c (p > 0.4), 4Non-specific symptoms are a common presentation to primary care in patients over 65 with and without diabetes. However we have shown that patients at high risk of hypoglycaemia – patients over the age of 65 who are insulin-treated and have had a recognised episode of hypoglycaemia, present to primary care on other occasions with unexplained non-specific symptoms which may represent unrecognised hypoglycaemia. Falls, unsteadiness and nausea are particularly worth noting.4.1This study examined the difficult question of whether additional episodes of hypoglycaemia might be being missed in the older population. Primary care consultation records from just one primary care practice were used, albeit a reasonably large one. This meant reliance on documentation (coding and free text) by a limited number of staff; however this may also have offered more internal consistency for recording and comparing the vague symptoms between patients in this population. The similar rates of “hypo clue” consultations seen in sulphonylurea, metformin and non-diabetic patients is reasonably reassuring for consistency of the approach for identification of these consultations.The “hypo clue” consultation definition used was deliberately all-embracing – hence high rates were seen in patients without diabetes. Even so, more “hypo clue” consultations were seen in insulin-treated patients who had also had a documented episode of hypoglycaemia. This rate may be artificially elevated as this group of patients could consult more often. We tried to address this by presenting the results comparing those with and without episodes of hypoglycaemia within treatment groups.The definition of hypoglycaemia, in contrast, was taken as a strict definition, i.e. only those episodes documented as having been confirmed in some way by a healthcare professional. This will under-estimate the frequency of hypoglycaemia, and may preferentially identify more “severe” episodes. This approach combined with the small study size may have limited the chance of identifying possible associations with those patients experiencing hypoglycaemia. In order to corroborate any relationships seen, a prospective study of possible “hypo clue” symptoms and hypoglycaemia could be undertaken.Although not captured in the current analysis, less “severe” episodes of hypoglycaemia (e.g. those which were self-treated and not reported to primary care) are obviously also important: they may potentially pose a risk for development of reduced hypoglycaemia awareness and a subsequent more “severe” event, in addition to as yet under-appreciated possible effects e.g. on long-term cognition. A study which also directly asked patients about their experience/frequency of hypoglycaemia may prove valuable, perhaps combined with a more intense but objective assessment of hypoglycaemia, e.g. using continuous glucose monitoring.Further study in a bigger dataset could be revealing: e.g. an “index” event of hypoglycaemia taken and preceding consultations analysed to see if “hypo clue” consultations preceded a recognised event – and thus potentially expose more robust “red flag” symptoms – or corroborate those suggested in the current study. A larger study would also allow more sophisticated analyses to be done, in particular corrections for factors which may have an impact on risk, such as age Finally, HbA1c analysis was limited as it was based on a single HbA1c level from the year; and therefore does not reflect potential variation  over the year. No apparent relationship with hypoglycaemia or “hypo clues” was seen in the current study. This particular practice had been subject to a similar audit previously, and thus it is possible that the frequency of patients with very low HbA1c was lower than average.4.2Consistent with published literature, we found that documented hypoglycaemia is more frequent in insulin-treated patients, and the finding that 34% of insulin-treated patients had a “definite” episode of hypoglycaemia confirmed by a healthcare professional over the year is consistent with the 7–46% in insulin-treated patients of different durations in the UK Hypoglycaemia Study 18/50 (36%) of patients without diabetes had a “hypo clue” consultation by our definition. As previously discussed, this was an all-embracing definition, which included many non-specific symptoms frequently presenting to primary care. Although not directly comparable, other primary care studies have found 22-48% patients presenting with symptoms which could not be given a same-day diagnosis Regarding presentation with non-specific symptoms, lethargy/fatigue, feeling “generally unwell”, falls, and light-headedness/dizziness were the most frequently reported, each in over 10% of these patients aged >65. However falls and unsteadiness, along with nausea, were reported significantly more frequently in those who had also had (on another occasion) a hypoglycaemia episode. Overall 21% of those with at least one episode of documented hypoglycaemia over the year had attended on another occasion with nausea without a documented diagnosis, in comparison to 5% of those without an episode of hypoglycaemia – and 22% vs 2% in those who were insulin-treated.It is perhaps not surprising falls were one of the most frequently presenting symptoms, as they are one of the most dramatic. However the difference of 30% vs 12% (or 33% vs 8% of insulin-treated) patients presenting with a fall in those who had/had not also had a documented hypoglycaemic episode on a different occasion is marked. Kachroo et al. Recognised symptoms associated with hypoglycaemia which could predispose to falls include shakiness, anxiety, confusion, lethargy/fatigue, tiredness, drowsiness, weakness, light-headedness, dizziness, unsteadiness, incoordination, and double or blurred vision In contrast to the current study, a large meta-analysis showed a 30% increase in severe hypoglycaemia with tight glycaemic control in people with Type 2 diabetes 4.3Patients >65 and who are insulin-treated are at the highest risk of hypoglycaemia documented in primary care records, as will be well-recognised by primary care practitioners. However, hypoglycaemia in older adults is associated with non-specific and less intense symptoms than in younger people This study suggests those who have had a recognised episode of hypoglycaemia seem more likely to present on another occasion with a non-specific symptom which could conceivably be due to hypoglycaemia, and nausea, falls and unsteadiness seem to be particularly notable. The likelihood of this is corroborated by other published data, and as such, insulin-treated patients presenting with these symptoms should be reviewed with hypoglycaemia in mind.More recent guidelines for older adults At the very least however, clinicians should be alert to the possibility of unrecognised hypoglycaemia in their older insulin-treated patients, and review them with this in mind.5Non-specific symptoms which can be symptoms of hypoglycaemia are common in a population over 65. However in insulin-treated patients at risk of hypoglycaemia, these “hypo clue” symptoms, in particular nausea, falls and unsteadiness, may represent episodes of hypoglycaemia not recognised by the patient. Thus GPs should consider a review, including of diabetes medication, when patients report or present with these symptoms.The authors state that they have no conflict of interest.ATH is an NIHR and a Wellcome Trust Senior Investigator.


Cystathionine-γ-lyase (CSE) was shown to have a regulatory role in glucose metabolism. Circulatory shock can induce metabolic stress, thereby leading to hyperglycemia and mitochondrial dysfunction. In vitro data suggest an effect of high glucose on CSE expression. Therefore, the aim of this study was to investigate the effects of hyperglycemia on CSE expression in resuscitated murine septic shock.n = 5-6 per group) underwent cecal ligation and puncture (CLP)-induced polymicrobial sepsis or sham procedure (n = 6 per group) and, 15 h afterwards, were anesthetized again, surgically instrumented and received intensive care treatment, including antibiotics, lung protective mechanical ventilation, circulatory support, and intravenous (i.v.) glucose infusion . Blood and breath gas were sampled hourly to quantify parameters of glucose metabolism. 5 h later, mice were sacrificed and organs were harvested. The liver mitochondrial respiratory activity was determined via high resolution respirometry; CSE, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), and adipocyte differentiation-related protein (ADRP) expression was immunohistochemically investigated.Normo- (80–150 mg/dl) and hyperglycemic (>200 mg/dl) male C57/BL6J mice (In sepsis combined with hyperglycemia the least CSE and PGC1α expression could be detected, along with reduced mitochondrial respiratory activity, and enhanced ADRP expression, a marker of lipid droplet formation, in the liver. A novel in vivo finding is the CSE translocation from the cytosol to the nucleus triggered by metabolic stress.A relationship between CSE and glucose metabolism was established, which, when dysregulated, may contribute to fatty liver disease and hepatic steatosis. Hyperglormation  resultinormation . All theormation . In factormation –16. The ormation . Consequfunction , and thefunction .All animal experiments were performed with the approval of the University Animal Care Committee and of the federal authorities for animal research of the Regierungspräsidium Tübingen, Baden-Württemberg and with adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals. We used the available liver tissue from a previous study  of normon = 6 for septic, n = 5 for sham) and normal  endpoint glycemia.Since mice, when critically ill, become hypoglycemic instead of hyperglycemic, due to their different metabolic phenotype –21, in t2 0.5, tidal volume from 6 to 8 μl/g , respiratory rate 160 breaths/min, inspiratory/expiratory time ratio 1:2, and PEEP 5 cmH2O. Catheters were inserted into the jugular vein, the carotid artery, and the bladder; a perivascular flow probe was placed around the portal vein in the liver organ bed. Microvascular perfusion, capillary hemoglobin concentration, and oxygen saturation were determined simultaneously every hour by a combined laser Doppler flowmetry and remission spectroscopy technique, as also described previously [The anesthesia, cecal ligation and puncture (CLP) procedure, and surgical instrumentation, all have been described in detail previously . Mice weeviously , 27. Aneeviously  was choseviously , 30. Sep13C6-glucose. For the measurement of blood glucose concentrations and 13C6-glucose tracer enrichment, plasma samples were spiked with 6,6-2H2-glucose for concentration determination, with an amount targeted to achieve a 2H2-tracer mole fraction of 50%. The spiked samples were derivatized with N-methyl-bis (trifluoroacetamide)  to obtain the trifluoroacetyl-glucose derivative. The latter was analyzed by gas chromatography/mass spectrometry (GC/MS) under electron impact determination, and the signals at (m/z) 319, 321, and 325 were recorded for the 2H2- and 13C6-tracer mole fraction determination. Expiratory gas, 1 ml, was intermittently collected as an aliquot from 60 to 80 respiratory cycles from the expiratory branch to determine both expiratory CO2 concentration and 13CO2 tracer enrichment using GC/MS (masses m/z 44 and m/z 45). CO2 production rates (VCO2) were calculated as the product of tidal volume, respiratory rate, and CO2 concentration. During steady-state conditions, the glucose rate of appearance (Ra) was derived from the arterial plasma isotope enrichment, while glucose oxidation can be determined from 13CO2 tracer enrichment in breath gas samples.Glucose metabolism was evaluated as described previously , 27. 50%2 · 6H2O, 60 mM Lactobionic acid, 20 mM Taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM Sucrose, and 1 g/l bovine serum albumin), and 2 mg of tissue were added to the Oxygraph chamber. By the addition of a defined sequence of substrates and inhibitors, various states of mitochondrial function could be assessed. Complex I activity was determined after the addition of 10 mM pyruvate, glutamate and 5 mM malate, and ADP. 10 μM cytochrome c was added to check for mitochondrial integrity. Maximum oxidative phosphorylation (max OxPhos) was evaluated after subsequent addition of 0.5 mM octanoyl-carnitine and 10 mM succinate, leak compensation was assessed after inhibition of the ATP-synthase by 5 μM oligomycin, followed by stepwise titration of the uncoupling agent Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone  to reach maximum respiratory activity of the electron transfer system in the uncoupled state (ETS). The activity of complex II could only be determined in the uncoupled state by the addition of 0.5 μM rotenone, an inhibitor of complex I. The measurement was finished after the addition of 5 μM of the complex III inhibitor antimycin A.Mitochondrial respiratory activity was determined via high-resolution respirometry with a Clark-electrode-based system  as also described previously . Post-mo2 fields were evaluated. Data are represented as mean densitometric sum red.Identical liver biopsies were sampled immediately post mortem and were fixed in formalin for a defined period of time, dehydrated and embedded in paraffin. Immunohistochemistry (IHC) was perfomed on 3-μm sections that were deparaffinized in xylene and rehydrated in graded mixtures of ethanol to deionized water. Heat-induced antigen retrieval was performed by microwaving in citrate buffer pH 6.0, followed by blocking with 10% normal goat or donkey serum, depending on the source of the secondary antibody, as described previously , 32. Thehttps://www.ptglab.com/). Furthermore, a gene database search confirms that CSE should be found in the nucleus with the same confidence as in the cytoplasm (www.genecards.org).IHC for cystathionine-β-synthase (CBS) and 3-mercaptopyruvate-sulfurtransferase (MST) were performed as well; expression levels for both enzymes were negligible (data not shown). Due to the surprisingly clear and specific CSE nuclear stain with the antibody, which had never been reported before, we decided to investigate it further. In spite of the fact that it has been shown by western blot (including knock-out) to be the most specific commercially available antibody , we requ−bx.All data are presented as median (quartiles) unless otherwise stated. After exclusion of normal distribution using the Kolmogorov-Smirnov test, differences between all groups were analyzed with a one way Kruskal-Wallis analysis of variance on ranks followed by a post hoc Dunn’s test for multiple comparisons. Quantitative relations, of pooled data sets from all experimental groups, between CSE expression and glucose production rates and CSE expression and PGC1α were evaluated by measuring the Pearson’s coefficient of correlation for linear relationships. The relation between CSE and ADRP expression was determined with the Spearman’s coefficient for non-linear relationships. For the latter, data were fitted to an exponential decay function according to the general equation f (x) = a · eTable p = 0.001). Glucose production was lowest in the hyperglycemic septic animals, but this difference did not reach statistical significance (p = 0.069). There were no differences in the glucose production rates between the other experimental groups. Whole body CO2 production was significantly elevated in the hyperglycemia sham arm in comparison to normoglycemia , but did not differ between normo- and hyperglycemia.Data on glucose metabolism are depicted in Fig. p = 0.024). Leak compensation flux did not differ between the groups, whereas the maximum respiratory activity was significantly reduced by hyperglycemia as well as sepsis (p = 0.009). Reduced mitochondrial activity was mostly associated to impaired function of complex II (p = 0.008), activity of complex I was only significantly reduced in the hyperglycemic septic animals in comparison to normoglycemia sham (p = 0.016).Oxygen consumption in various respiratory states of liver mitochondria is given in Fig. p = 0.001). The loss of CSE in the cytosol, particularly in regions of injury, was concomitant with an apparent translocation to the nucleus. Higher CSE expression correlated with higher glucose production rates  .The expression of the mitochondrial biogenic factor peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) showed a comparable pattern and did also correlate with CSE expression (p = 0.002). The hyperglycemic CLP animals had the highest ADRP levels. In particular the reduction of CSE and PGC1α staining around the central vein was concomitant with increased ADRP expression in the same region. Pooled data from all experimental groups revealed a negative exponential correlation between CSE and ADRP expression (p = 0.0003)  a significant downregulation of CSE in the liver of septic mice with hyperglycemia along with (ii) apparent nuclear translocation of the CSE protein under stress conditions (iii) reduced glucose production, (iv) reduction of mitochondrial respiratory activity, and (v) a significant downregulation of PGC1α, ultimately being associated with (vi) fat accumulation in the liver.2 production (VCO2), indicating that substrates were being cleared rather than accumulated, thus helping to explain the low levels of ADRP expression in the livers. In contrast to our study, all previous experiments that have shown a downregulation of CSE by high glucose have been performed in isolated cell systems in vitro [Hyperglycemia led to reduced mitochondrial activity in this study, characterized by decreased maximum respiration in the uncoupled state, which was due to complex II. Interestingly, the attenuated mitochondrial function reflected in complex II in the hyperglycemic sham arm, was similar to that observed in normoglycemic CLP animals. In hyperglycemic sham animals, we only observed a small downregulation of CSE by high glucose, albeit coinciding with elevated lactate levels, which did not reach statistical significance. Acute hyperglycemia, apparently due to the short duration of the glucose administration, had only minimal effects, in contrast to the effects shown for chronic hyperglycemia . The hypin vitro , 7, 36.2S in the induction of gluconeogenesis [The septic hyperglycemic group presented with a significant downregulation of liver CSE, which in turn coincided with compromised gluconeogenesis as evidenced through the lowest glucose rate of appearance, confirming the finding of Untereiner et al. . PGC1α eogenesis , was alsogenesis –41. Reduogenesis  and was ogenesis , as wellogenesis . This maogenesis ).The reduction of PGC1α may not only lead to impaired function but to a total decrease in the number of mitochondria . This reThese findings were corroborated by the physiological results, which demonstrated that the hyperglycemic septic animals had the highest norepinephrine requirements to maintain the targeted MAP, i.e., 1.5-fold higher than CLP in the presence of normoglycemia. Lactate, both documenting a more severe septic shock as well as reduced hepatic gluconeogenesis , was sig2 production. This might hint to a shift towards glucose oxidation and reduced fat utilization. Other authors refer septic shock-related liver dysfunction to reduced hepatic arterial blood flow [2 saturation differed between the experimental groups. Hence, the observed liver damage was most likely not due to malperfusion or tissue hypoxia, but may indeed, be due to the cellular inability to utilize oxygen [These results are consistent with the findings reported above that the septic hyperglycemic group presented with the lowest glucose rate of appearance. This group was further characterized by a modestly elevated glucose oxidation, which might be attributed to the administered norepinephrine , togetheood flow . We did e oxygen , as evinMice, when critically ill, become hypoglycemic instead of hyperglycemic, due to their different metabolic phenotype, which is why in our study animals received exogenous glucose administration , 21. ThuThe IHC evaluation of the tissue not only helped identify topography of protein expression in regions affected by septic shock, but also eliminated confounding results due to contamination with occult blood products, a common occurrence in processed tissue . We werepost hoc study demonstrated, for the first time, in vivo, in a clinically relevant resuscitated mouse model of septic shock, that there is a direct effect of glucose on CSE expression. Hyperglycemia in sepsis can downregulate CSE expression in the liver and thereby compromise PGC1α signaling, reduce mitochondrial activity, and promote lipid droplet formation—all early signs of liver damage, which may ultimately manifest as fatty liver disease and hepatic steatosis [This teatosis .