EPAS1 haplotype carrying selective sweep signals.Adaptive evolution in humans has rarely been characterized for its whole set of components, i.e. selective pressure, adaptive phenotype, beneficial alleles and realized fitness differential. We combined approaches for detecting polygenic adaptations and for mapping the genetic bases of physiological and fertility phenotypes in approximately 1000 indigenous ethnically Tibetan women from Nepal, adapted to high altitude. The results of genome-wide association analyses and tests for polygenic adaptations showed evidence of positive selection for alleles associated with more pregnancies and live births and evidence of negative selection for those associated with higher offspring mortality. Lower hemoglobin level did not show clear evidence for polygenic adaptation, despite its strong association with an  EGLN1 and EPAS1, the genetic variants in which were swept to high frequency in Tibetans due to strong positive natural selection. However, it still remains unclear if and how these and other genetic variants are connected to adaptive phenotypes and ultimately to fitness advantage. In this study, we collected genotype and phenotype information of 1,000 ethnically Tibetan women from the high Himalayan valleys in Nepal. Using both genome-wide association analysis and test for polygenic adaptations, we show that natural selection systematically altered frequency of alleles associated with reproductive outcomes to the direction of increasing fitness. That is, alleles associated with more pregnancies and live births, as well as those associated with lower offspring mortality, were under positive selection. Omitting the EPAS1 haplotype under selective sweep, the other variants associated with lower hemoglobin did not collectively show a clear signal for polygenic adaptation. Our study highlights the polygenic nature of human adaptive traits.The adaptations to high altitude environments in Tibetan populations have long been highlighted as an important case study of adaptive evolution in our species. Recent genetic studies found two genes,  Decades of research have accumulated evidence for widespread adaptive phenotypic evolution in nature, including correlations between phenotypes and environmental factors –3, and hA complementary set of approaches aims at assessing directly the effects of genotype on reproductive fitness . These dEGLN1 (egl-9 family hypoxia inducible factor 1) and EPAS1  .–39.EGLN1EPAS1, and find no loci with strong signatures of selective sweep beyond EPAS1  and EGLN1   with 1KGp = 0.002). We also found that Hb correlated positively with the numbers of stillbirths or miscarriages , as well as their proportions among pregnancies .A previous analysis of this sample of Tibetan women found strong relationships between physiological traits and reproductive success in this Tibetan sample by using a large set of covariates, including physiological, sociocultural, and socioeconomic variables  . Becausep = 2.02×10−5 and 2.76×10−5, respectively; p = 0.005) after correcting for a woman’s age at her last pregnancy, even if weaker. The pulse and fertility traits were previously shown to be marginally correlated if a larger set of physiological and sociocultural covariates was included in the model  [Another interesting finding was the negative correlations between pulse and most of the fertility phenotypes, with the strongest correlations found with the numbers of pregnancies and livebirths (ctively) .These results suggest that both low Hb and low pulse are associated with better reproductive outcomes at high altitude and raise the possibility that genetic variants decreasing these traits were selected for in Tibetans.EPAS1 and EGLN1 loci harbored the highest PBS values  ascertained from our Tibetan Hb and oxyHb GWAS, respectively. Compared to control SNPs [p = 0.047 and 0.018, respectively; p = 0.102 and 0.046 for Tibetans and Sherpa, respectively; EPAS1 SNP rs372272284 was excluded, no difference between the Hb- or oxyHb-associated SNPs and control SNPs was observed  for oxyHb, or Hb (poverdispersion = 0.201 and poutlier = 0.846 with no multiple test correction) when the EPAS1 SNP was excluded . Therefore, these analyses do not provide evidence that alleles associated with lower Hb levels were selected for, except for the EPAS1 locus. Given that the EPAS1 SNPs explain a small fraction of the total variation in Hb levels (2.7% in our cohort), these results raise the question of whether unelevated Hb per se was the adaptive trait in Tibetans.GWAS SNP effect sizes have been shown to be correlated between European and East Asian populations , implyinulations . We founexcluded . Similarpoutlier = 0.023 and ppairwise = 0.002; r = 0.441 compared to that with oxyHb r = 0.874). The alleles associated with higher deoxyHb in Tibetans were on average less common in Tibetans than in 1KGP CHB and the genetic values of deoxyHb in Tibetans and the Sherpa tended to be lower than those in 1KGP East Asians. Maximizing oxygen delivery while minimizing blood viscosity is likely to be beneficial in high-altitude environments; therefore, this advantage may underlie our signal of polygenic adaptation for lower deoxyHb.In contrast to Hb and oxyHb, deoxyHb showed significant polygenic adaptation signals, with both the outlier and the pairwise difference tests . Interestingly, we do not see significant signals in either overdispersion or outlier test for Tibetans or Sherpa (p > 0.05); instead, we see a strong signal in the outlier test for lowland East Asians toward high pulse  and the fertility fitness component . Furthermore, consistent with expectations, alleles increasing offspring mortality were selected against whereas those increasing offspring survival were positively selected for. A variable known to be directly linked to reproductive fitness, i.e. a woman’s age at her first childbirth, is also under selection, with earlier ages being advantageous, as expected. Twinning appears to have been selected against in Tibetan women. Although twinning may increase fitness, it is also associated with increased risks to mother and offspring due to limits on women’s ability to support adequate weight gain for two babies during the third trimester and to the lower birth weight of twins relative to singletons [The GWAS of reproductive traits allowed us to identify candidate variants that are currently being selected for in the sampled Tibetan population. In our results, none of the most strongly associated variants with reproductive outcomes showed strong signals of selective sweeps. However, if these variants affected reproductive fitness in the past in addition to the current generation, we might expect signals of polygenic adaptation. Indeed, a number of reproductive traits showed strong signatures of polygenic adaptations based on the outlier test; the pairwise population difference test, which uses less information and hence is likely to be less powerful, gives broadly consistent results, although at lower levels of significance . We see ngletons , which iEPAS1 gene, even though we confirmed a correlation between low Hb and better reproductive outcomes. Because Hb concentration is a polygenic trait, these results raise the question of whether lower hemoglobin is causally related to higher reproductive fitness.We identified several genome-wide significant associations with key physiological and fertility phenotypes in Tibetans , by analEGLN1 and EPAS1 SNPs, was not significantly associated with reproductive success probably due to low power: we estimated that the lowest selection coefficients that we had 80% power to detect were 6.6% and 7.4% at the EGLN1 and EPAS1 SNPs, respectively  and live in a mostly traditional society, where modern medical care, including in some regions contraception, has been introduced only very recently . The repectively . These sectively . BecauseEPAS1 gene [EPAS1 haplotype and low oxyHb, which correlates highly with total Hb. Interestingly, the association was stronger for oxyHb than for total Hb (p = 0.883 for rs372272284). This observation raises the possibility that it is the oxygen-carrying portion of total Hb that drives the well-replicated association between EPAS1 SNPs and Hb. We also found that SNPs associated with Hb did not show polygenic adaptation signals in our Tibetan sample, if the EPAS1 SNP was excluded from the analysis (p = 0.019 without multiple test correction). Based on our estimate of 0.386 g/dL per allele, and a mean allele frequency difference of 0.743 between high and lowlanders, we calculated that the EPAS1 SNPs can explain 52% of the 1.1 g/dL difference reported in [EPAS1 SNP explains only 2.7% of inter-individual variation in Hb: therefore, almost all within-population (97.3%) as well as a substantial portion of between-population (48%) variation remains unexplained.An attenuated erythropoietin and Hb concentration response to hypobaric hypoxia is a hallmark phenotype of the “Tibetan pattern” of high-altitude adaptations, which is markedly different from that of Andean highlanders , 67, 68.AS1 gene , 34 haveAS1 gene ; this hyAS1 gene . Our GWAtotal Hb , while ianalysis . IntriguEPAS1 and with the significant evidence for polygenic adaptations toward lower deoxyHb. This finding raises the question of whether unelevated Hb was the true target of selection in Tibetans rather than a mere correlate of the true adaptive trait. This scenario would be consistent with the observed correlation between low Hb and better reproductive outcomes because pleiotropy can induce a non-causal association between phenotypes. A recent study showed that the same EPAS1 SNP that is associated with Hb and other hematological traits is also associated with uric acid levels [EPAS1, a transcription factor with dozens of target genes, may affect multiple, seemingly unrelated phenotypes. Interestingly, the peak of our association signal for oxyHb at EPAS1 spans active enhancer (H3K27Ac) marks in human umbilical endothelial cells, as detected by the ENCODE project [EPAS1 expression in the endothelium with effects on vascularization, vasoconstriction, vasodilation and possibly beneficial effects in oxygen delivery at high altitudes. These findings suggest that the WHO altitude-adjusted elevated hemoglobin cut-off for detecting iron-deficiency anemia [Several scenarios could account for these results. Incomplete power in the Tibetan GWAS and/or in the polygenic adaptation tests could underlie the lack of clear evidence for polygenic adaptation for lower Hb levels, although we had sufficient power to detect polygenic adaptation signals for several other traits in the same samples. One possibility is that post-reproductive Hb levels are a poor proxy for the levels while women are reproductively active. Some, but not all, studies of Tibetans find an increase in Hb concentration with age –75, but d levels , suggest project , pointiny anemia  may be iADH locus [OCA2 gene (CHB = 0.59 and JPT = 0.57 versus Tibetan = 0.06 and Sherpa = 0.16). Therefore, the observed shift towards higher genetic values for pulse at low altitude could be the result of a selective event that similarly affected only low altitude populations. However, the correlation between low pulse and better reproductive outcomes in Tibetans suggests that low pulse is adaptive  at high altitude and raises the possibility that lower pulse was selected for when ancestral low altitude populations moved to high altitude.The lower genetic values for pulse in Tibetans compared to low altitude East Asians, coupled with the correlation between lower pulse and better reproductive outcomes in Tibetans, suggest an important role for cardiac function in pregnancy at high altitude. Intriguingly, tests of polygenic adaptation that use data from worldwide populations are consistent with selection favoring higher pulse in low altitude East Asians, but not in the closely related populations at high altitude. There is some prior evidence for selective events that took place in low altitude, but not high altitude East Asians. For example, the well-known selective sweep signal at the DH locus  in low aCCDC141 gene (TTN (titin) gene, which codes for a major component of cardiac muscle and has been linked to idiopathic dilated and peripartum cardiomyopathy and cardiac remodeling [Our GWAS of fertility phenotypes discovered three genome-wide significant associations . Those s141 gene , which imodeling , 81. Genmodeling , 82. AltPAPOLA gene encoding a poly-A tail polymerase that affects mRNA stability and nuclear export. Intriguingly, the product of this gene is inhibited by cordycepin, an adenosine analog (3’ deoxyadenosine), found in a fungus, “Yartsa gunbu” or Cordyceps sinensis, which is native to the highlands of Nepal and Tibet. Cordycepin is known to interfere with important biochemical and molecular processes, such as purine biosynthesis, DNA/RNA synthesis and mTOR  signaling transduction . TherefOLA gene , or throThis study was designed to extend the genetic study of human local adaptation beyond selective sweeps and candidate gene associations, by collecting genotype and physiological phenotype and reproductive history data for a large group of indigenous high-altitude Tibetan women in Nepal. Using this data set, we successfully identified several new genome-wide associations and signatures of polygenic adaptations. Our sample size of 1,000 participants is remarkably large for the genetic study of populations living in remote locations in a traditional society, but we acknowledge that is rather small for a modern-day GWAS. The census population size of ethnic Tibetans of villages in this region set the ultimate constraint on our sample size, which was obtained by recruiting virtually all inhabitants fitting our inclusion criteria. Despite this constraint, this study shows the necessity to study phenotypes of locally adapted populations in their native environments to correctly identify the adaptive phenotypes. With ever increasing throughput to generate genetic and phenotypic variation data, in-depth phenotyping of potentially adaptive features will help better understand how Tibetans and other populations living in extreme environments have adapted to their habitats.The study protocol was approved by: the University Hospitals Institutional Review Board, University Hospitals of Cleveland (protocol no. 12-15-27), the Nepal Health Research Council, Kathmandu, Nepal (protocol no. 38/2011), the Oxford Tropical Research Ethics Committee, Oxford, UK (protocol no. 23–11), the Dartmouth College Committee for the Protection of Human Subjects (protocol no. 23374) and the Human Research Protection Office, Washington University in St. Louis (protocol no. 201202114). A written informed consent was signed by each participant.2), and pulse rate (pulse/minute) were measured altogether using a non-invasive device Masimo Pronto-7 ©  as described in Cho et al. [2 as follows: oxyHb = Hb × SaO2 / 100 and deoxyHb = Hb–oxyHb. For each participant, an interview session was held to retrieve detailed reproductive history as well as to collect other potential covariates. A summary of the Tibetan samples and their phenotype measurements are presented in A total of 1,008 ethnic Tibetan participants were recruited from high-altitude villages in Mustang and Ghorka districts in Nepal in the spring and summer of 2012. All participants were women of age 39 or older and lifelong residents above 3000 m of altitude. The study communities in Nepal lie on the southern aspects of the Tibetan Plateau. Although they are citizens of Nepal, local people speak Tibetan dialects, practice forms of religion and social organization akin to those across the Tibetan Plateau, and retain the characteristic agro-pastoral and trading mode of subsistence common among highland Tibetans . An addio et al. . Two addEPAS1, EGLN1, HIF1A and NOS2 genes. Then, a subset of 344 unrelated Tibetans from the present study and all 103 Sherpa individuals were genotyped on 716,503 markers using the Illumina OmniExpress-24 v1.0 array to obtain denser genome-wide variation data. For each array platform, genotypes were called using the genotyping module in the Illumina Genome Studio with default parameters . Previously defined clusters, downloadable from the Illumina website, were applied for genotype calling. For the 2,553 custom markers we added to the HumanCore array, we retrieved intensity data from the Illumina Genome Studio and performed genotype calling using the OptiCall v0.6.4 [We generated new genome-wide genotype data for a total of 1,104 individuals indigenous to the high-altitude regions in the Himalayas in Nepal, including 1,001 ethnic Tibetans from the present study and 103 Sherpa . Array gl v0.6.4 . For 344EGLN1 gene, rs12097901 and rs188966510, in the set of 344 unrelated Tibetans. We used Epicenter FailSafe PCR system with the manufacturer’s recommended condition in buffers G and H, instead of using standard TAQ polymerases. We generated a 1,025 bp PCR fragment in an 11 ul reaction volume using a previously published primer pair PHD2-X1F (CCCCTATCTCTCTCCCCG) and PHD2-X1R (CCTGTCCAGCACAAACCC) [We separately genotyped two non-synonymous SNPs in the CAAACCC) . These PWe generated novel whole genome sequence data for 18 Sherpa and 35 Tibetans from the present study, all from Nepal. Seventeen individuals were sampled with known familial relationships (four Tibetans mother-daughter duos and three Sherpa parents-offspring trios), and sequenced to high-coverage  to generate high quality phased genome sequences. The remaining 36 individuals were chosen to be unrelated and sequenced to low-coverage targeting 5x autosomal coverage.For Sherpa, we began with 172 individuals, including 103 newly genotyped in this study and 69 previously published , and choFor Tibetans, we ran ADMIXTURE with K = 3 in a supervised mode, with 103 1KGP CHB, 103 1KGP GIH  and the 51 unrelated Sherpa as three reference groups. Pairwise relatedness was then calculated with the ADMIXTURE output using the RelateAdmix v0.08, controlling for population structure due to admixture . Among ihttp://hgdownload.soe.ucsc.edu/goldenPath/hg19/chromosomes/, using BWA backtrack v0.7.4 with -q15 option [http://broadinstitute.github.io/picard/) and were excluded from further analysis. Local realignment around indels and base quality score recalibration were performed using the GenomeAnalysis ToolKit (GATK) v2.8–1, following the best practice pipeline [Single-barcoded libraries for Illumina sequencing were constructed using the TruSeq library preparation kit. Libraries were pooled into multiple batches and sequenced in the Illumina HiSeq 2500 and 4000 machines for paired-end (PE) 100 and 125 bp designs . Sequenc5 option . PCR andpipeline –93. Finapipeline  by filteLD-aware variant and genotype calling was performed using the GotCloud pipeline  with defp-value ≥ 0.00001 and per-individual missing rate ≤ 0.03. Strand-ambiguous (A/T and G/C) SNPs were removed and only SNPs in autosomes or X chromosome were retained for imputation. The filtering process was performed using PLINK v1.90 [https://mathgen.stats.ox.ac.uk/impute/1000GP_Phase3.html, as imputation references by merging them with “-merge_ref_panels” flag in IMPUTE2. For other parameters, we used default values set by the program. Following imputation, genotypes with posterior probability ≥ 0.9 were accepted. Genotypes were assumed to be missing if none of three possible genotypes reached posterior probability threshold of 0.9. Then, we conducted an additional round of quality control by removing SNPs with missing rate higher than 0.05 or HWE p-value smaller than 10−6.We performed genotype imputation of Tibetan and Sherpa samples, which were array-genotyped either in the present or in our previous study  S1 Tabl. For eacNK v1.90 . GenotypNK v1.90 . We usedAmong 1,001 successfully genotyped and imputed Tibetan women, 991 individuals were included in our genome-wide association analysis (GWAS). Four individuals were excluded from the analysis because they were born below 3,000 m. Another individual was excluded from the analysis was a genetic outlier who clustered with individuals from the Indian subcontinent. The other five were excluded either because they had inconsistent reproductive record or because they were nuns who became celibate during their reproductive years.p-value obtained from the “lrtest” function in the R “lmtest” library was bigger than 0.05. The final sets of chosen covariates for physiological covariates are listed in a priori chosen set of four covariates: age, subdistrict, use of contraception and “continuously married (CM)” status. Use of contraception was categorized into three classes: never used, previously used, and currently in use. “Continuously married” status is a binary variable defined as being in a marital relationship throughout the ages of 25 and 40. It includes two who had experienced less than two years of gap before re-marriage following divorce or death of the husband. For physiological phenotypes, we chose relevant covariates by performing a stepwise model selection, allowing removal of a single covariate each step if likelihood ratio test (LRT) p-values from GEMMA were used to assess significance of genetic association. P-values of the full and subsample sets were highly correlated for each fertility phenotype .GWAS was performed using GEMMA v0.94.1 . Univarip ≤ 10−4 and lumped them into peaks by allowing maximum inter-SNP distance of 200 kb. Finally, we chose one SNP with the smallest association p-value for each peak to retrieve a set of independently associated SNPs. We also retrieved a set of SNPs associated with blood hemoglobin level (Hb) using summary statistics from a published large-scale GWAS meta-analysis [p ≤ 10−5. Then, we removed SNPs in LD: for each pair of SNPs with r2 > 0.2 in 1KGP CEU, we removed one with larger association p-value.To detect signatures of polygenic adaptation, we investigated systematic changes in allele frequencies of SNPs associated with each phenotype. For all of Tibetan GWAS phenotypes, we first took all SNPs with analysis . To obtap-value was calculated as the proportion of random SNP sets with their mean allele frequency difference equal to or more extreme than the observed one.After retrieving phenotype-associated SNPs with their effect size, we first compared mean frequency difference of trait-increasing alleles between Tibetans and 1KGP CHB. Following , we sampWe also looked into comprehensive signatures of polygenic adaptation using a machinery introduced by . It requp, have the mean absolute fitness W0, W0 (1+s) and W0 (1+2s). Using the observed mean phenotype value, Wm, we can get the per-allele effect size sW0 as a function of s, Wm and p:s, the statistical power to detect association was calculated using the “pwr.r.test” function in the R package “pwr”.To estimate the strength of positive selection necessary to generate a significant association between the fertility count phenotypes and genotype in our sample of the unrelateds, we assumed a simple additive model. That is, genotypes with 0, 1 and 2 adaptive alleles, with population frequency S1 FigGWAS quantile-quantile (QQ) and Manhattan plots for fertility phenotypes with genome-wide significant associations:  the number of pregnancies,  the number of live births,  the number of stillbirths, (G-J) the proportion of children born alive but died < 15 yr.  show GWAS results using the continuously married (“CM”) subset of individuals.(TIF)Click here for additional data file.S2 Fig2 , Pulse , oxyHb  and deoxyHb .GWAS quantile-quantile (QQ) and Manhattan plots for five physiological phenotypes in Tibetans: Hb , SaO(TIF)Click here for additional data file.S3 FigEPAS1 gene and (A) Hb and (B) oxyHb, conditional on the genotypes of the top EPAS1 SNP rs372272284. No residual association was observed.Locuszoom plots of the conditional association between markers around the (TIF)Click here for additional data file.S4 Figp ≤ 10−4),  42 oxyHb-associated SNPs after excluding the EPAS1 SNP rs372272284, and  45 deoxyHb-associated SNPs (p ≤ 10−4).  The mean frequency difference of trait-increasing alleles was presented (solid red line) together with the empirical null distribution of 10,000 sets of matched random SNPs.  The genetic values of populations (filled dots) and of regions  were plotted. The size of dots and the width of lines are proportional to the significance of the corresponding outlier test.Tests of polygenic adaptation of oxyHb- and deoxyHb-associated SNPs in Tibetans:  all 43 oxyHb-associated SNPs ((TIF)Click here for additional data file.S5 Figp ≤ 10−4; n = 52) or  in the UK Biobank data .  The mean frequency difference of trait-increasing alleles was presented (solid red line) together with the empirical null distribution of 10,000 sets of matched random SNPs.  The genetic values of populations (filled dots) and of regions  were plotted. The size of dots and the width of lines are proportional to the significance of the corresponding outlier test.Tests of polygenic adaptation of pulse-associated SNPs  in Tibetans ((TIF)Click here for additional data file.S6 Figp < 0.05) for both the mean frequency difference and the outlier tests:  the number of children surviving at 1 yr,  the number of live births,  the number of children born alive but died < 1 yr,  the number of children born alive but died < 15 yr,  the number of miscarriages, and  the number of twin births. (C-L) show GWAS results using the continuously married (“CM”) subset, while  show GWAS results using all individuals.Tests of polygenic adaptation of six fertility phenotypes reaching nominal significance ((TIF)Click here for additional data file.S7 Figlsqproject: YES” option. Numbers in the parenthesis in the axis label show the percentage of total genetic variation explained by each PC.The first two principal components (PCs) calculated from 1,295 individuals belonging to 132 groups  are plot(TIF)Click here for additional data file.S8 Figlsqproject: YES” option. Numbers in the parenthesis in the axis label show the percentage of total genetic variation explained by each PC.The first two principal components (PCs) calculated from 357 individuals belonging to 31 groups  are plot(TIF)Click here for additional data file.S9 Figf3 signals, measuring shared genetic drift from a common outgroup Mbuti, are plotted for (A) Nubri, (B) Tsum, (C) Lower Mustang and (D) Upper Mustang. (E-F) The extra genetic affinity with South Asians  of the Himalayan Tibetans compared to (E) Tibetans from Lhasa and (F) Sherpas are measured by f4 statistics. Thick and thin horizontal bars represent ± 1 and 3 standard errors, respectively, estimated by 5 cM block jackknifing.(A-D) The top 15 outgroup-(TIF)Click here for additional data file.S10 FigThe distribution of (A) altitude of residence and (B) South Asian ancestry proportion across individuals are summarized into a violin plot, using R package “vioplot”. South Asian ancestry proportion is estimated by fitting a two-way admixture model of Sherpa+Pathan using the qpAdm program. In panel (B), grey triangles show South Asian ancestry proportion estimated by group. Group-based estimates match well with the mean value of individual-based estimates.(TIF)Click here for additional data file.S11 Figβ = -1.835×10−5, p = 2.89×10−9).Each color-filled symbols represents a single individual. The red solid line shows a simple linear regression line. The observed negative correlation is highly significant even after controlling for the sub-district label (effect size (TIF)Click here for additional data file.S12 FigThe solid line curves show an exponential fit to the observed weighted admixture LD decay using ALDER. Point estimates ± one standard error estimates are shown in each panel. Standard errors are estimated by leave-one-chromosome-out approach, as implemented in ALDER.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Tableβ’s are calculated per alternative allele. SNPs with a derived allele shared between Tibetans and Denisovan are marked in bold face.POS column is for the genomic position in hg19. REF, ALT and DEN columns show reference, alternative and Denisovan alleles, respectively. TBN and CHB present alternative allele frequencies in our Tibetan data and 1KGP CHB, respectively. Effect size estimates (PDF)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Tablep ≤ 0.01 were marked with grey shades. Positive regression coefficients are marked in red color.Tests with (PDF)Click here for additional data file.S7 Tablep ≤ 0.01.Per-allele selection coefficient for 80% power to detect association in a single SNP test (α = 0.05) was estimated for fertility count phenotypes. No test showed (PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Tablef3 and f4 statistics. For each group, we show in which studies samples belonging to it were published in the “Study” column.This list includes 182 present-day and 58 ancient groups. “N” column shows the total number of individuals in each group. Four sub-district groups of the Himalayan Tibetans reported in this study are marked by yellow color. For each group, we assigned a unique three-letter code (“Symbol”) and used it across figures. “Eurasian” and “East Asian” PCA columns mark populations used for calculating PCs for each set. “F-statistics” column marks populations used for calculating world-wide outgroup-(XLSX)Click here for additional data file.S10 TableP2way ≥ 0.05). Coef1, Coef2 and SE columns represent estimated ancestry proportions from Ref1 (Sherpa) and Ref2 (South Asian), as well as their associated standard errors. Although the South Asian ancestry proportion is small (2.5–6.2%), it is necessary to explain the Himalayan Tibetans, as shown by the insufficiency of the Sherpa-only model (P1way << 0.05).Sherpas from the Khumbu region and a South Asian population are used as sources. All four sub-district groups are adequately approximated by the two-way model ((PDF)Click here for additional data file.S11 TableWe used effect size estimates from multiple studies, ranging from 0.386 to 1.676 g/dL per allele. Power was calculated using the observed allele frequency (maf = 0.336), sample size (n = 649) and standard deviation of Hb residuals after regressing out covariates (1.330 g/dL), assuming a single test (α = 0.05).(PDF)Click here for additional data file.S12 Tableind), the number of unrelated individuals (nunrelated), and Pearson’s correlation coefficient across overlapping SNPs (r). For fertility phenotypes, we present results both for all-sample set and for the continuously married subset.For each phenotype, we present the number of individuals included in the main GWAS (n(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S3 Text(PDF)Click here for additional data file.


Saccharomyces cerevisiae (SC) on nutrient intake, digestibility, nitrogen balance, rumen fluid pH, and serum concentrations of glucose and urea nitrogen in Awassi female lambs in a 2×2 factorial arrangement of treatments.The aim of this study was to evaluate the effect of feeding low (LO)- or high (HI)-fiber diets supplemented with Experimental diets were as follows: (1) LO-fiber diet with no SC supplementation (−LO), (2) LO-fiber diet supplemented with SC (+LO), (3) HI-fiber diet with no SC supplementation (−HI), or (4) HI-fiber diet supplemented with SC (+HI). Eight female lambs were used in a replicated 4×4 Latin square design with 15-day experimental periods (10-day adaptation period and 5-day collection period).A fiber×SC interaction (p≤0.05) was detected for dry matter (DM) and crude protein (CP) intake among diets showing greater DM and CP intake for +LO diet compared to +HI group supplemented with SC, whereas −LO and −HI were intermediate. A fiber×SC interaction (p=0.05) was also detected for the neutral detergent fiber (NDF) intake among diets. Intake of NDF was greater for the –HI diet compared with +LO and –LO diets. Similarly, NDF intake was greater for +HI diet than –LO diet. A tendency (p=0.07) for a fiber×SC interaction was detected for acid detergent fiber (ADF) intake among diets as well. ADF intake tended to be greater for HI-fiber diets. No difference was observed in the rumen fluid pH for lambs fed with the different diets. No fiber×SC interactions were detected for the digestibility of DM, CP, NDF, and ADF among dietary treatments. Digestibility of DM was greater  for LO versus HI fiber. However, NDF and ADF digestibilities were greater  for the HI-fiber than the LO-fiber diets.Results obtained in the current study indicate that SC supplementation has a minimal effect on the performance of Awassi female lambs fed with varying fiber levels. Saccharomyces cerevisiae (SC) has been found to improve ruminal fermentation [Nutritional programs for ruminant animals focus on providing the precise level of nutrients at the proper time, to obtain optimal productivity and profitability . Level aentation , enhanceMany studies showed beneficial effects of yeast on the ruminal environment, the number of ruminal microbes, and microbial activity -7. The nSupplementing yeast with high (HI)- and low (LO)-fiber diets in sheep can test the hypothesis that the level of the fiber in the diet can impact the efficacy of yeast on performance. Therefore, this study aimed to evaluate the influence of SC supplementation on diets containing LO versus HI neutral detergent fiber (NDF) on nutrient intakes and digestibility, nitrogen balance, and rumen fluid pH in Awassi female lambs.The study was conducted at the Agricultural Research and Training Unit at Jordan University of Science and Technology (JUST). All procedures used in the study were approved by the JUST Institutional Animal Care and Use Committee (16/3/3/222).®SC, Lallemand, France; 3×109/g) supplementation on the criteria listed above. Animals were housed in metabolic crates (1.05 m×0.80 m) and offered diets containing either LO or HI level of fiber (HI). Diets were as follows: (1) LO-fiber diet with no SC supplementation (−LO), (2) LO-fiber diet supplemented with SC (+LO), (3) HI-fiber diet with no SC supplementation (−HI), or (4) HI-fiber diet supplemented with SC (+HI). To ensure complete consumption, SC (0.25 g/head/day+10 g ground barley) was mixed with 50 g of the assigned diet throughout the experimental period. Diets were formulated to have similar crude protein (CP) contents . Diets were offered ad libitum (at 8:00 h) with free access to water throughout the study. The study was designed with four experimental periods; each period lasted for 15 days . Offered and refused feeds were recorded from days 10 to 15 of each experimental period to evaluate intake.Eight Awassi female lambs (6 months old) were used in 2×2 factorial arrangements to evaluate the effect of yeast , weighed, and recorded, and 5 g/100 g of each was kept for nitrogen analysis. On day 15 of each experimental period, 5 ml of rumen fluid was collected from the ventral sac using a syringe fitted with a G14 needle at 0, 2, and 4 h after feeding (at 12: 00 h) to evaluate rumen fluid pH.Following the Association of Official Analytical Chemists (AOAC)  procedur#976.06) using AOAC procedures [At the end of the experiment, feed and fecal samples were dried at 55°C in a forced air oven to constant weight and ground to pass a 1 mm sieve . These samples were analyzed for DM (100°C in an air-forced oven for 24 h), and nitrogen  after 1 h of collection. Serum was immediately harvested and stored at −20°C until the day of analysis. Serum concentrations of glucose and urea nitrogen were analyzed using commercial kits  and according to the manufacturer’s specifications. Absorbance was measured using a spectrophotometer .This experiment was designed to test the effect of fiber levels during the presence or absence of yeast  by running two 4×4 Latin squares. Four animals were used in each square and sampled over four experimental periods (15 days for each period). Data analyses were conducted on measurements collected from pooled samples during day 11–15 of the experimental periods. Experimental unit measurements were analyzed using the mixed model procedure of SAS  to account for the effects of square, animal within square, period, and treatment . Treatments were considered fixed effect; square, animal within square, and period were considered as random effects. Data are presented as least square means±standard error of the mean. Statistical significance was considered at p≤0.05.The effect of SC supplementation on nutrient intakes and rumen fluid pH is presented in The digestibility of DM, CP, NDF, and ADF is presented in The effects of SC supplementation in LO- or HI-fiber diets on nitrogen balance are shown in No fiber×SC supplementation interaction was detected for serum concentrations of glucose and urea nitrogen among diets . Similaret al. [The use of yeast as an additive for growing lambs is to optimize the use of dietary fiber, resulting in improved growth. Moreover, yeast may affect ruminal pH by reducing the activity of lactic acid producing bacteria and thus increasing its nutrient availability . In the et al.  reportedet al. [Moreover, an interaction between SC supplementation and daily feed intake of ADF and NDF was previously reported . CP intaet al.  observedet al. .et al. [et al. [Contrary to our finding, Hasunuma et al.  found th [et al.  reportedet al. [Results reported in the present study revealed no fiber×SC interactions with respect to digestibility of DM, CP, NDF, and ADF. Digestibility of nutrients is enhanced by the type of fiber used. Bueno et al.  reportedet al. . Other ret al. . Intake et al. .et al. [et al. [Digestibility of CP was not affected in lambs fed with diets supplemented with SC or by the type of fiber used, while NDF and ADF digestibilities improved in lambs fed with HI-fiber diets. Wiedmeier et al.  found hi [et al.  reported [et al. .et al. [et al. [et al. [A fiber×SC supplementation interaction was detected for the nitrogen intake among diets, showing that nitrogen intake was improved with the different fiber levels. These results contradict those of Bueno et al.  who foun [et al.  reported [et al.  reported [et al.  and/or det al. [et al. [The addition of SC to female lamb diets containing different fiber levels did not affect serum concentrations of glucose and urea nitrogen. Our results are in agreement with the results obtained by Khormizi et al.  who indi [et al. , on the Supplementing SC has a minimal effect on the performance  of Awassi female lambs fed with two levels of fiber. The relationships between SC supplementation and fiber levels are worth further investigation to determine the situations where supplemental SC is expected to improve animal performance.BSO: Designing, supervising, and writing the manuscript, KZM: Helping in statistical analysis and writing the manuscript, MDO: Drafting the manuscript, MA: Drafting the manuscript, RTK: Helping in technical writing, SGH: Drafting the manuscript, HHT: Drafting the manuscript, KIJ: Drafting the manuscript, HJA: Drafting The manuscript, HSS: Drafting the manuscript, SMH: Field work, MAAI: Doing the laboratory analyses. RAA: Field work. All authors read and approved the final manuscript.


Central venous catheters (CVCs) are necessary for critically ill patients, including those with hematological malignancies. However, CVC insertion is associated with inevitable risks for various adverse events. Whether ultrasound guidance decreases the risk of catheter-related infection remains unclear.We observed 395 consecutive CVC insertions between April 2009 and January 2013 in our hematological oncology unit. Because the routine use of ultrasound guidance upon CVC insertion was adopted based on our hospital guidelines implemented after 2012, the research period was divided into before December 2011 (early term) and after January 2012 (late term).Underlying diseases included hematological malignancies and immunological disorders. In total, 235 and 160 cases were included in the early- and late term groups, respectively. The median insertion duration was 26 days  and 18 days  in the early- and late term groups, respectively. The internal jugular, subclavian, and femoral veins were the sites of 22.6, 40.2, and 25.7% of the insertions in the early term group and 32.3, 16.9, and 25.4% of the insertions in the late term group, respectively. The frequency of catheter-related bloodstream infection (CRBSI) was 1.98/1000 catheter days and 2.17/1000 catheter days in the early- and late term groups, respectively. In the subgroup analysis, the detected causative pathogens of CRBSI did not differ between the two term groups; gram-positive cocci, gram-positive bacilli, and gram-negative bacilli were the causative pathogens in 68.9, 11.5, and 14.8% of the cases in the early term group and in 68.2, 11.4, and 18.2% of the cases in the late term group, respectively. In the multivariate analysis to determine the risk of CRBSI, only age was detected as an independent contributing factor; the indwelling catheter duration was detected as a marginal factor. A significant reduction in mechanical complications was associated with the use of ultrasound guidance.Ultrasound-guided CVC insertion did not decrease the incidence of CRBSI. The only identified risk factor for CRBSI was age in our cohort. However, we found that the introduction of ultrasound-guided insertion triggered an overall change in safety management with or without the physicians’ intent. Central venous catheters (CVCs) are a useful treatment modality for patients who require intensive critical care. A CVC allows intravenous administration of drugs and parenteral nutrition support. Although CVCs enable the delivery of medications and nutritional support that cannot be administered safely via a peripheral vein, their use is inevitably associated with adverse events, including mechanical complications, deep venous thrombosis, and catheter-related infections , 2. AccoWe conducted a retrospective cohort study to investigate the incidence of CRBSI following CVC placement with or without ultrasound guidance. We observed 395 consecutive CVC insertions performed between April 2009 and January 2013 (3 years and 10 months). The study population included patients with hematological or immunological diseases who required CVC replacement for nutritional support and/or intravenous drug treatment.The research period was divided into two terms based on substantial differences in the use of ultrasound-guided CVC insertion in the hematological oncology unit: before December 2011 (early term) and after January 2012 (late term). Between the early and the late terms, the insertion maneuvers changed from a blind approach to an ultrasound-guided approach after 2012.The following devices were used in this study: SMAC Plus MicroNeedle , Argyle peripherally inserted central catheter (PICC) kit , Arrow triple lumen , and Blood Access UK catheter kit, double .N = 24) all had > 4 years of experience  in CVC/PICC intervention performance. The residents (N = 32) had < 4 years of experience . We used 10% povidone iodine solution for skin decontamination for the preparation of CVC/PICC insertion. Chlorhexidine gluconate dressing (CHGD) was applied as a form of infection prevention for stem cell transplantation. Levofloxacin and azoles were used as prophylactic antimicrobials during chemotherapy. Other interventions were not applied.All of the practitioners involved in this study had attended a hospital training program providing information on the standard insertion technique. The need for a CVC was determined by each practitioner; the practitioners also determined the preferred CVC device and the insertion site for each patient. Maximal sterile barrier precautions were routinely adopted. A 10% tincture of povidone iodine was used for skin preparation, and Tegaderm Transparent Film Dressing  was used as the routine catheter site dressing unless otherwise specified. Study competency in CVC insertion was guaranteed by the training of the CVC providers. Our institute demands institutional certification to perform CVC/PICC insertion, which is only granted after the regulated orientation and training program and three instances of practical performance under a qualified instructor. All the CVC/PICC providers, who were a combination of staff and residents, were certified according to Kagawa University Hospital institutional regulation. The staff practitioners   was defifunction . The prit-test was used for the parametric analyzes, and the Wilcoxon signed-rank test was used for the non-parametric analyzes. The contributing risk factors for catheter-related infection were analyzed using the multivariate analysis method. For the multivariate analysis, we evaluated patient background  and catheter conditions  as independent variables, and the onset of CRBSI as a dependent variable using the regression model. All potential confounders were included as independent variables. A stepwise selection procedure was used to build the multivariable logistic regression model using the above background risk variables. The entry criterion was set at P < 0.15. Statistical significance was defined as P < 0.05. An interrupted time-series analysis (TSA) was performed to analyze the trends and test the significance over the study period [t-test or Chi-squared test was used to compare values between the two groups. For the comparison tests, P-values < 0.05 were considered significant. Statistical analyzes were performed using SPSS version 19.0 J software .We used basic statistics and described representative values for the patients’ backgrounds. To compare values between the two groups, a two-tailed paired Student’s y period . The t-tThis study was conducted in accordance with the ethical standards of the responsible committee on human experimentation  and with the Declaration of Helsinki  of the World Medical Association.n = 340), immunological disorders such as autoimmune diseases (n = 35), and solid malignancies (n = 20).The patients’ characteristics are presented in Table P < 0.0001; Fig. P = 0.09; Fig. P = 0.1215). The observed effect size d was calculated as 0.530, which was an intermediate effect size.Of the 395 cases recruited in this study, 235 cases were included in the early term and 160 in the late term. The median insertion duration was 26 days  in the early term and 18 days  in the late term (Table P = 0.0091); the indwelling duration was detected as a marginal factor (P = 0.0868)  to 0.000 (0/160) incidences/insertions after the introduction of the ultrasound-guided insertion approach. The cumulative incidence of mechanical comorbidities was 2.01 and 0.00% per insertion, respectively. The observed complications were grade 2 pneumothorax in two cases, grade 3 pneumothorax in eight cases, and grade 2 hematoma with grade 1 bleeding in three cases. In the subgroup analysis, the proportion of detected causative pathogens in the CRBSI cases did not differ between the two terms; gram-positive cocci, gram-positive bacilli, and gram-negative bacilli were the causative pathogens in 68.9, 11.5, and 14.8% of the cases, respectively, in the early term and in 68.2, 11.4, and 18.2% of the cases, respectively, in the late term. In the multivariate analysis to determine the risk of CRBSI, only age was detected as an independent contributing factor , but this difference was not significant (P = 0.1294). The overall incidence of bacteria including other organisms was comparable in the two groups at approximately 30% each . In cases without CHGD use, the documented organisms included Staphylococcus epidermidis (n = 6), methicillin-resistantS. aureus (n = 1), S. hominis (n = 2), S. simulans (n = 1), Staphylococcus species (n = 1), Streptococcus species (n = 1), Capnocytophaga species (n = 1), Stenotrophomonas maltophilia (n = 1), Escherichia coli (n = 1), and gram-negative bacilli (n = 1) (unidentified species). In cases with CHGD use, S. epidermidis (n = 1), Stenotrophomonas maltophilia (n = 1), and Enterococcus faecium (n = 1) were documented.The controlled-cohort study revealed that CHGD use decreased the incidence of CRBSI caused by Ultrasound-guided CVC insertion did not significantly decrease the incidence of CRBSI in this study. A previous study reported the incidence of CRBSI to be 24.5% in hematological malignancies, including in recipients undergoing stem-cell transplantation , which iThe ultrasound-guided CVC cannulation is a safer procedure than the landmark technique . PreviouOur survey demonstrated an intervention that triggered an overall shift in the management of CVC insertion to minimize the risks contributing to mechanical complications (pneumothorax and hematoma or other organ injuries) , which iWe found no definitive evidence of reduction in the incidence of CRBSI following ultrasound-guided CVC insertion. However, we found that the introduction of ultrasound-guided insertion triggered an overall change in safety management with or without the physicians’ intent.


Nine (2.2%) pediatricians scored above the cut-off value on the Trauma Screening Questionnaire, indicative of PTSD. In total, 7.3% (n = 30) and 14.1% (n = 58) scored above the cut-off values in the Hospital Anxiety and Depression Scale, indicative of depression and anxiety. Only 26.3% reported to have a peer support protocol available for emotional support following adverse events.The objective of this study was to study mental health, coping, and support after work-related adverse events among pediatricians. Physicians are frequently exposed to adverse events. It makes them at risk for posttraumatic stress disorder (PTSD), depression, and anxiety disorders. Besides the personal impact, physicians could pose a threat towards patients, as mental health problems are associated with medical errors. A questionnaire was sent to all members of the Pediatric Association of The Netherlands in October 2016. The questionnaire focused on adverse events, coping, and support. The Hospital Anxiety and Depression Scale and the Trauma Screening Questionnaire were included for evaluation of anxiety, depression, and posttraumatic stress. Four hundred ten questionnaires (18.9%) were eligible for analysis. Seventy-nine %  support should be implemented. Physicians are frequently exposed to adverse events, such as medical errors, patient safety incidents, violence, and complaints. Adverse events may lead to mental health problems including posttraumatic stress disorder (PTSD) , 2. AparThe 1-year prevalence of mood disorders  among Dutch citizens with a high income is 3.0%, and the 1-year prevalence of anxiety disorders is 6.0% . In physA cross-sectional questionnaire was conducted with members of the Pediatric Association of The Netherlands (NVK).Among the members of the NVK are residents, attending, non-practicing, and retired pediatricians. At the time of the questionnaire, there were 2160 members in total. All members received an invitation from the NVK for an online questionnaire and three reminders over the course of a 3-month period from October 2016. The questionnaire was sent through SurveyMonkey® using an anonymous (non-traceable) link.The questionnaire consisted of 56 questions and contained two validated screening instruments, the Trauma Screening Questionnaire (TSQ) and the Hospital Anxiety and Depression Scale  –16. The The TSQ is a ten-item screening instrument based on items from the PTSD Symptom Scale-Self Report and has five items concerning re-experiencing and five items concerning arousal. Answers can be “yes” or “no” . The cutThe HADS is a 14-item screening instrument for depression and anxiety, where both subscales contain seven questions each. Each question contains an answer consisting of a four-point Likert scale. The cut-off value of the Dutch version for clinically relevant depressive symptoms (HADS-D) or anxiety symptoms (HADS-A) is equal to or bigger than eight. The total HADS cut-off value for psychological distress is equal to or bigger than 12.Further questions were added to the questionnaire to explore demographics , assess adverse events at work, way of coping with adverse events and how pediatricians learned to cope, satisfaction with current support, and if and how the current support system should change (Appendix).P values were calculated with binomial tests. Differences in categorical outcomes between residents and attending physicians were tested with either chi-square tests (χ2) or Fisher’s exact. In continuous data, independent t or Mann-Whitney U tests were used. A two-sided P value of 0.05 or smaller was considered statistically significant.Statistical analysis was performed with IBM Statistical Package for the Social Sciences . Only completed questionnaires were analyzed. Open questions were categorized by two independent contributors (GY and EJ), the overall inter-rater agreement was calculated with Cohen’s kappa. Demographic data and multiple-choice questions were analyzed using descriptive statistics, and This study was exempted from ethical approval by the Medical Research Ethics Committees United (MEC-U) and registered under the number W18.096.A total of 456 questionnaires 21.1%) were completed, of which 410 questionnaires (18.9%) were eligible. Figure .1% were Table Selected quotes of the respondents are added in Table n = 292), suspicion of child abuse , doubts about making the right decision , death of a patient , and critically ill children  of the respondents indicated that they actually perceived an event as an adverse event, of which 277 described this event. Aggressive behavior of parents towards the physician was most commonly named as an adverse event .The following events were experienced as high emotional impact stressors at work by the respondents (multiple answers possible): missing a diagnosis  reported having experienced this event during their work more than 4 weeks ago.Table P = 0.02).The mean score of the TSQ was significantly lower in the group of participants where a peer support protocol was present for adverse events (0.77 ± 1.06) compared to the group where no protocol was present or where it was not used .Outcomes of the HADS are shown in Table n = 353), seeking support from friends and family , seeking some other form of distraction , or doing sports . Respondents learned their coping strategies (multiple answers possible) during residency , as an attending , during clerkships , and 21.0% (n = 86) reported to having never learnt to cope with adverse events.The most common coping strategies after adverse events were (multiple answers possible) seeking support from colleagues  has seriously considered quitting their job at some point in their career. Most common reasons for this were (multiple answers possible) disbalance between work and private life , high workload , disutility , too much stress , and too much responsibility  considered quitting because of a complaint to the disciplinary board.Of the respondents, 41.0% (n = 26) of the respondents admitted that they were dealing with adverse events by drinking more alcohol and 1.2% (n = 5) by taking new medication. Professional psychological help was sought by 9.8% (n = 44) and 16.1% (n = 66) stifled emotions.Six percent (n = 76) of the respondents adjusted their work. Most common ways to do this were (multiple answers possible) performing more diagnostic tests , calling a colleague earlier , work less , and starting treatment faster . Over time, 40.5% (n = 166) of the respondents reported to have become more defensive.After being exposed to a work-related adverse event, 18.5% (n = 108) indicated that there was a protocol for support in the case of an adverse event in their current working environment, 34.2% indicated that there was no protocol, and the remainder did not know whether there was a protocol. Furthermore, 50.2% (n = 206) thought that a culture change is necessary concerning coping with adverse events. When asked what the standardized support system involved (multiple answers possible), respondents indicated that discussing the situation with the present team  and self-initiated peer support with direct colleagues  were used. Of the respondents, 16.3% (n = 67) indicated that there was no support system at all and 41.5% (n = 170) confided that there is not enough opportunity to discuss adverse events and express emotions.Of the respondents, 26.3% (n = 313), peer support from direct colleagues , and professionally organized peer support . Even though 28.5% (n = 117) would prefer to get help from a psychologist or coach, only 9.8% (n = 40) actually sought out this kind of help.According to the respondents, the preferred form of support would be (multiple answers possible) discussing the situation with the present team ) focus on how to cope with aggression , 20. AnoThe point prevalence of PTSD in The Netherlands is 1.3%, making that the point prevalence of work-related PTSD is expected to be even lower . In our After experiencing an adverse event, a little over a quarter of all respondents indicated that there was a protocol regarding adverse events, but over half of the respondents do think a culture change is needed. Therefore, pediatricians have the need for a better support system. When more than half of the pediatricians perceive that care surrounding adverse events is not sufficient, this could lead to unnecessary stress. Physicians might experience more barriers than non-physicians to seek out professional help for mental health problems due to their fear of losing their license, denial of problems, and embarrassment . When phThus far, little research has been done on this topic, specifically concerning mental health problems in relation to institutional or peer support. This study, which included validated questionnaires, allowed for detailed data collection. Allowing respondents to fill out examples of their experiences gives further insight in the way they experience their problems.Limitations of the study are the response rate, with 18.9% lower than the average e-mail response rate of 25–30% . ReasonsIn conclusion, work-related stressors in Dutch pediatricians are high and can subsequently lead to posttraumatic stress disorder. Parental aggression towards pediatricians seems to be a common problem, something that should be addressed, for example with CMD training programs . In this


In the absence of head-to-head trials comparing axitinib with cabozantinib or everolimus, the aim of this study was to conduct an indirect comparison of their relative efficacy in patients with metastatic renal cell carcinoma (mRCC), using data from the AXIS and METEOR trials.Progression-free survival (PFS) and overall survival (OS) in prior sunitinib-treated patients with mRCC were compared by conducting matching-adjusted indirect comparison (MAIC) analyses, including base-case and sensitivity analyses. Individual patient-level data from prior sunitinib-treated patients who received axitinib in AXIS were weighted to match published baseline characteristics of prior sunitinib-treated patients who received either cabozantinib or everolimus in METEOR.There was no statistically significant difference in PFS (aHR [adjusted hazard ratio] = 1.15 [CI: 0.82–1.63]) and OS (aHR = 1.00 [CI: 0.69–1.46]) between axitinib versus cabozantinib in the base-case analysis. In the sensitivity analysis, PFS (aHR = 1.39 [CI: 1.00–1.92]) and OS (aHR = 1.35 [CI: 0.95–1.92]) were shorter for axitinib compared with cabozantinib; however, the OS difference was not statistically significant. Axitinib was associated with significantly longer PFS compared with everolimus in the base-case (aHR = 0.53 [CI: 0.36–0.80]) and sensitivity analyses (aHR = 0.63 [CI: 0.45–0.88]), respectively. Results suggested an OS benefit for axitinib versus everolimus in base-case analyses (aHR = 0.63 [CI: 0.42–0.96]); however, the difference in OS in the sensitivity analysis was not statistically significant (aHR = 0.84 [CI: 0.59–1.18]).MAIC analyses suggest PFS and OS for axitinib and cabozantinib are dependent on the Memorial Sloan Kettering Cancer Center definition used; in the base-case analysis, there was no significant difference in PFS and OS between axitinib and cabozantinib. In the sensitivity analysis, PFS in favour of cabozantinib was significant; however, the trend for prolonged OS with cabozantinib was not significant. For axitinib and everolimus, MAIC analyses indicate patients treated with axitinib may have an improved PFS and OS benefit when compared to everolimus. Disparities between the base-case and sensitivity analyses in this study underscore the importance of adjusting for the differences in baseline characteristics and that naïve indirect comparisons are not appropriate. Each year, approximately 214,000 patients are diagnosed with renal cell carcinoma (RCC) worldwide . The treFor patients with mRCC who progress on prior targeted therapy, mostly with sunitinib, treatment options available as second-line and/or subsequent systemic therapies include axitinib, everolimus, lenvatinib in combination with everolimus, cabozantinib, and nivolumab. With an increasing number of targeted agents being approved for the treatment of mRCC, data from comparative studies of these agents would help attending physicians and patients to make decisions based on individualized treatment algorithms. Due to the limited number of head-to-head clinical trials that directly evaluate these targeted agents as second-line and/or subsequent therapy for mRCC, an indirect treatment comparison analysis is needed. Axitinib was a well-established targeted agent in the second-line setting before other targeted therapies were introduced. Axitinib has been compared with sorafenib in the Axitinib as Second Line Therapy for Metastatic Renal Cell Carcinoma (AXIS) trial , 5. EverThe MAIC technique has been acknowledged in health technology assessments in oncology , 11 and The aim of this study was to compare progression-free survival (PFS) and overall survival (OS) in prior sunitinib-treated patients who received axitinib in the AXIS trial  with priThe AXIS trial was a randomised, phase 3 trial of axitinib compared with sorafenib, a multi-targeted tyrosine kinase inhibitor (TKI), in previously treated patients with mRCC . The triIn the AXIS trial, eligible patients had mRCC with a clear cell component, measurable disease by Response Evaluation Criteria in Solid Tumours (RECIST) v1.0, Eastern Cooperative Oncology Group performance status (ECOG PS) 0 or 1, and progressed as assessed by investigators following one prior systemic first-line therapy with sunitinib, bevacizumab plus interferon-alpha, temsirolimus, or cytokines . PatientEligible patients in the METEOR trial had mRCC with a clear cell component, measurable disease by RECIST v1.1, and Karnofsky performance status (KPS) score ≥ 70%. Patients must have received previous therapy with at least one vascular endothelial growth factor receptor (VEGFR)-targeting TKI . They must also have had radiographic progression during therapy or within 6 months after the last dose of VEGFR inhibitors , 19. PatThe primary outcome in both trials was PFS; it was defined as the time from randomisation to either first documentation of RECIST-defined disease progression or death due to any cause, whichever came first. PFS was evaluated by independent review committee using the RECIST v1.0 (AXIS) or v1.1 (METEOR) criteria. A secondary outcome in both trials was OS, defined as the time from randomisation to death. Published METEOR data on PFS and OS were extracted using Engauge Digitizer, and patient-level data for each curve were generated using the methods described by Guyot et al. . The accA compatibility assessment was performed to determine the feasibility of conducting MAIC analyses with the available data through a comparative review of the trial design, population profiles, and outcome measures of the AXIS and METEOR trials.Following the compatibility assessment, the MAIC was conducted as outlined by Signorovitch et al. . Weightswi)2/(∑wi2), where wi represents weights for the ith patient. A low ESS may indicate an irregular distribution of weights across patients, with a large fraction of patients with very small weights.A propensity-score logistic regression equation was used to reweight the data from the prior sunitinib-treated patients who were treated with axitinib in the AXIS trial, so that their aggregate characteristics matched exactly with those in the METEOR trial for all baseline characteristics that were available in both studies. Race was not included in the matching process as it was strongly correlated with geographic region. Time since diagnosis, number of metastases, and duration of the first TKI treatment were not included, as they were not reported for the subgroup of prior sunitinib-treated patients in the METEOR trial. Effective sample size (ESS) was derived as  with 95% confidence interval (CI) in the MAIC. The aHRs were obtained using a Cox proportional hazard regression analysis based on the weighted patient-level data in the AXIS trial and derived patient-level data for PFS and OS in the METEOR trial; the 95% CI for the aHR estimate took the ESS into account. Median PFS, median OS, and 95% CIs were estimated based on the weighted Kaplan–Meier analysis.The AXIS and METEOR trials were generally similar in design. Both were phase 3 trials with comparable inclusion/exclusion criteria and geographic coverage (most patients enrolled from the United States and Europe). In addition, both trials required disease progression on prior treatment before enrolling, and had similar definitions of PFS and OS.However, there were important differences in study design between the two trials. First, MSKCC score in the AXIS trial was calculated using ECOG PS, whereas KPS was used for this calculation in the METEOR trial. Secondly, the AXIS trial enrolled patients who progressed after one prior first-line systemic therapy, including sunitinib, bevacizumab plus interferon-alpha, temsirolimus, or cytokines; the METEOR trial enrolled patients who received prior therapy with at least one VEGFR-targeted TKI and experienced progression, resulting in 30% of the patients having ≥2 prior VEGFR therapies. For the current analysis, only prior sunitinib-treated patient subgroups from both trials were used. Finally, PFS assessment schedules differed between the trials .Prior sunitinib-treated patients who were subsequently treated with axitinib in the AXIS trial, and those treated with everolimus or cabozantinib in the METEOR trial, were generally similar in terms of age, sex, geographic region, previous nephrectomy, and histology Table . Prior tMore patients in the AXIS trial received post-study subsequent therapies than those in the METEOR trial, and the composition of post-study therapies were different between the trials Table . A smallThe naïve indirect comparative analysis before adjustments showed a shorter PFS for axitinib than cabozantinib: median PFS was 4.8 months (95% CI 4.5–6.5) for axitinib versus 9.1 months (95% CI 6.4–9.4) for cabozantinib Table . For axiAfter matching, baseline characteristics were balanced between prior sunitinib-treated patients who received axitinib in the AXIS trial and those who received either cabozantinib or everolimus in the METEOR trial Table . The ESSp = 0.423); and estimated median PFS was 6.5 months (95% CI 4.7–10.4) for axitinib versus 9.1 months (95% CI 6.4–9.4) for cabozantinib (Table p = 0.002); and estimated median PFS was 6.5 months (95% CI 4.7–11.0) for axitinib versus 3.7 months (95% CI 3.5–4.4) for everolimus ; and estimated median (95% CI) PFS was 4.8 months (95% CI 4.2–6.7) for axitinib versus 9.1 months (95% CI 6.4–9.4) for cabozantinib (Table p = 0.007); and estimated median PFS was 6.5 months (95% CI 4.6–7.8) for axitinib versus 3.7 months (95% CI 3.5–4.4) for everolimus  for axitinib versus 21.5 months (95% CI 17.1–not estimable) for cabozantinib Table . OS betwp = 0.983); and estimated median OS was 21.5 months (95% CI 15.7–27.3) for axitinib versus 21.5 months (95% CI 17.1–not estimable) for cabozantinib (Table p = 0.032); and estimated median OS was 21.7 months (95% CI 13.5–28.3) for axitinib versus 16.5 months (95% CI 13.3–19.1) for everolimus ; and estimated median OS was 15.7 months (95% CI 12.8–21.5) for axitinib versus 21.5 months (95% CI 17.1–not estimable) for cabozantinib (Table p = 0.309); and estimated median OS was 15.5 months (95% CI 12.8–23.1) for axitinib versus 16.5 months (95% CI 13.3–19.1) for everolimus  and sensitivity (p = 0.007) analyses. The benefit of axitinib compared with everolimus was also seen for OS in the base-case analysis (p = 0.032), but not in the sensitivity analysis (p = 0.309). When comparing axitinib with cabozantinib, the base-case analysis suggested no difference in PFS (p = 0.423) or OS (p = 0.983) between the two treatments. The sensitivity analysis suggested a marginal benefit for cabozantinib versus axitinib for PFS (p = 0.050); however, the differences in OS did not reach statistical significance (p = 0.096). Different conclusions in the base-case and sensitivity analyses for comparison of axitinib with cabozantinib arise from differences in how the MSKCC score was calculated in AXIS in these two analyses. The disparities between the base-case and sensitivity analyses in this study underscore the importance of adjusting for the differences in baseline characteristics, and that the naïve indirect comparisons are potentially problematic and should be avoided.Using the MAIC analyses , prior sThe OS results in the current analyses should nevertheless be interpreted with caution due to the difference in maturity of the OS data across the trials. In addition, OS is impacted by study treatments and by post-study treatments. Indeed, a higher percentage of axitinib-treated patients in the AXIS trial received post-study therapies compared with everolimus- or cabozantinib-treated patients in the METEOR trial ; this may be explained, in part, by the difference in the maturity of the trials. The most common post-study systemic treatments in axitinib-treated patients were everolimus (43%) and sorafenib (20%). In the METEOR trial, the most common post-study treatments were axitinib (39%), followed by either pazopanib (7%) or sorafenib (7%) in everolimus-treated patients; and everolimus (25%) and axitinib (18%) in cabozantinib-treated patients. The OS results would have been confounded by the observed imbalance in the composition of subsequent systemic therapies between the trials, which could not be corrected for, and, more importantly, the fact that 39% of everolimus-treated patients and 18% of cabozantinib-treated patients in the METEOR trial received post-study axitinib, and 43% of axitinib-treated patients in the AXIS trial received post-study everolimus (none received cabozantinib).n = 43) and included patients who were sunitinib-intolerant. Similarly, in a retrospective chart review of the axitinib versus everolimus cohort study by Vogelzang et al. [The study results are not in agreement with the findings of three studies that compared survival between axitinib, everolimus, and cabozantinib in the second-line setting using different analytical methods –24. In ag et al. , no diffThere are inherent limitations to our study. Although the clinical trial design, inclusion criteria, and outcomes definitions were comparable between the AXIS and METEOR trials, some differences were noted that could have potentially impacted the comparison. Firstly, differences in timing of PFS assessments by an independent review committee may have led to overestimation of PFS for everolimus and cabozantinib  compared with axitinib . Secondly, the difference between the MSKCC scores derived in the AXIS versus METEOR trial could not be fully adjusted for in our analyses. Thirdly, there was a potential for residual confounding due to omitting some patient characteristics from analyses , since these were not available for the prior sunitinib-treated patient subgroups in the METEOR trial. Although the current study adjusted for the main prognostic factors –27, the Despite these limitations, the MAIC analysis offers advantages over a naïve comparison since it attempts to adjust for differences in baseline patient characteristics, and thus allows for a more comprehensive comparison of the treatment effects.Although assumptions were required, an indirect comparison using MAIC based on the AXIS and METEOR trials suggested no differences in PFS and OS in the base-case analysis between axitinib and cabozantinib in prior sunitinib-treated patients. Sensitivity analyses suggested a significant PFS gain with cabozantinib compared with axitinib; however, no significant difference in OS was observed. For axitinib versus everolimus, a beneficial treatment effect was observed for PFS, and potentially for OS, in patients with mRCC previously treated with sunitinib. Disparities in the base-case and sensitivity analyses between axitinib and cabozantinib highlight the importance of adjusting for differences in trial populations for indirect treatment comparisons. Additionally, these analyses demonstrate that MAIC can improve the reliability of indirect comparisons compared with using aggregate data alone; however, a randomised, head-to-head, controlled trial is needed if definitive conclusions are to be made.


In the present paper, we aimed to test the value of the red cell distribution width (RDW) coefficient of variation as a candidate biomarker for pulmonary arterial hypertension (PAH) in patients with connective tissue disorders (CTD), correlating it with the degree of cardiopulmonary impairment in these patients. N = 141 patients with CTD and N = 59 patients affected by pulmonary hypertension of other etiologies, all referred to the Pulmonary Hypertension Clinic of the Cardiology Division of an Academic Hospital in Northern Italy for evaluation (including right catheterization). Clinical, instrumental, and laboratory data were collected and related to RDW and other full blood count indexes.  The study population included p = 0.02) and a lower platelet count (205 (177‐240) × 109/l vs. 244 (197.5‐304.2) × 109/l; p = 0.005). Moreover, with respect to CTD patients without PAH, RDW was significantly larger also in PH of other etiologies. In contrast, the platelet count was significantly lower only in CTD-related PAH, with a value > 276 × 109/l being 100% sensitive in ruling out PAH. Finally, RDW, but not the platelet count, was related directly to systolic pulmonary arterial pressure  and right ventricle diameter  and inversely to diffusing capacity of the lung for carbon monoxide .  Twenty out of 141 CTD patients (14%) received a diagnosis of PAH. In comparison to those without PAH, CTD patients with PAH displayed a larger RDW (14.9% (13.5-17.2) vs. 13.8% (13.1-15.0);  RDW is a promising candidate biomarker for the screening and the prognostic stratification of PAH in CTD patients. Pulmonary hypertension (PH) is a clinical entity that comprises multiple conditions of different etiologies and pathophysiology, defined by the presence of a mean pulmonary arterial pressure (mPAP) equal to or greater than 25 mmHg, measured during invasive right heart catheterization (RHC) at rest . AlthougConsensus exists that these high-risk patients should be screened for PAH, since CTD-related PAH has a severe prognosis, even poorer than the idiopathic form ; in factThe red cell distribution width (RDW) coefficient of variation, a measure of the variability in size of circulating erythrocytes, is routinely reported as a component of the complete blood count and has been used in the differential diagnosis of anemia . RecentlIn the present paper, we aimed to evaluate RDW as a candidate biomarker for PAH in CTD patients and to correlate it with the severity of cardiopulmonary impairment.st, 2016, to April 20th, 2018, we recruited 141 consecutive patients, affected by CTD and referred to the Pulmonary Hypertension Outpatient Clinic of the Cardiology Department, A.O.U. “Maggiore della Carità,” Novara, Italy.We performed a cross-sectional observational study. From October 1Age < 18 yearsRefusal to participate in the studyImpossibility to undergo the required cardiopulmonary assessmentWe applied the following exclusion criteria:We also recruited 59 patients affected by PH of other etiologies, applying the same exclusion criteria, to check whether the predictive factors identified in CTD-related PAH were valid in other clinical settings.Following echocardiography estimation of systolic pulmonary artery pressure (sPAP), right heart catheterization (RHC) was performed to confirm PH, when appropriate. PAH was defined by mean pulmonary artery pressure (mPAP) ≥ 25 mmHg, pulmonary capillary wedge pressure ≤ 15 mmHg, and pulmonary vascular resistance > 3 Wood Units. Whenever contraindications to RHC occurred, PH was diagnosed based on echocardiography-estimated sPAP ≥ 35 mmHg and additional high-probability criteria, according to 2015 ESC/ESR guidelines. In the control group, PH etiology was carefully searched for, and patients were classified according to the Nice 2013 criteria [All patients underwent a comprehensive medical history and a thorough physical examination, aimed at assessing the presence and severity of signs and symptoms compatible with PH. Cardiovascular risk factors and other comorbidities were investigated. Past medical history and drug history were recorded. Rheumatologic disease history was recalled through an extensive review of the medical records.12-lead electrocardiogram with 6 limb and 6 precordial leads with paper speed set at the standard rate of 25 mm/ssix-minute walking distance (6MWD), which measures the distance that the patient could quickly walk over a total of six minutes on a hard, flat surfaceposteroanterior and lateral chest X-rays and, when necessary, high-resolution computed tomography (HRCT) pulmonary function tests (PFTs), performed on the same day the patient attended the PH clinic. The spirometry was performed using standardized equipment and technique with a spirometer, which measured the amount of air the subject exhaled and the rate at which it was exhaled. The device was connected to a computer employing “Medisoft ExpAir 1.28.20” to convert the signals into numerical values and graphics. The following standardized measurements were evaluated: forced vital capacity (FVC), forced expiratory volume in the 1st second (FEV1), FEV1/FVC (%) 4). Then, the patient was asked to hold his/her breath for about 10 seconds and exhale afterwards. During the expiration, alveolar air was analyzed: the ratio between carbon monoxide in inspired gas and carbon monoxide in exhaled air determined the diffusion of carbon monoxide. Predicted DLCO corrected for haemoglobin and alveolar volume was assessedthe diffusing capacity of the lung for carbon monoxide (DLCO), measured with the single-breath Jones-Meade protocol. Once the mouthpiece and the nose clip were in place, the subject made a maximal expiration and then, with a maximal inspiration, inhaled a gas blend containing carbon monoxide (0.3%) and other inert gases (0.3% CH2 + RAP). RAP was estimated with the respiratory motion and the size of the inferior vena cava (IVC) from the subcostal view. TR jet was graded according to recommendations for the quantification of native valvular regurgitationtransthoracic echocardiography, performed using the Vivid 7 or E9 cardiovascular ultrasound machine by GE Medical Systems  with a 1.7/3.4 MHz tissue harmonic transducer. All data were obtained in standardized patient positions, according to the guidelines of the American Echocardiography Society. The exam was performed by an echocardiography expert in PH. The following parameters were studied : sPAP, rright ventricle systolic function was evaluated by estimating the Tricuspid Annular Plane Systolic Excursion (TAPSE)Complete blood count (CBC), serum creatinine, C-reactive protein (CRP), and brain natriuretic peptide (BNP) were measured. The serum autoantibody profile was examined. Further assessment of these patients consisted ofThe study protocol was approved by the institutional ethical committee and conducted in strict accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all individual participants included in the study.χ2 test were used, as appropriate, to explore the associations of categorical variables. To test the diagnostic performance of RDW and platelet count, receiver operating characteristic curves were built, with calculation of the respective areas under the curve (AUC). The level of significance chosen for all statistical tests was 0.05 (two-tailed).Anthropometric, clinical, and biochemical data were recorded in a database and analyzed by the statistical software package MedCalc v.18.10.2 . The measures of centrality and dispersion of data chosen were medians and interquartile range (IQR). Continuous variables were compared between groups by the Mann–Whitney and Kruskal-Wallis (K-W) tests. Exact Fischer's test and Pearson's N = 102, 72.3%), SLE , SS , PM/DM , MCTD , and undifferentiated CTD . Out of 141, N = 20 received a diagnosis of PAH (14%): in N = 10 patients, the diagnosis was based on RHC data.Among the 141 patients with CTD, the following diagnoses were recorded: SSc (p = 0.015). A platelet count ≤ 276 × 109/l was 100.0% sensitive and 36.3% specific for the diagnosis of PAH, with an AUC 0.697 . 5/20 patients were affected by chronic respiratory failure requiring continuous oxygen supplementation. This subset of patients had a trend, though not statistically significant, towards a larger RDW (17.7% (15.4-18.2) vs. 14.2% (13.4-16.0)) when compared to those with PAH and no chronic respiratory failure. Platelet count was not different between groups (data not shown).In p < 0.001). On the contrary, the platelet count was significantly lower only in CTD-related PAH (205 (177‐240) × 109/l; K-W 7.30; p = 0.02), being similar between PH of other etiologies (222 (184‐266) × 109/l) and CTD not complicated by PAH (242 (194‐302) × 109/l)  1, and IL-6 [The rationale of the present study stems from previous reports, suggesting that red blood cells and platelets are possibly involved in the pathogenesis of several inflammatory diseases and may serve as diagnostic and prognostic biomarkers in this context . Growingand IL-6 .We also reported a significantly lower platelet count in CTD patients with PAH; this observation might be explained by an intravascular thrombosis due to endothelial damage, leading to the local consumption of platelets . HoweverIt might be argued that the increased RDW and reduced platelet count belong to the development of hypoxia, leading to increased erythropoiesis at the expense of megakaryopoiesis. We have no data about blood gas analysis of our patients; however, those on oxygen supplementation because of chronic respiratory failure were not characterized by a different platelet count. However, a trend towards a larger RDW was observed. Hypoxia is, therefore, an intriguing potential explanation of these associations, deserving consideration in further studies.A main limitation of the present study is related to the small sample size of our cohort which, being representative of a relatively rare complication of a rare disease, allowed us to enrol only 20 patients with CTD-related PAH. This significantly limits our possibility to draw definitive conclusions, particularly with regard to the identification of diagnostic thresholds. Therefore, the ROC curves presented have the value of an exploratory analysis, requiring confirmation in larger cohorts.Finally, for the present study, the diagnosis of PAH was based on the 2015 ESC/ESR guidelines which defined pulmonary arterial hypertension in the presence of a mPAP ≥ 25 mmHg; this cut-off has been recently revised during the 2018 PH World Symposium. The new proposed definition of PAH is based on the detection of a mPAP ≥ 20 mmHg with a pulmonary vascular resistance ≥ 3 WU at RHC . Our finIn conclusion, RDW is a promising tool for the screening and the prognostic stratification of PAH in CTD patients; its role in complementing and refining the approach by which we currently screen for PAH on these high-risk patients is worth to be established in future studies.


PRNP). Although Korean native goats have been consumed as meat and health food, the evaluation of the susceptibility to scrapie in these goats has not been performed thus far. Therefore, we investigated the genotype and allele frequencies of PRNP polymorphisms in 211 Korean native goats and compared them with those in scrapie-affected animals from previous studies. We found a total of 12 single nucleotide polymorphisms (SNPs) including 10 nonsynonymous and 2 synonymous SNPs in Korean native goats. Significant differences in allele frequencies of PRNP codons 143 and 146 were found between scrapie-affected goats and Korean native goats (p < 0.01). By contrast, in PRNP codons 168, 211 and 222, there were no significant differences in the genotype and allele frequencies between scrapie-affected animals and Korean native goats. To evaluate structural changes caused by nonsynonymous SNPs, PolyPhen-2, PROVEAN and AMYCO analyses were performed. PolyPhen-2 predicted “possibly damaging” for W102G and R154H, “probably damaging” for G127S. AMYCO predicted relatively low for amyloid propensity of prion protein in Korean native black goats. This is the first study to evaluate the scrapie sensitivity and the first in silico evaluation of nonsynonymous SNPs in Korean native black goats.Small ruminants, including sheep and goats are natural hosts of scrapie, and the progression of scrapie pathogenesis is strongly influenced by polymorphisms in the prion protein gene ( TSEs are characterized by neurodegenerative symptoms in brain tissue and are attributed to the conformational change from the normal prion protein (PrPC) to the deleterious isoform of prion protein (PrPSc), which entails distributional changes in secondary structure of PrPC 8.Small ruminants, including sheep and goat, are natural hosts of scrapie belonging to the group of transmissible spongiform encephalopathies (TSEs), which also includes bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and kuru, fatal familial insomnia (FFI), Gerstmann-Sträussler-Scheinker syndrome (GSS) and Creutzfeldt-Jakob disease (CJD) in humansPRNP) polymorphisms have a critical effect on prion disease susceptibility among a wide range of hosts10. In our previous studies, PRNP polymorphisms in Korean people, chickens, horses and cattle have been reported, and evaluation for susceptibility to prion diseases was performed19. In addition, several polymorphisms in paralogs of the PRNP gene have been reported in cattle and goats23. In humans, distributions of the single nucleotide polymorphisms (SNPs) at codons 129 and 219 of the PRNP gene are correlated with the susceptibility to sporadic and variant CJD11. For example, M129V and E219K heterozygotes are protective against the development of sporadic CJD. In addition, all variant CJD patients were 129MM homozygous24. In cattle, insertion/deletion polymorphisms in the regulatory region of the PRNP gene may influence the expression level of PrPC, leading to different incubation periods and an increase in BSE susceptibility26. According to recent studies, 23-bp insertion/deletion polymorphisms in the promoter region and 12-bp insertion/deletion polymorphisms in the promoter region of intron 1 of the PRNP gene are associated with BSE susceptibility27.Prion protein gene  2015 survey, Korean native black goats are known as the only breed raised in Korea. In addition, 300,000 heads of Korean native black goats were raised in 9,400 farm houses and were consumed as meat and health food. To date, scrapie in goat has not been reported in Korea. In addition, the estimation of the susceptibility to scrapie has not been investigated in Korean native black goats thus far.Korean native black goats are the only Korean indigenous breed that has been farmed for over 2,000 years. According to the Statistics Korea  and analyzed haplotypes of the PRNP polymorphisms. Furthermore, we also evaluated the biological impact, such as the protein structure and functions of nonsynonymous SNPs, using PolyPhen-2, PROVEAN and AMYCO analyses.The purpose of this study was to evaluate the degree of potential scrapie susceptibility in Korean native black goats. Thus, we investigated the genotype and allele frequencies of PRNP gene in 211 Korean native black goats. The sequenced ORF in the Korean native black goats was 771 bp in length and homologous with the PRNP gene of Capra hircus registered in the GenBank website (Gene ID: EU870890.1). We found a total of 12 SNPs, including 10 nonsynonymous SNPs. The genotype and allele frequencies of the caprine PRNP gene are shown in Table We performed automatic direct sequencing at the open reading frame (ORF) of the PRNP gene by analyzing Lewontin’s D’ (|D’|) values . In addition, a significant difference in allele frequencies of PRNP codons 143 and 146 was found between scrapie-affected Greek goats and Korean native black goats (p < 0.01) . Interestingly, the allele frequencies of this SNP were not significantly different between scrapie-affected French goats and Korean native black goats (p = 1.0) . Interestingly, the allele frequencies of this SNP were not significantly different between scrapie-affected Greek goats and Korean native black goats (p = 0.109) . There was no significant difference in the allele frequencies between French scrapie-affected goats and Korean native black goats   between Korean native black goats and scrapie-affected goats in other countries. These 6 scrapie-associated SNPs of the caprine 01) Fig. . The allats Fig. . In PRNP01) Fig. .Figure 1PRNP haplotypes of codons 136, 154 and 171 in Korean native goats. In addition, the PRNP haplotypes were compared with those previously reported in 4 countries, namely, Pakistan, China, Japan and France45. In all countries, the ARQ haplotype was distributed over 97% of goats. In Pakistani goats, Chinese goats and Japanese goats, the frequency of the detected AHQ haplotype was less than 1%. Notably, ARR was found in over 1% of Japanese goats, and AHQ was found in 2.39% of French goats. In French goats, a significant difference was found in the allele distribution of the AHQ haplotype between scrapie-affected goats and healthy goats (p < 0.001)33. In addition, more than 95% of the ARQ/ARQ genotype was distributed in all countries, and significant differences in the distribution of the ARQ/AHQ genotype in French goats were found between scrapie-affected goats and healthy goats. (p < 0.001). There were few ARQ/ARR genotypes in Korea (0.47%) and Japan (1.34%), and the ARR/ARR genotype was found only in Japan (0.67%)  H143R (0.129), N146S (0.024), Q171R (0.035), R211Q (0.447), I218L (0.023); “possibly damaging”: W102G (0.603), R154H (0.934); “probably damaging”: G127S (0.992)  was highly resistant to conformational change for becoming deleterious form of prion protein. Thus, we analyzed the prion protein based on those alleles. RSHQK haplotype was measured with 0.27 values by AMYCO. In addition, we analyzed the prion protein of Korean native black goats. Amino sequences of prion protein in Korean native black goats were classified to 4 haplotypes  based on alleles of nonsynonymous SNPs. HNRRQ and HSRRQ haplotypes were measured with 0.27 values. RNRRQ haplotype was measured with 0.24 values  and 4.78% for the ARQ/AHQ genotype (p < 0.001). The haplotypes and genotype distributions of French goats were 100% of ARQ and 100% of ARQ/ARQ, respectively, in scrapie-affected animals. The haplotype distribution in Korean native black goats was similar to that in Pakistani goats, Chinese goats and Japanese goats, and the genotype distribution in Korean native black goats was similar to that in Pakistani goats and Chinese goats  was ‘probably damaging’ and W102G (0.603) and R154H (0.934) were ‘possibly damaging’. Interestingly, all 10 nonsynonymous SNPs of PRNP gene were predicted to be ‘neutral’ by PROVEAN. The inconsistencies in the results of PolyPhen-2 and PROVEAN are due to the differences in algorithms that translate the impact on the protein function55. Because the PolyPhen-2 analysis is based on the structural influence of nonsynonymous SNPs on the protein and the PROVEAN analysis is based on phylogenetic differences among species, we presume that two nonsynonymous SNPs may have an effect on the structure of goat prion protein. Point mutation at the codon 102 of human PRNP gene (codon 105 in goat) has been linked to GSS of human familial prion diseases. In addition, polymorphism at the codon 154 of ovine PRNP gene (codon 154 in goat) has been associated with the susceptibility of scrapie in sheep. Thus, further investigation of caprine PRNP at the codons 102 and 154 is highly desirable in the future.In addition, we predicted the effects of nonsynonymous SNPs on caprine PRNP gene. Interestingly, the degree of amyloid formation of prion protein according to haplotypes of the PRNP gene of the Korean native black goats was lower than or equal to that of prion protein which are known to be resistance to prion diseases. Since scrapie in Korean native black goats has not been reported thus far, this result seems likely to be consistent with the data of AMYCO analysis. Thus, to the best our knowledge Korean native black goat presumed to have resistance to scrapie thus far. However, to verify the nonsynonymous effect of the goat prion protein, it is necessary to perform in vivo or in vitro experiments using models that contain these two polymorphisms.Next, we evaluated potential scrapie susceptibility according to alleles of nonsynonymous SNPs of caprine PRNP gene and scrapie susceptibility in various countries; however, Korean native black goats have not been tested for scrapie susceptibility thus far. Here, we suggest that the multiple PRNP alleles of Korean native black goats can predict potential scrapie susceptibility and may contribute to the onset of the disease. In addition, to exclude the probability of quite different frequencies of PRNP gene polymorphisms in other regions of Korea, we collected 41 blood samples of Korean native black goats from a slaughter house of another region, which is located in Jecheon-si, Chungcheongbuk-do, Republic of Korea and investigated genotype, allele and haplotype frequencies of caprine PRNP gene. The genotype, allele and haplotype frequencies of caprine PRNP gene showed similar distributions between Chungcheongbuk-do goats and Jeollanam-do goats . All experiments using Korean native black goats were performed in accordance with the Korea Experimental Animal Protection Act.Blood samples of 211 Korean native black goats were collected from a slaughter house of Hwasun-gun, Jeollanam-do, the Republic of Korea. These samples were provided from 8 farms, which is located in Jeollanam-do. We collected samples 5 times from March 2016 to June 2016. The sample size used in the present study may be enough to identify rare polymorphisms, including below 1% genotype frequency. In addition, the sample size can also represent the total population of Korean native black goats with a 95% confidence level and a confidence interval of 7. Whole blood samples were treated with ethylenediaminetetraacetic acid (EDTA) and were frozen at −80 °C prior to analysis. Genomic DNA was purified from 200 μl frozen blood using the QIAamp DNA Blood Mini Kit  following the instructions from the supplier.PRNP gene, PCR was performed with gene-specific primers as follows: caprine PRNP-Forward (5′-ATTTTGCAGAGAAGTCATCATGGTGA-3′) and caprine PRNP-Reverse (5′-AACAGGAAGGTTGCCCCTATCCTA-3′). The primers were designed based on the genomic sequence of the caprine PRNP gene, which was registered in the GenBank website (Gene ID: EU870890.1). The PCR mixture contained 10 μM of each primer, 2.5 μl of 10 × Taq polymerase reaction buffer containing 25 mM of MgCl2, 2.5 mM of each dNTP mixture, and 2.5 units of SolgTMTaq DNA polymerase . The PCR was carried out as follows: predenaturation at 95 °C for 2 min, 30 cycles of denaturation at 95 °C for 20 sec, annealing at 58 °C for 40 sec, extension at 72 °C for 1 min, and final extension at 72 °C for 5 min. The purification of PCR products for sequencing analysis was performed with a QIAquick Gel Extraction Kit . The PCR products were directly sequenced by an ABI 3730XL sequencer .To amplify the caprine PRNP gene in Korean native black goats were performed using Haploview Version 4.2 . Differences in genotype, allele and haplotype frequencies of the PRNP gene were tested by chi-squire test (χ2) or Fisher’s exact test using SAS 9.4 Software .Analysis of HWE, LD and haplotype distributions of the PRNP gene using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and PROVEAN (http://provean.jcvi.org/index.php). PolyPhen-2 utilizes a naive Bayes classifier to provide independent count (PSIC) scores using information from sequence alignment and protein structural properties47. PolyPhen-2 predictions are subdivided into three types, “benign”, “probably damaging”, and “possibly damaging”, according to a numerical score ranging from 0.0 to 1.0. PROVEAN calculates the impact score by predicting the functional effect on all classes of protein functions for protein sequence changes such as deletion and multiple substitution as well as single amino acid substitutions47. The PROVEAN program predicts the biological impacts of nonsynonymous SNPs, including effects on protein function and structure. Score predictions form PROVEAN have two types of threshold; scores below −2.5 are considered “deleterious”, and scores above −2.5 are considered “neutral”. Amyloid propensity of caprine prion protein according to alleles of PRNP SNPs was analyzed by AMYCO (http://bioinf.uab.cat/amyco)57. AMYCO is the algorithm used to predict amyloid fibril propensity from amino acid sequences.We evaluated a total of 10 nonsynonymous SNPs of the Supplementary Table 1