Thus, they do not seem to model the mechanisms for the decrease in telomerase activity in fetal brains. Our results suggest that decreased hTR levels, as well as transient increase in α-spliced hTERT, might both contribute to downregulation of telomerase activity during early human brain development between 6 and 17 pcw.Changes in hTERT splice variant expression have been proposed to facilitate the decrease of telomerase activity during fetal development in various human tissues. Here, we analyzed the expression of telomerase RNA (hTR), wild type and α-spliced hTERT in developing human fetal brain  and in young and old cortices using qPCR and correlated it to telomerase activity measured by TRAP assay. Decrease of telomerase activity occurred early during brain development and correlated strongest to decreased hTR expression. The expression of α-spliced hTERT increased between pcw 10 and 19, while that of wild type hTERT remained unchanged. Lack of expression differences between young and old cortices suggests that most changes seem to occur early during human brain development. Using  Telomerase is a ribonucleoprotein comprised of the TERT (telomerase reverse transcriptase) protein and a TR (telomerase RNA) subunit. Its main and best understood function is telomere maintenance. In humans, telomerase activity is high during early fetal development but down regulated soon afterwards in most somatic cell types. However, telomerase is still active in some adult tissues such as many immune cells, endothelium and also inducible in adult stem cells. It is also known that the TERT protein has various telomere-independent functions (for review see ). One ofIn addition to wild type hTERT, various catalytically inactive splice variants of hTERT with splice sites across the reverse transcriptase region have been described . AlternaWhile around 21 different hTERT splice forms have been discovered across the 16 exons of hTERT, the best studied are the α and β splice variants ,14,15,16HTERT expression does not always correlate with telomerase activity and various non-canonical functions for hTERT have been described recently . We demonstrate that TA and hTR both decrease during fetal brain development, while WT hTERT and α-spliced TERT seemed to co-exist in adult human brain. However, while TA was also downregulated during in vitro differentiation of NPSCs no changes in the three telomerase-related genes were found suggesting a different mechanism for the decrease in TA compared to human fetal brain development.Here, we investigated the regulation of hTR, wild type and α-spliced hTERT expression during human fetal brain development, young and old adult frontal cortices as well as during The fetal brains were obtained from the MRC/Wellcome Trust Human Developmental Biology Resource (HDBR), United Kingdom . We used 12 human fetal brains at different stages of development (from pcw 6 until 20) with normal karyotypes . HealthySH-SY5Y neuroblastoma cells were obtained from ECACC. HeLa cervix carcinoma cells were obtained from ATCC. Both cell lines were used as positive controls in the TRAP assay and contained equally high amounts of telomerase activity.et al., 2015 . ThuCorrelation of TA with wild type, α-spliced and β-spliced hTERT differs across human fetal heart, liver and kidney development ,9 see a. In fetaIn contrast, in the fetal heart, TA, WT and α-spliced hTERT were all simultaneously downregulated by gestational week 13/pcw 11 . In liveIn vitro differentiation of NPSCs usually generates a mix of neurons (70%) and astrocytes (30%) after two weeks [in vitro expansion of these NPSCs during their in vitro cultivation via neurospheres prior to their differentiation [et al. in primary NPSCs derived from gestational week 12 [Neural stem cells emerge very early during embryonic development and produce all of the different cells of the brain and central nervous system . In ordewo weeks . During ntiation , exhaust week 12 .in vitro differentiation process of hNPSCs. This result suggests that brain development in vivo might involve different mechanisms of downregulation of telomerase activity than in NPSCs during in vitro differentiation. However, the downregulation of TA in vitro in human NPSCS is comparable to that found during in vitro culture of mouse neuronal cells from embryonic day 15 that declined within the same time frame (after day eight and undetectable at day 14) [We did not find any changes in the expression of telomerase-related genes that could explain the decrease in telomerase activity during the  day 14) . HoweverIt is known that a decrease in telomerase activity correlates to differentiation of human embryonic stem cells (hESCs) . We demoin vitro differentiation of NPSCs did not model the expression kinetics of telomerase-related genes during downregulation of telomerase activity.In general, telomerase activity is regulated by many different endogenous and exogenous factors (for review see ). DownreIn conclusion, the main finding of our study is that telomerase activity is downregulated between pcw 6 and 14/17 during human fetal brain development. Our data suggest that a strong decrease in hTR expression might causally contribute to the downregulation of telomerase activity. In addition, both hTERT variants (wild type and α-spliced hTERT) seem to co-exist in neurons. It would be interesting to determine whether α-spliced hTERT is able to exert a protective function similar to those demonstrated for other hTERT splice versions previously .in vitro does not seem to be regulated by the same mechanisms as during brain development in vivo.Furthermore, our results also suggest that downregulation of TA in NPSCs


To date we have little knowledge of how accurate next-generation sequencing (NGS) technologies are in sequencing repetitive sequences beyond known limitations to accurately sequence homopolymers. Only a handful of previous reports have evaluated the potential of NGS for sequencing short tandem repeats (microsatellites) and no empirical study has compared and evaluated the performance of more than one NGS platform with the same dataset. Here we examined yeast microsatellite variants from both long-read (454-sequencing) and short-read (Illumina) NGS platforms and compared these to data derived through Sanger sequencing. In addition, we investigated any locus-specific biases and differences that might have resulted from variability in microsatellite repeat number, repeat motif or type of mutation. Out of 112 insertion/deletion variants identified among 45 microsatellite amplicons in our study, we found 87.5% agreement between the 454-platform and Sanger sequencing in frequency of variant detection after Benjamini-Hochberg correction for multiple tests. For a subset of 21 microsatellite amplicons derived from Illumina sequencing, the results of short-read platform were highly consistent with the other two platforms, with 100% agreement with 454-sequencing and 93.6% agreement with the Sanger method after Benjamini-Hochberg correction. We found that the microsatellite attributes copy number, repeat motif and type of mutation did not have a significant effect on differences seen between the sequencing platforms. We show that both long-read and short-read NGS platforms can be used to sequence short tandem repeats accurately, which makes it feasible to consider the use of these platforms in high-throughput genotyping. It appears the major requirement for achieving both high accuracy and rare variant detection in microsatellite genotyping is sufficient read depth coverage. This might be a challenge because each platform generates a consistent pattern of non-uniform sequence coverage, which, as our study suggests, may affect some types of tandem repeats more than others. Recent technical developments and improvements in next-generation sequencing (NGS) platforms have revolutionized genetic and genomic research by increasing the throughput and lowering the cost of DNA sequencing. NGS technology makes it possible to sequence millions of individual DNA strands in DNA mixtures such as fragmented genomic DNA or multiplexed amplicons in relatively short time de novo genome assembly and RNA-seq analysis. They focused on repeat classes with sequence lengths of at least 100 bp, that occur two or more times in the genome and exhibit>97% identity to at least one other copy of themselves, and they concluded that such repeats result in ambiguities in alignment and assembly that may lead to biases and errors in interpretation. While not the focus of that work, the difficulties posed by repeats and the computational errors they generate would surely be exacerbated by any intrinsic errors in the sequencing of these repeats. However, to date we have little knowledge of how accurate NGS approaches are in sequencing repetitive sequences beyond known limitations accurately sequencing homopolymer runs One of the biggest technical challenges that are associated with NGS technologies are repetitive DNA sequences Microsatellites are short  DNA sequences in which motifs of 1–6 bp are tandemly repeated. They are highly polymorphic in their length, which results from variability in repeat number The analysis of microsatellite polymorphism has traditionally been based on locus specific PCR-amplification followed by fluorescence capillary electrophoresis to identify fragment length differences Despite rapid and ubiquitous implementation of NGS platforms in genetics and genomics, only a handful of previous reports have evaluated the accuracy of these platforms for sequencing tandem repeats and their potential for microsatellite genotyping Due to the short length of sequencing reads obtainable from the Illumina platform (150–250 bp), this NGS technology had been assumed impractical for microsatellite genotyping. However, Bornman et al. Here we undertook the first comprehensive empirical study that examines and compares the performance of multiple NGS platforms for short tandem repeat sequencing. Using a reference set of yeast DNA samples we evaluate both long-read (454-sequencing) and short-read (Illumina) NGS platforms and compare these to data derived through Sanger sequencing. Unlike previous studies, our experimental design allowed us to (1) examine microsatellite variants from both amplicon and whole genome next-generation sequencing and (2) investigate any locus-specific biases and differences that might have resulted from variability in microsatellite copy number, repeat motif or type of mutation.Saccharomyces cerevisiae derived from the haploid DH89 α, ho strain, which in turn derives from the laboratory Y55 wildtype strain We used 16 populations of S. cerevisiae genome  using a program written in C as described in Bagshaw et al. ftp://ftp.sanger.ac.uk/pub/users/dmc/yeast/latest/). Locus-specific PCR primers, designed for all microsatellite loci using Primer 3 We identified microsatellites with 2–6 bp repeat motifs in the To facilitate GS FLX 454-sequencing (Roche Diagnostics), the primers were modified into fusion primers, which consisted of: (1) locus-specific microsatellite primers; (2) multiplex identifier sequence ; and (3) the Roche GS FLX 454-sequencing adaptors 4 reaction buffer (16 mM (NH4)2SO4, 67 mM Tris-HCl pH 8.8, 0.01% stabilizer; supplied with BIOTAQ DNA Polymerase), 3 mM MgCl2, 1 µM of each primer , 200 µM of each dNTP and 1 Unit BIOTAQ DNA Polymerase (Bioline). PCR conditions were 94°C for 5 min followed by 32 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min. The PCR products were verified on a 1% agarose gel stained with SYBR Safe.For each sample, each microsatellite locus was amplified by individual polymerase chain reaction (PCR). All amplification reactions were performed in 25 µl volume containing 10 ng of template genomic DNA, 1x NHAll successfully amplified PCR products were purified using Agencourt AMPure XP DNA purification kit following the manufacturer's instructions (Beckman Coulter). DNA concentrations of the purified amplicons were measured using Quant-iT PicoGreen dsDNA Assay kit .From our sample of 16 yeast populations, 12 of these were also sequenced through the Illumina FastTrack Service . Whole genome sequencing using 100 bp paired-end reads of libraries with an average insert size 300 bp was undertaken on one lane of an Illumina Genome Analyzer IIx using chemistry version 5. This design expected around 40-fold average coverage depth per genome.For GS FLX 454 sequencing, equimolar amounts of all purified barcoded amplicons (i.e. amplified using the fusion primers) were pooled and submitted for 454-sequencing at the High Throughput Sequencing Service .A subset of 50 purified PCR products that had been amplified using the locus-specific primers were submitted for cloning and Sanger sequencing at the Central Analytical Facility  and Macrogen Inc. . Ten clones per amplicon were Sanger sequenced in both forward and reverse directions, which provided 20 unambiguous sequences per PCR product.ftp://ftp.sanger.ac.uk/pub/users/dmc/yeast/latest/) using Burrows-Wheeler Alignment Tool version 0.5.9-r16 with default settings CLC bio). Aligned reads were mapped to the 255 microsatellite reference loci. The CLC mapping parameters were set to the following: mismatch, insertion and deletion cost  = 2, length fraction  = 0.75, similarity fraction  = 0.9 and non-specific matches were set to ignored. This filtering included in the final mappings only read alignments that had greater than 90% similarity with the reference in at least 75% of their read length and non-specific matches, i.e. the reads that would align at more than one position with an equally good score, were excluded from the final mappings. It is important to note that the CLC Genomics Workbench mapping parameters do not filter reads based on the read length per se, thus reads of various lengths may be included in the final mappings that are then used for variant detection. Nevertheless, insertion and deletion variants are only detected if they are spanned by reads, which means that the reads have to align both before and after the variant (CLC bio). The quality filters for the quality-based variant detection were set to neighborhood radius  = 5, maximum gap and mismatch count  = 2 and minimum central and neighboring Phred quality score  = 20. The significance thresholds were set to minimum coverage  = 5 reads and a minimum variant frequency  = 10%. Thus, low frequency variants/alleles that may result from sequencing errors For each of the 12 samples, the quality of obtained reads was first examined using FastQC software (Babraham Institute) with default settings. Low quality bases were trimmed off using Dynamic Trim software with default settings CLC bio). Reads per each sample were mapped against the 255 microsatellite reference loci. The CLC mapping parameters and the criteria for the quality-based variant detection were almost identical to those used for the Illumina sequencing reads described above, but the minimum coverage was set to 25 reads and the minimum variant frequency set to 5%. These adjustments accounted for the higher average coverage per locus obtained from the amplicon 454-sequencing compared to the whole genome Illumina sequencing and aimed to achieve a similar level of variant detection across the platforms. Only InDel variants with length of 2 bp and greater were further considered. In addition, the variants in homopolymers were excluded from further analyses, as 454-sequencing is known to have difficulties in accurately sequencing these regions The reads obtained from GS FLX 454-sequencing were initially examined using the Genome Sequencer FLX System Software version 2.3 and its SFF Tools commands (Roche Diagnostics). Only reads that contained complete MID barcodes with no mismatches were extracted for the analysis. The extracted Standard Flowgram Format (sff) files were then processed using CLC Genomics Workbench 6.0.5 (CLC bio). The analyses were performed as described with the following modifications: using the functions specific to Sanger sequence data, the sequences for each amplicon were assembled against the relevant microsatellite reference locus using default parameters. The criteria for the quality-based variant detection were the same as those used for the Illumina sequencing reads described above, apart from minimum coverage, which was set to 10 reads and a minimum variant frequency set to 5%. These modifications were again derived from the average coverage per locus obtained from our Sanger sequencing and aimed to achieve a similar level of variant detection across the platforms. Only InDel variants with length of 2 bp and greater were further considered in our analyses.To eliminate alignment software bias or errors, the microsatellite sequences obtained from Sanger sequencing were also analyzed using the CLC Genomics Workbench 6.0.5  that were also successfully sequenced using the Sanger method. These amplicons represented 21 microsatellite loci, with an average amplicon length 138.8±24.7 bp (mean ± SD). In order to assess how feasible and accurate NGS platforms are for high-throughput parallel microsatellite-amplicon sequencing we compared InDel variant data obtained from the NGS platforms to those obtained from the conventional Sanger sequencing. Statistical tests were performed using functions available in R statistical software version 3.0.3 The two NGS strategies, amplicon (454-sequencing) and whole genome (Illumina) sequencing differed in the number of microsatellite loci that passed our quality criteria for InDel variant detection analysis. This was largely caused by differences in coverage, as the average coverage for loci sequenced by 454-platform and analyzed in this study was 77.2 reads, while the average coverage for loci sequenced by Illumina in this study was 13.8 reads. As a result of this, less than half of the variants detected by both Sanger and 454-sequencing were detected by Illumina platform.Therefore, our first point of comparison in validating microsatellite mutation detection by NGS platforms was between 454-sequencing and Sanger sequencing. Among the 45 amplicons that represented 21 yeast microsatellite loci, we detected a total of 112 InDel variants using the two sequencing methods . There w2 = 4.06, p = 0.54), InDel type defined as deletion or insertion , microsatellite repeat number  or microsatellite motif . We also found no significant results repeating these tests and comparing the subset of variants, for which detection differed among platforms after Bonferroni and Benjamini-Hochberg corrections, with remaining variants.We looked further for locus-specific biases by comparing attributes/contexts of the variants for which significant differences were found between platforms with those for which no difference was found. Initially, we compared the 29 variants showing nominally significant detection frequency differences between the two platforms with the remaining 83 variants. We found no significant differences with respect to InDel type defined as number of repeats deleted or inserted .Sanger coverage was relatively uniform across all variants (18±3.2), but 454-sequencing coverage varied substantially (77.2±55). Searching for modifiers of 454-platform coverage over all detected variants, we found that motif type was not a significant predictor of coverage . However, we did find that coverage of AC microsatellite variants (n = 17) was significantly lower, with an average of 47.3 reads, compared to AT microsatellites variants (n = 87) showing an average of 82.1 reads . It was also apparent that the 454-platform may have been somewhat poorer at detecting AC microsatellite variants independently of coverage, as the observed frequencies of these variants were nearly twice as high on average using Sanger sequencing, but this was not significant  was included in this analysis . Thus, oIllumina sequencing gave more uniform coverage than 454-sequencing  and short-read (Illumina) NGS platforms and compared these to Sanger sequencing. The 454-platform was used for high-throughput parallel sequencing of yeast microsatellite-specific amplicons, while the Illumina platform was used for yeast whole-genome sequencing from which we subsequently extracted mapped microsatellite loci of interest. Data from all sequencing techniques were analysed with the same mapping/InDel detection software and the parameters used were standardized in order to limit discrepancies that might arise from the data analysis Our experimental designs for Illumina sequencing expected around 40-fold average coverage depth per genome. From our obtained data, the mean raw coverage depth per genome was 38.1 reads, which decreased to a mean of 29.3 reads after trimming and alignment. As previously shown, however, each NGS technology generates a pattern of non-uniform sequence coverage Out of 112 InDel variants identified among 45 microsatellite amplicons in our study, we found 87.5% agreement between the 454-platform and Sanger sequencing after Benjamini-Hochberg correction for multiple tests and 92% after Bonferroni correction. Out of 14 significant disagreements following Benjamini-Hochberg correction, six resulted from detection by Sanger sequencing but not by 454-sequencing. To further understand the cause of this discrepancy, we scrutinized the NGS mappings by eye. In all but one case we found that the variants were in fact present in the mappings, but had gone undetected due to their low frequency (<5%) in our 454-data. Similar discrepancies were previously reported and attributed to low coverage combined with allelic amplification bias Although our dataset for the Illumina microsatellite genotyping analysis was considerably smaller than that of the 454-platform, the short-read platform appeared to be highly accurate in InDel variant detection, with over 100% agreement with 454-sequencing and over 93% agreement with Sanger sequencing after Benjamini-Hochberg correction. Similar results were also demonstrated by the study of Bornman et al. In contrast to those few studies that have previously evaluated application of NGS for microsatellite genotyping Interestingly, searching for modifiers of 454-platform coverage over all detected mutations, our study indicates that the 454-platform may have been somewhat poorer at sequencing AC microsatellite variants, as these had on average significantly lower coverage than AT microsatellite variants. On the other hand, microsatellite repeat number, the attribute that might have been expected to affect the NGS coverage the most due to secondary structures formed by longer microsatellites We have demonstrated that both short-read and long-read NGS platforms can be used to sequence short tandem repeats accurately, which makes it feasible to consider the use of these platforms in high-throughput genotyping. Nevertheless, the use of each platform is accompanied by a variety of challenges that should be considered when scoping such an experiment. First, it appears the major consideration for achieving high accuracy and rare microsatellite variant detection is sufficient read depth coverage. This might be a challenge because each platform generates a consistent pattern of non-uniform sequence coverage Table S1Indel variant number refers to the number of repeat units deleted or inserted; negative number refers to a deletion; positive number refers to an insertion. Var refers to the observed number of reads for the particular variant, while non-var refers to the number of remaining reads covering the variant position; i.e. total coverage minus variant reads at the given position. P used refers to a standard chi-square test, except when (i) expected read counts were less than one and we used Monte-Carlo simulation to calculate P values or (ii) the observed number of reads minus the expected number was ≥ the expected number and we used a log-likelihood ratio goodness of fit test. P values for the Illumina platform are for three-way comparisons between Sanger, Illumina and 454-sequencing. Significance: * statistically significant values unadjusted for multiple tests (P<0.05), ** statistically significant values after Benjamini-Hochberg correction for multiple tests, *** statistically significant values after Bonferroni correction.Summary of microsatellite variant data and statistics from Sanger, GS-FLX and Illumina platforms. (XLSX)Click here for additional data file.


AGRN) gene, encoding an essential neural regulator that induces the aggregation of acetylcholine receptors (AChRs), is known. In 2015, three genetically related cases of generalized AMC affecting Red dairy calves were diagnosed in Denmark.Bovine arthrogryposis multiplex congenita (AMC) is a syndromic term for a congenital condition characterized by multiple joint contractures. Rare inherited forms of bovine AMC have been reported in different breeds. For AMC in Angus cattle a causative genomic deletion encompassing the agrin (MPRIP) and the cholinergic receptor nicotinic beta 1 subunit gene (CHRNB1), were perfectly associated with the AMC phenotype. Previously described CHRNB1 variants in humans lead to a congenital myasthenic syndrome with impaired neuromuscular transmission. The cattle variant represents a single base deletion in the first exon of CHRNB1 (c.55delG) introducing a premature stop codon (p.Ala19Profs47*) in the second exon, truncating 96 % of the protein.The family history of three affected calves suggested an autosomal recessive inheritance. Single nucleotide polymorphism (SNP) genotyping showed a single genomic region of extended homozygosity of 21.5 Mb on chromosome 19. Linkage analysis revealed a maximal parametric LOD score of 1.8 at this region. By whole genome re-sequencing of the three cases, two private homozygous non-synonymous variants were detected in the critical interval. Both variants, located in the myosin phosphatase Rho interacting protein  contains supplementary material, which is available to authorized users. ISG15) gene, the 5’ regulatory region of the hairy and enhancer split 4 (HES4) gene, and the first two exons of the agrin (AGRN) gene (OMIA 001465-9913)  is a syndromic term for a congenital condition characterized by arthrogryposis (from Greek for ‘curved or hooked joints’) or joint contracture of multiple joints involving more than just one part of the body . Most liOMIA 00145-9913  284; Case 2: a female delivered at GD 275; and Case 3: a female delivered at GD 258. The three cases originated from different herds. Two cases (Cases 1 and 2) were sired by the same artificial insemination bull. Initially, ethylenediaminetetraacetic acid (EDTA) stabilized blood samples were obtained from all three dams and semen samples were collected of both bulls. Later on, semen samples of three ancestral sires were obtained.Initially full body computed tomography (CT) scanning using a single slice helical CT machine  with a slice thickness of 3 mm was performed to obtain a full view of the bone malformations. The calves were then necropsied. Specimens of brain, lung and spleen for virology and pleural effusion for serology were sampled in separate plastic containers and stored at -20 °C until analysis. Specimens of heart, lung, liver, kidney, spleen, adrenal gland, thymus, skeletal muscle, brain and spinal cord were fixed in 10 % neutral buffered formalin for histology and then processed by routine methods, embedded in paraffin, sectioned at 2–3 μm and finally stained with hematoxylin and eosin.The herds were declared free from bovine virus diarrhea virus (BVDV) based on regular bulk tank milk analyses as part of the national BVDV surveillance. Fetal tissues were examined for SBV by real-time quantitative reverse transcription polymerase chain reaction (PCR) . ExaminaFive-generation pedigrees were constructed based on information obtained from the Danish Cattle Database and breeding associations and analyzed for inbreeding loops and shared ancestors. Initial investigation of pedigrees showed a close genetic relationship between the parents of all three cases.Genomic DNA was extracted from samples of eight animals (three cases and their respective dams and the two sires) and used for genotyping with the BovineHD BeadChip (illumina), including 777,962 evenly distributed SNPs, at Geneseek . Genomic DNA from additional three normal male ancestors was used for targeted genotyping of candidate variants during mutation analysis. To identify extended homozygous regions with allele sharing across cases, the following PLINK software commands were used: --maf 0, --max-maf 1.0, --geno 0.01, --hwe 0, --mind 0.15, --homozyg, --homozyg-match 1, --homozyg-group . All givIndividual PCR-free fragment libraries with average insert sizes of 400 base pairs (bp) were prepared from the three affected calves which were sequenced to ~15x coverage on two lanes of an Illumina HiSeq3000 instrument using 2 x 150 bp paired-end reads. The mapping to the UMD 3.1 bovine reference genome assembly  and variCHRNB1 variant, and a 221-bp PCR product using a forward primer (5-GCACTGGTTTTTGCACATTC-3) and a reverse primer (5-TGTCTTTTTGCCTGCAGTTG-3) flanking the MPRIP variant. The PCR products were amplified with AmpliTaqGold360Mastermix (Life Technologies), and the products were directly sequenced using the PCR primers on an ABI 3730 capillary sequencer (Life Technologies). The sequence data were analyzed using Sequencher 5.1 (GeneCodes).Genotyping of the two candidate variants for AMC was performed by re-sequencing a 247-bp PCR product using a forward primer (5-CCAATAACAGGTGCACATTCC-3) and a reverse primer (5-GCCTGGAGGAGGAAAGAACT-3) flanking the All cases had reduced body weight  and displayed severe generalized contracture of joints of the spine and limbs with flexion of all joints, except the phalangeal joints that were extended. Vertebral lesions consisted of combinations of torticollis and kypho-scoliosis affecting the entire spine. Case 3 also had mild lordosis. Cases 1 and 2 had an almost 180° scoliosis of the thoraco-lumbar spine, so that the head approached the caudal part of the calf , dilated urinary bladder with diverticulae (case 2) and edema and a subcutaneous cyst in the ventral neck (case 3). The brain and spinal cord appeared grossly normal.CT scanning confirmed the skeletal lesions found at necropsy, but provided a more detailed morphology of skeleton, which was difficult to assess in detail due to the generalized ankylotic arthrogryposis. The fusion between adjacent vertebrae resulted in a rigid structure and deformities in the thoracic and lumbar vertebrae combined together formed a twisted, open helical shaped spine. The ribs were thickened. Fusion of the dorsal spinous processes was seen in all vertebral regions, while the size and shape of the lumbar transverse processes were asymmetrical. The pelvis was deformed, and attached asymmetrically to the sacral vertebrae Fig. . A CT scHistology showed complete lipomatous muscular atrophy Fig.  and congExamination for SBV and antibodies against SBV and BTV were negative.The pedigree of the established Red dairy cattle family segregating for AMC was consistent with monogenic autosomal recessive inheritance Fig. . In all To obtain a comprehensive overview of all variants in the critical interval, the whole genome of all three available AMC cases were re-sequenced. SNPs and indel variants were called with respect to the reference genome of a presumably non-affected Hereford cow. Due to the recessive inheritance and the lethal effect of the mutation we hypothesized that most likely a non-synonymous loss of function mutation affecting the coding sequence of a gene would be responsible for AMC. Therefore subsequently only variants that were homozygous in the affected calves are reported. The visual inspection of the BAM files of the three sequenced cases revealed no evidence for the homozygous presence of a larger structural variant affecting coding regions of the critical interval on chromosome 19.CHRNB1 c.55delG and Chr19 35573959C > T or MPRIP c.2030G > A. Both coding variants were confirmed by Sanger sequencing and genotyped in the parents of the affected calves and additional three family members  and other postsynaptic proteins on muscle fibers and is crucial for the formation and maintenance of the neuromuscular junction (OMIM 103320). Acetylcholine receptor expression at the motoric end-plates is not noticeably reduced in human agrin deficiency, a rare form of human congenital myasthenic syndrome (CMS) [So far a genetic cause for bovine AMC has been reported only in Angus cattle  .CHRNA1, CHRNB1, or CHRND cannot survive because no substituting sub-units exist and hence these individuals probably die in utero [CHRNB1 gene encoding the cholinergic receptor nicotinic beta 1 subunit are known to cause dominant and recessive forms of CMS type 2 (OMIM 616313 and OMIM 616314). Mice lacking AChR beta-subunit, the target of agrin, show abnormal neuromuscular synapse morphology [The AChR controls electrical signalling between nerve and muscle cells by opening and closing a gate, membrane-spanning pore to trigger muscle contraction. It has five subunits of four different types: two alpha and one each of beta, gamma (or epsilon), and delta subunits . Mutatioin utero . Patientin utero . Non-synrphology .MPRIP gene encodes myosin phosphatase Rho interacting protein, which is localized to actin myofilaments and binds to the myosin binding subunit of myosin phosphatase in vascular smooth muscle cells [CHRNB1:c.55delG variant is much more likely to cause AMC in Red dairy cattle than the MPRIP missense variant (p.Arg677Gln). As the identified p.Ala19Profs47* premature stop codon is predicted to lead to a 96 % truncation of the bovine CHRNB1 protein, and the mutant mRNA is very likely targeted by the non-sense-mediated decay pathway, this bovine variant represents the most likely candidate causative mutation.In contrast, the le cells . The MPRle cells  and is ile cells . Thus weHere we demonstrate that an intensive surveillance programme identifying calves with similar disease phenotype in combination with the typical genetic structure of cattle populations and the availability of whole genome re-sequencing methods enables the identification of pathogenic variants causing inherited defects before a high number of diseased calves occurred. The identified genetic syndrome is associated with significant welfare issues and economic losses. If born alive, the syndrome is lethal to the offspring due to impaired respiration associated with musculoskeletal malformations or the offspring needs to be euthanized for welfare reasons. However, the generalized skeletal malformations may also cause dystocia requiring either assisted delivery or Caesarean section for the cow to survive. The magnitude of these issues may be significant for the Red dairy cattle breed unless preventive breeding measures are implemented as the common ancestor, which carries the mutation, has been used extensively in several countries.The study therefore provides another example that the widespread use of elite sires by means of artificial insemination in livestock breeding leads to the frequent emergence of recessive genetic defects, which cause significant economic and animal welfare concerns .CHRNB1 loss of function mutation associated with a lethal neuromuscular phenotype in mammals. Finally, the findings provide a gene test to improve selection against this deleterious mutation in Red dairy cattle.This study provides the first phenotypically and genetically characterized example that bovine familial AMC represents an inherited neuromuscular disorder corresponding to human CMS. Bovine familial AMC is confirmed to be associated with mutations in genes of importance for the neuromuscular junction. It is also the first reported AGRN, agrin gene; AMC, arthrogryposis multiplex congenita; BAM, binary version of a sequence alignment/map (SAM) file; Bp, base pairs; BTV, bluetongue virus; BVDV, bovine virus diarrhea virus; CMS, congenital myasthenic syndrome; CT, computed tomography; EDTA, ethylenediaminetetraacetic acid; GD, gestation day; HES4, hairy and enhancer split 4 gene; IBD, identical by descent; ISG15, ISG15 ubiquitin-like modifier gene; LOD, logarithm of the odds; PCR, polymerase chain reaction; SBV, Schmallenberg virus; SNP, single nucleotide polymorphism.AChRs, acetylcholine receptors;


Most adolescents do not achieve the recommended levels of moderate-to-vigorous physical activity (MVPA), placing them at increased risk for a diverse array of chronic diseases in adulthood. There is a great need for scalable and effective interventions that can increase MVPA in adolescents. Here we report the results of a measurement validation study and a preliminary proof-of-concept experiment testing the impact of Zamzee, an accelerometer-linked online intervention system that combines proximal performance feedback and incentive motivation features to promote MVPA. In a calibration study that parametrically varied levels of physical activity in 31 12-14 year-old children, the Zamzee activity meter was shown to provide a valid measure of MVPA . In a subsequent randomized controlled multi-site experiment involving 182 middle school-aged children assessed for MVPA over 6 wks, intent-to-treat analyses found that those who received access to the Zamzee intervention had average MVPA levels 54% greater than those of a passive control group (p < 0.0001) and 68% greater than those of an active control group that received access to a commercially available active videogame (p < .0001). Zamzee’s effects on MVPA did not diminish significantly over the course of the 6-wk study period, and were statistically significant in both females and males, and in normal- vs. high-BMI subgroups. These results provide promising initial indications that combining the Zamzee activity meter with online proximal performance feedback and incentive motivation features can positively impact MVPA levels in adolescents. Sub-optimal physical activity in youth has been identified as a major public health challenge –3. RecenExisting interventions to increase physical activity in adolescents range from curriculum-based programs to the use of active video games . Recent literature reviews suggest that no current intervention strategies have been able to consistently induce the sustained changes in MVPA needed to have health benefits in general population samples . HoweverIn the present studies, we evaluated a new metrics-driven incentive motivation program called “Zamzee” which seeks to increase MVPA in adolescents by combining proximal feedback on physical activity levels with frequent small scale incentives ,26. ThesAll study procedures were reviewed and approved by institutional review boards . Prior to study enrollment, all study participants provided written informed assent in conjunction with parental written informed consent.http://fireflydesign.com/) that implements standard procedures for accelerometer-based activity measurement [x)2 + (ay)2 + (az)2].5, where ax, ay, and az represent output from each of the 3 orthogonal accelerometers) .5, where ax, ay, and az represent output from each of the 3 orthogonal accelerometers). Correspondence between RT3 and Zamzee estimates of activity intensity was quantified by Spearman rank correlation coefficients in both the raw 10 s data epochs in which data were collected and in an aggregated dataset that averaged over repeated measurements on each individual for each activity bout (to control for autocorrelation across repeated epochs from the same person performing the same activity). Ancillary mixed effect linear model analyses were also conducted to relate Zamzee VMa values to criterion RT3 activity counts while controlling for potential correlation of residuals resulting from repeated measurements. Agreement between METs values estimated by Zamzee vs. RT3 was assessed by Bland-Altman plots and related linear regression of agreement (log10 Zamzee METs—log10 RT3 METs) on average estimated activity intensity ([log10 Zamzee METs + log10 RT3 METs]/2) as previously described [To validate the technical performance of the Zamzee meter as a measure of physical activity intensity, analyses first quantified relationship between benchmark RT3 activity counts and Zamzee VMa values , receiver operating characteristic (ROC) analyses were conducted in the context of logistic regression analyses relating VMa values to METs values for each activity bout ,42. AnalTo test for potential impact of the Zamzee meter/website system on MVPA levels in 11–14 year-old children, we conducted a 6-week multi-site randomized controlled pilot experiment  involving 3 study arms: 1) a passive control group that received Zamzee activity meters but no other intervention , 2) an active control group that received Zamzee activity meters and the Dance Dance Revolution (DDR) active video game , which has previously been shown to increase MVPA ,44, reduIn all study arms, participants were asked to wear their Zamzee activity meters close to the trunk of their bodies (e.g. clipped to their waistband or in their pants pocket) during all waking hours (except when in water). Participants were asked to charge the meter periodically (every couple of days) using a provided AC charger or by connecting it to a computer USB port. To facilitate activity data collection, participants were asked to periodically connect their Zamzee activity meters to the USB port of an internet-enabled computer  on which Zamzee upload center software had been installed.Passive and active control participants saw a study participation acknowledgement webpage when they initially uploaded data, but saw no information about their physical activity levels (no feedback) and had no access to any other intervention stimuli (no rewards or other incentives). Active control group participants received a version of DDR  specific to whatever game console they reported to have available at home in a pre-study survey . Participants who did not own a DDR-compatible computer or videogame console were loaned a Play Station console  to support DDR use for the length of the study. The DDR dance mats were equipped with tracking software to record the date, time, and duration of each DDR game play session. Participants were asked to use the assigned program (Zamzee website or DDR game) as little or as much as they wanted to over the course of the 6-wk study.The primary outcome analyzed was average daily MVPA levels aggregated weekly over the 6-wk study. Daily MVPA was quantified as both absolute duration of MVPA per day  and as the MVPA rate, or the ratio of MVPA duration to the total duration of time monitored per day . Data were analyzed by mixed effect linear models (SAS PROC MIXED) using a 3 (Group) x 6 (Time) factorial design. In the event of a significant omnibus test statistic for the overall Group main effect or the Group X Time interaction, follow-up contrast analyses assessed the statistical significance of pairwise differences among groups. Analyses were conducted on an intent-to-treat basis, with all available data analyzed for any day in which VMa values exceeded the “meter worn” threshold for > 10 min. Days in which VMa exceeded the “meter worn” threshold for < 10 min were treated as missing data . To ensure that individual differences in meter wear times did not bias results, analyses of MVPA duration controlled for individual differences in meter wear time as a covariate, and analyses of MVPA rates  were weighted by daily meter wear duration. All analyses also controlled for study site to address potential clustering. In addition to primary analyses of general experimental group effects, ancillary analyses tested for potential differences in the magnitude of experimental effects as a function of participant sex (self-reported at study entry) and adiposity (determined by research staff measurement of height and weight at study entry). BMI was computed based on measured height and weight, converted to age- and sex-specific percentile values, and classified as normal BMI for values < 95% or high BMI for percentiles ≥ 95% (the CDC-recommended cut-off for youth BMI classification) . In the At study completion, all participants received compensation deemed appropriate by institutional review boards in consultation with local site administrators . On average, Zamzee participants received an additional $7.55 in non-cash activity rewards .To assess the Zamzee system’s potential impact on MVPA in adolescents, 182 middle school-aged children were recruited from 3 study sites and randomized into a passive control condition , an active control condition , or an intervention group that received access to the Zamzee online incentive rewards system (n = 62). Total and site-specific sample characteristics are presented in th percentile = $1.60; 75th percentile = $8.30).Study protocol adherence was high, with 96% (175/182) of participants providing physical activity data over the course of the 6-wk study. On average, participants provided valid activity data on 63% of the study days (mean = 26.4 ± SE 13.2 days) and wore their activity meters for 8.9 ± 0.1 hrs / day. Measurement non-adherence rates were similar across study groups: participants who provided no activity data included n = 3 in the intervention group, n = 3 in the passive control group, and n = 1 in the active control group. In the Zamzee intervention group, 82% of participants visited the intervention website at least once. In the active control group, electronic tracking of DDR dance pad activity indicated that 51% of participants used the assigned game at least once. Intervention group participants earned a median US $5.00 / wk in incentive rewards (25Weekly average MVPA duration (min/day) and MVPA rate (min/monitored duration) were analyzed in a 3 (Group) x 6 (Week) mixed effect linear model, with planned contrasts assessing pair-wise differences between groups in average MVPA over the entire 6-wk period. As shown in X2). Results showed 38% greater MVPA levels in general for males vs. females (p < .0001), as well as a continued significant effect of Zamzee in promoting overall MVPA rates (p < .0001). Results also identified a statistically significant sex difference in the magnitude of Zamzee intervention effects (p = .0384), with female Zamzee participants showing an average 39% increase in MVPA (p = .0049), and male Zamzee participants showing an average 76% increase in MVPA (p < .0001).To determine whether Zamzee intervention effects might differ for females vs. males, ancillary analyses were conducted in a 2 (Sex) x 3 (Group) x 6 (Week) factorial design. Forty-eight percent of participants were female, and the prevalence of female participants was similar across experimental conditions , and the prevalence of high-BMI participants was similar across experimental conditions . Results identified no significant BMI x Group interaction for MVPA (p = .9146), and Zamzee intervention effects on MVPA remained highly significant in analyses that included BMI group as a factor (p < .0001).To determine whether Zamzee intervention effects might differ for individuals who were overweight at study entry, additional ancillary analyses were conducted in a 2  x 3 (Group) x 6 (Week) factorial design. Sixty-four percent of participants had normal BMI-for-age/gender percentiles , and that the Zamzee system (activity meter with an online incentive motivation system) can increase MVPA rates in 11–14 year-old children by > 50% over a 6-wk period. Zamzee intervention effects on MVPA did not diminish significantly over the course of the 6-wk study period, and were statistically significant in both males and females and in both normal- and high-BMI subgroups. These findings provide a promising proof-of-concept indication that the Zamzee system’s combination of comprehensive activity monitoring, online feedback, and incentive motivation can increase MVPA in a community sample of middle school-aged American youth.The present studies were intended to provide a “real world” assessment of the Zamzee system’s performance. Although intervention study participants were not required to use the Zamzee website, > 80% of participants given access to the Zamzee website chose to use it (a rate notably greater than the 51% who chose to use the DDR active video game utilized in the active control condition). The Zamzee activity monitor also appears to be a relatively acceptable device for measuring physical activity in adolescents, as indicated by the fact that valid activity data were obtained for more than 60% of total study days . Previous studies using technology-based interventions to promote physical activity have found that maintaining user engagement can be a significant challenge ,48. The Our results suggest that the Zamzee system may provide an effective system for promoting MVPA in adolescents, but the specific psychological mechanisms mediating the observed effects remain to be identified in future research. Provision of website-mediated financial rewards (in the form of gift cards) seems likely to have played a significant role in these effects, as previous studies have shown similar impacts of financial incentives on other health behaviors such as medication adherence, smoking cessation, and weight loss ,20,49. HThe present meter validation studies and randomized experimental results are promising, but these findings remain preliminary and are limited in several respects. The meter validation studies involved brief experimental manipulations of physical activity intensity in relatively small samples. Future validation studies using additional physical activity exposures   in largeDespite the early stage of the Zamzee system’s development, and the limited data available so far, the present studies do suggest that the Zamzee activity meter can provide a valid and engaging instrument for measuring MVPA in adolescents, and that integrating this meter with an online feedback and incentive motivation system can significantly increase MVPA levels over a 6-wk period in a community sample of normal adolescents. Given the public health significance of maintaining healthy levels of physical activity in young people –3 and thS1 File(DOC)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.


Relationship between structural variants of enzymes and metabolic phenotypes in human population was investigated based on the association study of metabolite quantitative traits with whole genome sequence data for 512 individuals from a population cohort. We identified five significant associations between metabolites and non-synonymous variants. Four of these non-synonymous variants are located in enzymes involved in metabolic disorders, and structural analyses of these moderate non-synonymous variants demonstrate that they are located in peripheral regions of the catalytic sites or related regulatory domains. In contrast, two individuals with larger changes of metabolite levels were also identified, and these individuals retained rare variants, which caused non-synonymous variants located near the catalytic site. These results are the first demonstrations that variant frequency, structural location, and effect for phenotype correlate with each other in human population, and imply that metabolic individuality and susceptibility for diseases may be elicited from the moderate variants and much more deleterious but rare variants. Metabolomics is emerging as an indispensable method for investigating the causes of diseases, because metabolite levels in biofluids are significantly influenced by various genetic and environmental factors. Recent technological advances have made it possible to implement the genome wide association study (GWAS) of metabolic traits to investigate genetic effects on blood metabolite levels12345Tohoku Medical Megabank organization (ToMMo) by nuclear magnetic resonance (NMR) spectroscopy. We obtained the metabolite profiling of plasma and analyzed the correlation among the quantified metabolites. We also performed an association study of plasma metabolites using whole-genome sequence dataset from all participantsAs elucidation of the relationship between quantitative diversity of human blood metabolites and structural diversity of enzymes caused by variants in human population is critically important to understand the mechanisms how human metabolic individuality is defined, in this study we performed a large-scale cohort association study of metabolomics-genomics to investigate the relationship between structural variations in enzymes and metabolic phenotypes in human. We analyzed the metabolites of plasma collected from 512 participants in a population cohort conducted by We identified five metabolites associated with non-synonymous variants in five metabolic enzymes, four of which were previously reported to be involved in metabolic diseases. To clarify the relationship between the variants and functional activity of related enzymes, we performed structural analysis of the five non-synonymous variants and found that they are not located in catalytic center regions, but located in peripheral regions or in regulatory domains, indicating that these variants retain only moderate impact on their corresponding enzymatic activities. Therefore, we further analyzed variants in these enzymes and found two individuals with larger changes of metabolite levels. These individuals have much more rare variants of one enzyme gene that cause non-synonymous variants located in closer proximity to the catalytic site, indicating that they cause larger functional impacts than the moderate variant. Whereas many studies have been conducted to clarify the relationship between gene variants and functional activities of proteins, our results unequivocally demonstrate that variant frequency, structural location, and effect for phenotype correlate with each other in human population. We expect that our approach is versatile to discover further associations of metabolites with diseases, as even a moderate variant can be detected as being associated with a significant change of plasma metabolite levels.Human plasma samples from 512 participants in the Community-Based Cohort Study executed by ToMMo were analyzed by using NMR spectroscopy. Sample characteristics in this study are summarized in We conducted correlation network analyses of plasma metabolites, and found that most of the quantified metabolites are correlated with one another . The corWe identified several metabolites correlated with time after eating in the questionnaire items . Ketone The concentration of plasma glucose quantified by NMR is highly correlated with the blood test value of plasma glucose, indicating the high precision of metabolite quantification by NMR . These rWe performed association studies of the quantified plasma metabolites and SNPs data derived from the whole genome analysis of 512 cohort participants. We applied 512 high-resolution whole genome sequencesASPG, also known as 60-kDa lysophospholipase), which product asparaginase (ASPG) catalyzes the hydrolysis of L-asparagine to L-aspartate and ammoniaOne of the four SNPs associated with amino acids, rs8012505 is associated with asparagine, and this association has not been reported . This SNguinea pig ASPG shows that the Ser344 residue is located at the edge of the C-terminal region of the domain, away from the catalytic site  cases, the P-value of another neighboring SNP rs61997624  was slightly more significant than that of rs8012505. The SNP rs61997624 is located in an intergenic region downstream of PAH) gene and causes the non-synonymous variant R53H. PAH catalyzes hydroxylation of phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolismWe also identified an association between the SNP rs118092776 and phenylalanine . This SN4), a cofactor of PAH4, the PAH activity of R53H mutant increased from 63% to 139%, compared with wild type4 levels recovered the stability of the PAH dimer.Based on preceding structural analyses1113i.e., a high activity tetramer and a low activity tetramer, which are interconverted  and V379A (SNP rs746203167). Both participants are heterozygotes, and their plasma phenylalanine levels were significantly higher than the average level (84.2 μM for R413P and 79.9 μM for V379A: PRODH) (1-pyrroline-5-carboxylate (P5C). Deficiency of PRODH causes type I hyperprolinemia, manifesting high level of plasma proline, and is known to associate with schizophreniaWe also detected association of plasma proline levels with rs5747933, a non-synonymous variant resulting in the T275N (isoform 1: T116N for isoform 2) substitution in proline dehydrogenase gene (PRODH) . PRODH cOur results revealed that this T275N variant causes an increase in plasma proline concentration, suggesting that the variant decreases the PRODH activity. As shown in E. coli PRODH shows that this variant is located at the edge of the dehydrogenase domain, away from the catalytic site  gene and causes the non-synonymous variant T1406N. The minor allele variant of this SNP increases the plasma levels of glycine  causing the non-synonymous variant A222V  variant on MTHFR and demonstrated that this variant did not affect Km and kcat but resulted in a thermolabile enzyme that loses its FAD cofactor more readily than does the wild type. These results indicate that this variant does not affect the catalytic site directly, but destabilizes the enzyme. Although the biological relationship between MTHFR and formate is unclear, formate is one of the one-carbon sources in folate metabolism and a precursor for the formation of 10-formyl-THF, indicating that the reduced activity of MTHFR observed with the A222V variant facilitates the consumption of formate as a one-carbon source.This SNP is associated with many types of cancer and cardiovascular disease, and individuals with the homozygous variant have elevated plasma homocysteine levels2633tic site 23. GuentMYLK) gene. The substitution of G to A at rs820336 increased the plasma creatinine levels (MYLK)MYLK, has a slightly lower P-value compared with the case of rs820336, though whether rs33262 regulates MYLK or not remains to be elucidated.Finally, we investigated the association of metabolites with SNPs in non-coding regions, using whole genome sequencing data. We found that several SNPs in non-coding regions were probably associated with metabolites, although biological significance of these associations was not clear (data not shown). Hence, we selected the SNPs whose biological functions were already reported. Among these SNPs, we identified that one SNP rs820336 was associated with plasma creatinine levels, although not at a genome-wide significance level . This ase levels . This vaIt has been demonstrated in several cohort studies that the combination of NMR-based metabolite analysis with GWAS is a robust way to identify loci influencing blood metabolite levels3Of note, while these studies directly measured serum metabolite levels without extraction and identified not only low-molecular weight metabolites but also many types of lipids and/or lipoproteins, in this study we measured plasma metabolite levels with an extraction protocol and quantified only hydrophilic metabolites. Although the direct measurement approach has an advantage to examine serum levels of lipids and lipoproteins, it is difficult to measure low-molecular weight metabolites bound to proteins or relatively low concentration metabolites, because the signals from these metabolites are suppressed by the effect of NMR pulse sequence or are overlapped with residual large protein/lipid signals. The direct measurement approach and extraction approach give rise to substantial difference. For instance, we have quantified 19 of the 20 standard amino acids, whereas the direct measurements were able to analyze only 8 and 12 amino acids3The latter study has demonstrated that the combination of GWAS and exome sequence analyses is quite effective for identifying causal variants compared with GWAS alonei.e., MYLK rs820336 for creatinine), the effect of the variant appears not to be clear, perhaps because this association is detected only for female but the number of female samples used is not enough. We are planning to conduct a more detailed replication study after finishing our cohort data cleaning process.In order to confirm the results of our association study, we performed a replication study using the other 230 samples . We found that the five missense variants are clearly associated with respective metabolites (data not shown). In the case of the regulatory variant  whose whole genome sequences were already sequenced in our previous study (Whole-genome reference panel of Japanese (1KJPN) in Tohoku Medical Megabank organization)® 2D screw tube (Thermo Scientific). Metabolites were extracted using a standard methanol extraction procedure using 200 μL of plasma per sample. 4-times volume of 100% cold methanol (−30 °C) was added to plasma and the solution was mixed for 2 min, incubated for 10 min on ice, and centrifuged at 15,000 g for 10 min at 4 °C. The supernatant was transferred to a new tube and evaporated. Each dried sample was suspended in a 200 μL solution of 100 mM sodium phosphate buffer (pH 7.4) in 100% D2O containing 200 μM d6-DSS. 190 μL of solution was transferred to a 3 mm Bruker SampleJet NMR tube.Blood samples were collected from 512 cohort participants using vacutainer tubes containing EDTA-2Na . Blood was immediately stored at 4 °C and transported to the ToMMo BioBank laboratory. Plasma was prepared and stored at −80 °C using MATRIX1H,13C-HSQC experiments were collected for several samples. Spiking experiments were also performed to confirm the identifications.All NMR experiments were performed at 298 K on a Bruker Avance 600 MHz spectrometer equipped with a CryoProbe and a SampleJet sample changer. Standard 1D NOESY and CPMG (Carr-Purcell-Meiboom-Gill) spectra were obtained for each plasma sampleSpearman correlations between all pairs of metabolite concentrations were calculated by the R Hmisc package−9), female dataset (7.03 × 10−9), and both dataset (7.08 × 10−9).From the 27,490,104 pre-filtering SNVs of the 1KJPN reference panel with 1,070 Japanese individuals43How to cite this article: Koshiba, S. et al. The structural origin of metabolic quantitative diversity. Sci. Rep.6, 31463; doi: 10.1038/srep31463 (2016).


Transcript levels of goblet cell differentiation factors SPDEF and Math1 were increased in the distal colon of Ednrb−/− mice. Both distal colon from Ednrb mice and biopsies from HSCR patients showed reduced Muc4 expression as compared to controls, but similar expression of Muc2. Particle tracking studies showed that mucus from Ednrb−/− mice provided a more significant barrier to diffusion of 200 nm nanoparticles as compared to wild-type mice. These results suggest that aganglionosis is associated with increased goblet cell proliferation and differentiation and subsequent altered surface mucus properties, prior to the development of inflammation in the distal colon epithelium. Restoration of normal goblet cell function and mucus layer properties in the colonic epithelium may represent a therapeutic strategy for prevention of HAEC.Hirschsprung disease-associated enterocolitis (HAEC) leads to significant mortality and morbidity, but its pathogenesis remains unknown. Changes in the colonic epithelium related to goblet cells and the luminal mucus layer have been postulated to play a key role. Here we show that the colonic epithelium of both aganglionic and ganglionic segments are altered in patients and in mice with Hirschsprung disease (HSCR). Structurally, goblet cells were altered with increased goblet cell number and reduced intracellular mucins in the distal colon of biopsies from patients with HSCR. Endothelin receptor B (Ednrb) mutant mice showed increased goblet cell number and size and increased cell proliferation compared to wild-type mice in aganglionic segments, and reduced goblet cell size and number in ganglionic segments. Functionally, compared to littermates, Ednrb Hirschsprung disease (HSCR) is a congenital disorder of the intestinal tract that is characterized by variable lengths of colorectal aganglionosis resulting from the failure of neural crest-derived cells to form the distal enteric nervous system (ENS) A luminal mucus layer coats the surface of the epithelium throughout the colon and is thought to be an important barrier against bacterial and viral infection and an important factor for maintaining the integrity of the epithelium −/− mice die at age 3–5 weeks from either distal intestinal obstruction or HAEC-associated sepsis −/− mice exhibit early alterations in the normal colonic and fecal microbiome prior to the onset of significant colitis −/− mice and from biopsies obtained from HSCR patients without active inflammation, to investigate epithelial changes associated with aganglionosis. Our findings reveal early alterations in goblet cell differentiation and proliferation in aganglionic colon in HD patients and Ednrb−/− mice. We also show that colonic barrier function, fluid absorption and secretion is initially unimpaired in the aganglionic colon. We therefore suggest that the production of a functionally altered mucus layer in the setting of colonic aganglionosis, likely in conjunction with alterations in the colonic microbiome, may play a key role in the pathogenesis of HAEC.Endothelin receptor B (Ednrb) mutant mice are a well-described model of colorectal aganglionosis that exhibit many of the features of human HSCR including megacolon and development of HAEC −/− mice. In agreement with our initial observations in human HSCR, the distal colon of Ednrb−/− mice showed increased thickness of the mucosal layer compared to WT littermates. Analysis of crypt proliferation in Ednrb−/− mice was performed by PCNA staining of tissue samples. We found a significant expansion of the proliferative zone in the distal colon of Ednrb−/− mice at both P7 and P18 as compared to WT littermates  and P18   . In cont p<0.05) .Analysis of histology from human HSCR colon showed that goblet cell numbers were increased compared to controls , while o−/− mice at P18, prior to the development of enterocolitis. As shown in −/− mice. Interestingly, an opposite effect was seen in the ganglionic proximal colon, where a reduction in goblet cell number and size was observed. Histological grading of inflammation and expression of the inflammatory cytokine IL-6, measured by qPCR, was not different between WT and Ednrb−/− mice (data not shown), confirming that the Ednrb−/− colon at this early time point did not exhibit inflammation.In order to investigate the colonic epithelium more closely, distal and proximal sections of colon were analyzed from Ednrb−/− and WT mice at P18–24. Mouse models in general show variable degrees in the extent of aganglionosis along the length of the intestinal tract. Previous studies have shown that the distal 3 cm of colon in Ednrb−/− mice is aganglionic and we therefore used distal colonic tissue <3 cm from the rectum (1) and harvested our proximal colon tissue 1–4 cm distal to the cecum to ensure ganglionic colon tissue for comparison. In order to look at overall barrier function in the colon, transepithelial resistance (TER) measurements were performed. −/− distal colon (6.3 vs 5.1 µA/cm2).Given the changes seen in the structure of the epithelium, we examined the functional properties of the colonic mucosa. Electrophysiological measurements were performed on whole mount distal and proximal colon from Ednrb−/− and WT colon after stimulation with either carbachol (Ca2+) or forskolin (cAMP). −/− mice have significantly reduced stool water content compared to WT , SPDEF expression was the same. No difference was observed in transcript levels of the cystic fibrosis transmembrane conductance regulator (CFTR).As goblet cell parameters and overall proliferation appeared to be an early difference in Ednrb−/− and WT mice. In contrast, the membrane-bound mucin Muc4 was significantly decreased (3.1-fold) in mutant colon.Since mucins represent the major protein product of mature goblet cells, the expression of specific mucin subtypes was analysed. The two major categories of mucin proteins are secreted gel-forming mucins , of which Muc2 is the most important in the mouse colon, and the membrane-bound mucins . −/− mice, Muc2 staining was similar in colonic crypts between normal and HSCR tissue (not shown), whereas Muc4 staining appeared reduced in HSCR goblet and epithelial cells  technique. In MPT, microscopic motion of hundreds of individual particles is tracked and analyzed to reveal important information pertaining to particle-environment interactions −/− mucus relative to WT, as evidenced by constrained trajectory profiles (eff)  . The low−/− mice prior to the onset of inflammation or any phenotypic signs of enterocolitis. Ednrb−/− mice exhibit many of the features found in patients with HSCR, including defective colonic motility and development of enterocolitis The overall aim of this study was to investigate changes to the colonic epithelium in the setting of congenital aganglionosis that may suggest possible etiologies for HAEC. A number of theories have emerged to explain the pathogenesis of HAEC. As with other inflammatory bowel conditions such as Crohn’s disease and ulcerative colitis, these theories have primarily focused on defects in colonic barrier function, innate immunity and the microbial milieu. The occurrence of enterocolitis both in HSCR patients and in mouse models of colonic aganglionosis suggest that there are fundamental changes to colonic structure and function that occur either in conjunction with defective neural crest migration or that result from the absence of enteric neuronal or glial function in the mucosa and submucosa. We obtained biopsy tissue from HSCR patients with minimal or no inflammation to see if there were obvious structural changes associated with aganglionosis. A common feature of the human tissues was increased numbers of goblet cells. Although the mean ages of the HSCR and control patients were similar, given the range of ages and clinical conditions in the patient biopsies we wanted to investigate possible epithelial differences without confounding by developmental or condition-specific changes. We therefore undertook a detailed analysis of the colon from neonatal Ednrb−/− distal colon, suggesting that fluid absorptive function remained intact. It seems likely that the reduction in stool water content is due to increased resident time in the colon secondary to impaired motility in mutant mice, rather than increased fluid absorption per se. However, a previous report found increased sodium and chloride transport across the rectum of HSCR patients, suggesting that there may be enhanced salvage of fluid and electrolytes in HCSR −/− distal colon. The carbachol response indicates that the epithelium retains the appropriate muscarinic receptors (M2) required for cholinergic chloride secretion. Although there is no reported data on epithelial muscarinic receptors in HSCR, studies have shown reduced intestinal muscle expression of certain subtypes of muscarinic receptors in keeping with the finding of hypertrophied acetylcholinesterase positive fibers in HSCR −/− mouse colon agree with a previous study that showed an intact colonic carbachol mediated chloride secretion in HCSR patients −/− mouse model appears to be a useful model to study the details of colonic epithelial absorptive and secretory function in the absence of the ENS.The primary function of the mammalian distal colon is the salvage of water and electrolytes from the luminal intestinal contents and the dehydration and storage of feces. We found increased fecal dehydration (reduced stool water content) in Ednrb−/− colon.Changes in barrier function properties have been proposed as a possible intrinsic defect in HSCR colon. Barrier function is comprised of the luminal mucus layer as well as the epithelium itself. Transepithelial electrical resistance (TER) has been widely used as a proxy for epithelial barrier function and there are no published data on progression of TER in either mouse models or human HSCR. Surpisingly, TER was higher in the aganglionic mouse colon, suggesting that electrical integrity, which is largely a function of epithelial tight junction properties, was not only intact in the presence of aganglionosis, but was even enhanced. A number of possibilities could explain this result, including changes in tight junction protein composition, faster maturation of epithelial junctions in the neonatal mutant mouse and others. A useful next step will be a detailed study of the progression of epithelial resistance, including macromolecular paracellular permeability, in the Ednrb−/− distal colon was increased goblet cell numbers. Additionally, goblet cell maturation appeared to be altered as suggested by the loss of the membrane bound mucin Muc4. These changes were accompanied by increases in Math1 and SPDEF, important intestinal epithelial secretory cell differentiation factors, suggesting that aganglionosis is associated with changes to the normal developmental program that drives the generation and constant renewal of the colonic crypt epithelium. Goblet cell parameters were conversely lower in the proximal colon, with increased Math1 but similar SPDEF expression, although it is unclear how the balance of proliferation versus differentiation of goblet and epithelial cells relates to HAEC susceptibility. While we did not find Muc2 levels altered in the epithelium, in contrast to a previous study The main structural difference found in our study in both HSCR patient tissues and EdnrbThe mucus layer overlying the colonic epithelium has multiple functional roles, including providing an initial barrier to pathogens, altering selection pressures on commensal microbiota and lubrication of intestinal contents. The layer itself resembles a mesh of mucin glycoproteins, and anti-bacterial and signaling proteins within an aqueous environment. The alterations in goblet cell number and function found in mutant mice appear to produce a significantly different mucus layer environment, as shown by the altered diffusivity of 200 nm particles found in the particle tracking studies. These changes may be due to altered macro-scale visco-elastic properties and/or nano-scale changes in mucin interactions resulting in a reduced effective mesh pore size. Alternatively, the changes may reflect altered interaction between mucus proteins and the carboxylate surface groups of the particles. Our previous studies have shown significant alterations in the commensal milieu in aganglionic colon −/− mice. Further studies will aim to elucidate the mechanisms by which defective goblet cell function in conjunction with an altered colonic microbiome and/or innate immune signaling pathways result in HAEC. Restoration of normal goblet cell function or mucus layer properties in both innervated and non-innervated colonic epithelium may represent a therapeutic strategy for prevention of HAEC.In summary, these results suggest that aganglionosis is associated with changes to goblet cell differentiation and functional expression of mucin sub-types prior to significant inflammation in the distal colonic epithelium. Additionally, these changes result in a functionally altered mucus layer with significantly hindered movement of nanoparticles. Despite these changes, distal colonic function in terms of fluid absorption, secretion and epithelial barrier function, are largely preserved in EdnrbAll human tissue samples were obtained from the archives of the Department of Pathology at Massachusetts General Hospital with approval from the Massachusetts General Hospital Institutional Review Board (Partners Human Research committee). Protocol number: 2003-P-000460/20. The Institutional Review Board waived written donor consent. Colonic samples from patients with Hirschsprung’s disease (n = 13) were from the resected aganglionic segment at the time of their pull-through operation. Samples from age-matched controls (n = 4) were from rectal biopsies obtained during a work-up for constipation to rule-out Hirschsprung’s disease. The ages and extent of aganglionosis of the patients are summarized in Sections of 6 µm thickness were prepared from formalin-fixed, paraffin-embedded tissues. H&E, alcian blue, PAS, and Muc4 stains on human tissue sections were done using standard histology protocols using automated sectioning and staining equipment  Anti-Muc4 antibody was from Santa Cruz Biotech.tm1Ywa mice on a hybrid C57BL/6J-129Sv background (JAX#003295) were maintained on a 12-h light–dark cycle at 25°C and had unlimited access to water and standard rodent chow . Homozygous mutant mice are identified by their white coat color and the genotype confirmed by polymerase chain reaction. Mice were weaned at postnatal day 21 (P21). Controls consisted of colon tissue from homozygous WT animals. Mice were euthanized by CO2 asphyxiation.Experiments were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee . EdnrbThe colon was removed and fixed with 4% paraformaldehyde. Cryosections (10 µm thick) were collected on Probe-on poly-L-lysine-coated slides . For analysis of cell division, immunohistochemistry was performed using primary antibody against PCNA , biotinylated goat anti-mouse IgG secondary antibody , avidin-biotinylated peroxidise complex , and the Perodixase Substrate Kit . Slides were examined under a Nikon 80i microscope and photographed with a Spot camera.For goblet cell labelling, sections were incubated in 3% acetic acid (Fisher Scientific), followed by Alcian blue  for 30 minutes at room temperature. After rinsing, Nuclear Fast Red (Vector Labs) was added for 5 minutes at room temperature, and then the slides were rinsed in water followed by 95% and 100% alcohol.−/− mice: For PCNA staining, individual crypts were analysed from images (20x) obtained from stained sections. At least 3 crypts per image and 3 images per section were examined by a blinded assessor from n = 5 mice per genotype. Goblet cell parameters were analyzed using a square region of interest (ROI) of arbitary size (512×512 pixels). Numbers of goblet cells within the ROI and size of the ten largest goblet cells were recorded.EdnrbHCSR patient samples: Goblet cells per crypt were counted and normalized to total crypt length. At least 3 crypts per image and 3 images per section were examined from patient samples. Size of goblet cells was measured from all counted goblet cells and averaged. Analysis was done blinded to section origin and patient information. All images were analyzed using Image J image analysis software .2 asphyxiation and cervical dislocation. The colon was washed with ice-cold Krebs buffer, opened along the mesenteric border. Distal colonic tissue (<3 cm from the rectum) and proximal colon tissue  was mounted in a micro-Ussing chambers . Hemichambers were filled with oxygenated Krebs-bicarbonate solution and tissues were incubated for 30 mins prior to recording measurement. Short-circuit current was recorded using a DVC-1000 voltage-clamp (World Precision Instruments) with Ag/AgCl electrodes and 1 mol/L KCl agar bridges. Agonists/inhibitors  were added to hemichambers. Transepithelial resistance was calculated from measured current change after 5 mV voltage pulses.Mouse colon was harvested after COStools were collected from the distal colon of P17–P22 WT (n = 7) and mutant (n = 7) mice at the time of sacrifice. Stools were weighed then dehydrated overnight at 55°C and weighed again. The percentage of water content  =  [(wet weight-dry weight)/wet weight]*100.Gapdh. Samples were analyzed in triplicate from 3 different mouse colon specimens.Total RNA was extracted using TRIzol Reagent  and cDNA generated with ThermoScript (Invitrogen). The specificity of amplification products was verified by electrophoresis on an agarose gel. qPCR was performed on a CFX96 Real-Time PCR Detection System. The expression level of each gene was normalized to The following oligonucleotide primer pairs were used:Muc2: (F) CAAGTGATTGTGTTTCAGGCTC; (R) TGGAGATGTTCTTGGTGCAGMuc4: (F) GACAAAGCACCAATTCCATCC; (R) CCTTAGAGTTGCTGGTGATCTCSpdef: (F) ACGTTGGATGAGCACTCG; (R) CCATAAAAGCCACTTCTGCACMath1: (F) GCTTATCCCCTTCGTTGAACTG; (R) CTCTTTTACCTCAGCCCACTCCftr: (F) CCGATCAGTTCTCAGTAAGGC; (R) ATCGCTTCTATCCTGTGTTCACGapdh: (F) CTTTGTCAAGCTCATTTCCTGG; (R) TCTTGCTCAGTGTCCTTGC2, 65 mM NaCl, 40 mM NaOH and 3 mM NaN3 mimicking gastrointestinal tract contents to yield a final particle concentration of 0.0025% wt/vol.Particle suspensions were prepared using 200 nm carboxylate-modified yellow-green fluorescent microspheres . Microsphere solutions (2% solids in distilled water with 2 mM azide) were diluted in maleate buffer consisting of 100 mM Trizma, 10 mM CaCl−/− type mice ranging in age from P11–P20. The most proximal and most distal 1 cm of colon were removed and placed in a chamber on a glass slide formed by attached silicone isolators . Colon sections were cut open such that the mucosal surface was exposed, and 5 µl of diluted particle solutions carefully pipetted onto the mucus surface. Samples were covered in a humid chamber, protected from light, and left at room temperature for 90 min before microscopy.Intestinal tissue segments were collected from WT and Ednrbeff) were determined as previously described Particles were tracked using an Olympus DP70 digital color camera  mounted on an inverted Olympus IX51 microscope with attached X-Cite 120 fluorescence illumination system . 20 second particle diffusion videos were captured using Olympus DP imaging software, with n≥100 particles analyzed in each excised section  of the colon. Particle trajectories were generated using tracking algorithm of the ParticleTracker ImageJ plugin Experimental groups were analyzed by unpaired t-test for single comparisons and ANOVA with post-hoc Tukey-Kramer analysis for multiple comparisons. p-values are indicated in the text and figure legends.


Glucose-6-phosphate dehydrogenase (G6PD) deficiency exhibits considerable allelic heterogeneity which manifests with variable biochemical and clinical penetrance. It has long been thought that G6PD deficiency confers partial protection against severe malaria, however prior genetic association studies have disagreed with regard to the strength and specificity of a protective effect, which might reflect differences in the host genetic background, environmental influences, or in the specific clinical phenotypes considered.A case-control association study of severe malaria was conducted in The Gambia, a region in West Africa where there is considerable allelic heterogeneity underlying expression of G6PD deficiency trait, evaluating the three major nonsynonymous polymorphisms known to be associated with enzyme deficiency  in a cohort of 3836 controls and 2379 severe malaria cases.Each deficiency allele exhibited a similar trend toward protection against severe malaria overall (15–26 % reduced risk); however, in stratifying severe malaria to two of its constituent clinical subphenotypes, severe malarial anaemia (SMA) and cerebral malaria (CM), the three deficiency alleles exhibited trends of opposing effect, with risk conferred to SMA and protection with respect to CM. To assess the overall effect of G6PD deficiency trait, deficiency alleles found across all three loci were pooled. G6PD deficiency trait was found to be significantly associated with protection from severe malaria overall (OR 0.83 [0.75–0.92], In a part of Africa where multiple alleles contribute to expression of G6PD deficiency trait, these findings clarify and extend previous work done in populations where a single variant predominates, and taken together suggest a causal role for G6PD deficiency trait itself with respect to severe malaria, with opposing effects seen on two major clinical subphenotypes. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzyme disorder with a global distribution, affecting hundreds of millions of people worldwide . It is aGenetic epidemiological evidence examining this hypothesis has been lacking in clarity, however, with past case-control association studies differing with respect to the strength and specificity of a protective effect –18, and The most common G6PD deficiency allele in sub-Saharan Africa is the C202T (A-) variant, and studies of this mutation have driven most association studies in this region. However, while the C202T allele is predominant in east Africa , in WestEthics approval was granted by The Gambia Government/Medical Research Council Joint Ethics Committee and by the Oxford Tropical Research Ethics Committee, and written informed consent was obtained from a parent or guardian prior to enrolment.The case-control cohort studied is comprised of 3836 controls and 2379 severe malaria cases, recruited at the Royal Victoria Hospital in Banjul. Malaria cases were confirmed by thin blood smear, and designated as ‘severe’ following WHO criteria, with two partially overlapping clinical syndromes identified in the majority of cases: severe malarial anaemia and cerebral malaria. Severe malarial anaemia (SMA) is defined here as packed cell volume mentclass2pt{minimGenotyping was performed on genomic DNA that was whole genome amplified via either primer extension pre-amplification or multiple displacement amplification reaction. Single nucleotide polymorphisms (SNPs) were then assayed via multiplexed genotyping on the Sequenom MASSarray platform. Automated genotype calls were manually curated via visual examination of cluster plots and mass spectra, with ambiguous genotypes re-coded as missing.Data curation was performed using custom Perl scripts and the PLINK software package . Gender Association testing was conducted using custom scripts written in R . A logisComposite G6PD deficiency trait genotype was defined as the sum of deficiency alleles present at all deficiency loci (968G + 542A + 202T) for an individual. Thus, a male was coded as 0 or 2, based on the absence or presence of a single deficiency allele across the three loci, while a female with a total of one deficiency allele present was coded as 1, and two deficiency alleles as 2 . It should be noted that this composite genotype was never greater than two because this scenario would require two deficiency alleles to co-occur on the same chromosome. Full expression of the G6PD deficiency trait, i.e. complete enzyme deficiency, was a binary genotype (0 or 1) inferred according to the absence or presence of one  or two  deficiency alleles in total across the three loci. Thus, a 202T male hemizgygote, 968G/202T female compound heterozygote, and a 542AA female homozygote would all be scored as 1 for full G6PD deficiency trait expression.Estimated odds ratios (OR) were adjusted by including the following covariates: HbS (sickle locus) genotype, self-reported ethnicity, and sex (omitted in sex-stratified tests). All odds ratios are presented here as ‘OR [95 % CI]’.G6PD known to be associated with enzyme deficiency, including A968G, T542A, C680A, and C202T  and cerebral malaria (CM). Although these subphenotype tests were not equally powered to detect signals of association due to sample size differences  was compiled, where presence of the trait required either one  or two  deficiency alleles in total across the three loci. In these association tests Table ; Fig. 1,nsortium . ImportaAs G6PD deficiency is an X-linked trait, and the subject of gender-specificity has been conflicting in previous association studies of severe malaria –15, sex-In examining multiple G6PD deficiency alleles in The Gambia, it was found that such alleles were associated with protection from severe malaria overall, but exhibited differential effects with respect to two important clinical presentations of severe malaria, conferring protection from cerebral malaria (CM) and increasing risk for severe malarial anaemia (SMA). Taken together with prior work from populations where a single variant, C202T (A-), predominates, there is compelling evidence for a direct, causal role for G6PD deficiency trait in modification of clinical malaria subphenotypes, confirming past results demonstrating protection from CM , 22, as As this study was limited to just one country, with its own distinct genetic background and environmental exposure history, including its own unique pattern of exposure to malaria, it will be important that further investigation be conducted in as many study populations in as diverse a range of geographic areas as possible, and there is ongoing work toward this end being conducted by the MalariaGEN consortium (manuscript in preparation). As there is substantial variation in the biochemical penetrance of G6PD deficiency alleles, it will also be useful for future work to include enzyme activity assays when feasible.G6PD should distinguish between the different presentations of severe malaria.The present study provides the somewhat paradoxical result that a single gene can be associated with protection from and susceptibility to disease from the same infectious agent. This highlights the fact that severe malaria comprises distinct clinical presentations, and the extent to which these syndromes differ from one another. These findings are not unexpected, then, given that G6PD deficiency is known to predispose individuals to haemolytic anaemia in the presence of oxidant stresses from infection, drugs, and diet . As the The differential association patterns seen here also suggest possible mechanistic differences between sickle cell trait (HbS) and G6PD deficiency in the modulation of malaria clinical phenotypes.in vitro [Sickle cell homozygous and heterozygous states have been variously reported to affect invasion and growth in vitro , 40, or in vitro –43 and ein vitro , 44 settin vitro , impairein vitro  or reducin vitro  may alsoin vitro growth phenotypes [in vivo is mixed for both Plasmodium vivax [Plasmodium falciparum [Studies of parasite phenotypes inside the G6PD-deficient erythrocyte have been more equivocal. While some have found evidence of invasion preferences  or aberrenotypes , others enotypes , 51. Simum vivax , 21 and lciparum , 14, 20.lciparum , 53), orlciparum ), might in vitro experiments relevant to severe manifestations of malaria. Ultimately, if knowledge gained from investigations surrounding the malaria protection hypothesis is to inform clinical treatment, a better understanding of the molecular mechanisms as they relate to severe disease will be crucial.Importantly, differences in the protection afforded by HbS and G6PD deficiency highlight the need to study clinical biomarkers and conduct In a region of West Africa with significant functional allelic heterogeneity, these data confirm that G6PD deficiency state is protective against severe malaria, but in contrast to sickle cell trait, this protection is limited to the CM subphenotype, with possible increased risk conferred to SMA. These results support the malaria protection hypothesis, but also suggest important mechanistic differences between the malaria-protective mechanisms of G6PD deficiency versus sickle cell trait.


Airway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma. Since the equine bronchial fibroblasts (EBF) have not been isolated and characterized yet, the use of defined medium was investigated.Primary EBF were cultured on non-collagen coated flasks without serum or in the presence of fetal bovine serum (FBS) or horse serum (HS) or in serum depleted medium. EBF cultured in serum-free basal media and those serum deprived were not able to proliferate and even exhibited considerable cell death. In media containing FBS or HS, proliferation of the cells was reproducible between different primary cultures and cells demonstrated expression of vimentin. Large variations were found in the ability of FBS and HS to support growth and differentiation of EBF in monolayer culture. Indications of growth-promoting actions, increasing passage number as well as maintaining fibroblast morphology were found rather in FBS than in HS. EBF culturing in HS needed longer doubling and confluence time. The protein content of the cell pellets was higher in EBF cultured in medium containing HS than FBS. Alpha-smooth muscle actin seemed to be less expressed in EBF cultured in medium containing FBS than those in HS.In sum, serum addition to basal EBF medium enhanced EBF differentiation into myofibroblasts, and these findings are useful to develop in vitro fibroblast culture models that mimic in vivo physiological processes and to study airway disease mechanisms and remodeling. Chronic airway diseases like human bronchial asthma and chronic obstructive pulmonary disease (COPD) and the equine recurrent airway obstruction (RAO) principally characterized by bronchial hyperreactivity and airflow obstruction result from several factors including structural alterations of the airway wall and cell function. Such airway remodelling is a common feature of abnormal deposition of extracellular matrix (ECM) components in airway mesenchymal layer associated with airway wall thickness -3. AirwaThere are several well characterized human fetal lung fibroblast cell lines that are cultured in defined media and for use as in vitro cell model to study airway diseases and remodeling, for example, HFL-1 , IMR-90 in vitro, either primary cultures or continuous cell lines, appropriate culture conditions are required that mimic the physiological conditions in vivo and situ. In fact, it is well known that serum represents a fundamental source of nutrients, cytokines and adhesive molecules necessary for in vitro cell growth, metabolism and to stimulate proliferation -thymidine incorporation assay [3H]-thymidine (37 kBq/well)  was added to the culture medium. 24 hours after incubation of cells with [3H]-thymidine, cells were washed in ice-cold PBS and incorporated radioactivity was determined by liquid scintillation counting (Beckman LS 6500 Scintillation Counter). Also, in thawed and cultured EBF, proliferation experiments were performed about 1-week after thawing, to avoid a major increase in the number of cumulative population doublings compared to experiments done with fresh cells.Furthermore, cell proliferation was measured by [on assay . EBF  were subjected to SDS-PAGE; samples were separated on 12% acrylamide gel under reducing conditions and transferred to a nitrocellulose membrane . The membrane was then blocked in 3% BSA (PAA Laboratories) with TBST  for 1 h at room temperature. α-SMA was detected following an overnight incubation of samples with mouse monoclonal antibody against human anti-α-SMA  and β-actin was detected using mouse monoclonal antibody against human anti-β-actin  in 3% BSA-TBST at 4°C. Band visualisation was performed using alkaline phosphatase conjugated secondary anti-mouse igG antibody  over 1 h at room temperature. Enzyme activity was detected using Western Blue stabilised substrate for alkaline phosphate (Promega). The membranes were digitalized and quantitated by densitometry analysis .Primary EBF grown in 6-well plates under different culture conditions were harvested and suspended in sample buffer  and boiled for 5 min. Equal amounts of whole cell lysate . P < 0.05 was considered significant.All data are expressed as means ± SEM. Statistical significance of differences was evaluated by paired two-tailed student’s All authors declare that no Competing interests exist.JF, VA, CZ and GA designed the research and wrote the paper. JF and VA performed the experiment and analyzed the data. JF analyzed the data and wrote the paper. GA analyzed the data, wrote and approved the paper. All authors read and approved the final manuscript.