Slave Trajanoski

Alteration of intestinal dysbiosis by fecal microbiota transplantation does not induce remission in patients with chronic active ulcerative colitis.

Background: In patients with ulcerative colitis (UC), alterations of the intestinal microbiota, termed dysbiosis, have been postulated to contribute to intestinal inflammation. Fecal microbiota transplantation (FMT) has been used as effective therapy for recurrent Clostridium difficile colitis also caused by dysbiosis. The aims of the present study were to investigate if patients with UC benefit from FMT and if dysbiosis can be reversed. Methods: Six patients with chronic active UC nonresponsive to standard medical therapy were treated with FMT by colonoscopic administration. Changes in the colonic microbiota were assessed by 16S rDNA–based microbial community profiling using high-throughput pyrosequencing from mucosal and stool samples. Results: All patients experienced short-term clinical improvement within the first 2 weeks after FMT. However, none of the patients achieved clinical remission. Microbiota profiling showed differences in the modification of the intestinal microbiota between individual patients after FMT. In 3 patients, the colonic microbiota changed toward the donor microbiota; however, this did not correlate with clinical response. On phylum level, there was a significant reduction of Proteobacteria and an increase in Bacteroidetes after FMT. Conclusions: FMT by a single colonoscopic donor stool application is not effective in inducing remission in chronic active therapy–refractory UC. Changes in the composition of the intestinal microbiota were significant and resulted in a partial improvement of UC-associated dysbiosis. The results suggest that dysbiosis in UC is at least in part a secondary phenomenon induced by inflammation and diarrhea rather than being causative for inflammation in this disease.

Targeted High-Throughput Sequencing Identifies Mutations in atlastin-1 as a Cause of Hereditary Sensory Neuropathy Type I

Hereditary sensory neuropathy type I (HSN I) is an axonal form of autosomal-dominant hereditary motor and sensory neuropathy distinguished by prominent sensory loss that leads to painless injuries. Unrecognized, these can result in delayed wound healing and osteomyelitis, necessitating distal amputations. To elucidate the genetic basis of an HSN I subtype in a family in which mutations in the few known HSN I genes had been excluded, we employed massive parallel exon sequencing of the 14.3 Mb disease interval on chromosome 14q. We detected a missense mutation (c.1065C>A, p.Asn355Lys) in atlastin-1 (ATL1), a gene that is known to be mutated in early-onset hereditary spastic paraplegia SPG3A and that encodes the large dynamin-related GTPase atlastin-1. The mutant protein exhibited reduced GTPase activity and prominently disrupted ER network morphology when expressed in COS7 cells, strongly supporting pathogenicity. An expanded screen in 115 additional HSN I patients identified two further dominant ATL1 mutations (c.196G>C [p.Glu66Gln] and c.976 delG [p.Val326TrpfsX8]). This study highlights an unexpected major role for atlastin-1 in the function of sensory neurons and identifies HSN I and SPG3A as allelic disorders.

Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation

In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype, and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription, and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord, and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a BM cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13, and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs), as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age, with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.

Deep sequencing reveals highly complex dynamics of human cytomegalovirus genotypes in transplant patients over time.

ABSTRACT In lung transplant patients undergoing immunosuppression, more than one human cytomegalovirus (HCMV) genotype may emerge during follow-up, and this could be critical for the outcome of HCMV infection. Up to now, many cases of infection with multiple HCMV genotypes were probably overlooked due to the limitations of the current genotyping approaches. We have now analyzed mixed-genotype infections in 17 clinical samples from 9 lung transplant patients using the highly sensitive ultradeep-pyrosequencing (UDPS) technology. UDPS genotyping was performed at three variable HCMV genes, coding for glycoprotein N (gN), glycoprotein O (gO), and UL139. Simultaneous analysis of a mean of 10,430 sequence reads per amplicon allowed the relative amounts of distinct genotypes in the samples to be determined down to 0.1% to 1% abundance. Complex mixtures of up to six different HCMV genotypes per sample were observed. In all samples, no more than two major genotypes accounted for at least 88% of the HCMV DNA load, and these were often accompanied by up to four low-abundance genotypes at frequencies of 0.1% to 8.6%. No evidence for the emergence of new genotypes or sequence changes over time was observed. However, analysis of different samples withdrawn from the same patients at different time points revealed that the relative levels of replication of the individual HCMV genotypes changed within a mixed-genotype population upon reemergence of the virus. Our data show for the first time that, similar to what has been hypothesized for the murine model, HCMV reactivation in humans seems to occur stochastically.

The lymphocyte/monocyte ratio predicts poor clinical outcome and improves the predictive accuracy in patients with soft tissue sarcomas

Increasing evidence indicates the involvement of inflammation and coagulation in cancer progression and metastases. Inflammatory biomarkers hold great promise for improving the predictive ability of existing prognostic tools in cancer patients. In the present study, we investigated several inflammatory indices with regard to their prognostic relevance for predicting clinical outcome in soft tissue sarcoma (STS) patients. Three hundred and forty STS patients were divided into a training set (n = 170) and a validation set (n = 170). Besides well‐established clinico‐pathological prognostic factors, we evaluated the prognostic value of the neutrophil/lymphocyte (N/L) ratio, the lymphocyte/monocyte (L/M) ratio and the platelet/lymphocyte (P/L) ratio using Kaplan–Meier curves and univariate as well as multivariate Cox regression models. Additionally, we developed a nomogram by supplementing the L/M ratio to the well‐established Kattan nomogram and evaluated the predictive accuracy of this novel nomogram by applying calibration and Harrells concordance index (c‐index). In multivariate analysis, a low L/M ratio was significantly associated with decreased CSS and DFS (HR = 0.41, 95% CI = 0.18–0.97, p = 0.043; HR = 0.39, 95% CI = 0.16–0.91, p = 0.031, respectively) in the training set. Using the validation set for confirmation, we found also in multivariate analysis an independent value for CSS (HR = 0.33, 95% CI = 0.12–0.90, p = 0.03) and for DFS (HR = 0.36, 95% CI = 0.16–0.79, p = 0.01). The estimated c‐index was 0.74 using the original Kattan nomogram and 0.78 when the L/M ratio was added. Our study reports for the first time that the pre‐operative L/M ratio represents a novel independent prognostic factor for prediction the clinical outcome in STS patients. This easily determinable biomarker might be helpful in improved individual risk assessment.

Alterations in the colonic microbiota in response to osmotic diarrhea.

Background & Aims Diseases of the human gastrointestinal (GI) tract are often accompanied by diarrhea with profound alterations in the GI microbiota termed dysbiosis. Whether dysbiosis is due to the disease itself or to the accompanying diarrhea remains elusive. With this study we characterized the net effects of osmotic diarrhea on the composition of the GI microbiota in the absence of disease. Methods We induced osmotic diarrhea in four healthy adults by oral administration of polyethylene glycol 4000 (PEG). Stool as well as mucosa specimens were collected before, during and after diarrhea and 16S rDNA-based microbial community profiling was used to assess the microbial community structure. Results Stool and mucosal microbiotas were strikingly different, with Firmicutes dominating the mucosa and Bacteroidetes the stools. Osmotic diarrhea decreased phylotype richness and showed a strong tendency to equalize the otherwise individualized microbiotas on the mucosa. Moreover, diarrhea led to significant relative shifts in the phyla Bacteroidetes and Firmicutes and to a relative increase in the abundance of Proteobacteria on the mucosa, a phenomenon also noted in several inflammatory and diarrheal GI diseases. Conclusions Changes in microbial community structure induced by osmotic diarrhea are profound and show similarities to changes observed in other GI diseases including IBD. These effects so must be considered when specimens from diarrheal diseases (i.e. obtained by stratification of samples according to diarrheal status) or conditions wherein bowel preparations like PEG (i.e. specimens obtained during endoscopy) are used.

Exome Sequencing Identifies a REEP1 Mutation Involved in Distal Hereditary Motor Neuropathy Type V

The distal hereditary motor neuropathies (dHMNs) are a heterogeneous group of neurodegenerative disorders affecting the lower motoneuron. In a family with both autosomal-dominant dHMN and dHMN type V (dHMN/dHMN-V) present in three generations, we excluded mutations in all genes known to be associated with a dHMN phenotype through Sanger sequencing and defined three potential loci through linkage analysis. Whole-exome sequencing of two affected individuals revealed a single candidate variant within the linking regions, i.e., a splice-site alteration in REEP1 (c.304-2A>G). A minigene assay confirmed complete loss of splice-acceptor functionality and skipping of the in-frame exon 5. The resulting mRNA is predicted to be expressed at normal levels and to encode an internally shortened protein (p.102_139del). Loss-of-function REEP1 mutations have previously been identified in dominant hereditary spastic paraplegia (HSP), a disease associated with upper-motoneuron pathology. Consistent with our clinical-genetic data, we show that REEP1 is strongly expressed in the lower motoneurons as well. Upon exogeneous overexpression in cell lines we observe a subcellular localization defect for p.102_139del that differs from that observed for the known HSP-associated missense mutation c.59C>A (p.Ala20Glu). Moreover, we show that p.102_139del, but not p.Ala20Glu, recruits atlastin-1, i.e., one of the REEP1 binding partners, to the altered sites of localization. These data corroborate the loss-of-function nature of REEP1 mutations in HSP and suggest that a different mechanism applies in REEP1-associated dHMN.

The impact of PCR-generated recombination on diversity estimation of mixed viral populations by deep sequencing

Ultra-deep pyrosequencing (UDPS) of targeted amplicons allows to determine a large number of individual sequence reads from a single PCR product, and this approach is thus extremely valuable for analysis of mixed viral populations. A mixture of genetically distinct DNA templates, however, may lead to the generation of recombinant sequences during the initial PCR amplification step. In the present study, the frequency at which these misleading PCR artefacts are formed has been estimated by using artificial mixtures of genetically distinct human cytomegalovirus (HCMV) DNA templates for a given cycling profile. The presence of similar copy numbers of non-identical HCMV target sequences, combined with high levels of input HCMV DNA, as is found in some clinical samples, favored the formation of recombinant PCR products. Thus, to estimate the full natural diversity within mixed viral populations using UDPS, artificial chimeras generated during the PCR step should be taken into account as a potential artefact.

Fibulin-5 mutations link inherited neuropathies, age-related macular degeneration and hyperelastic skin

To identify the disease-causing gene responsible for an autosomal dominantly inherited Charcot-Marie-Tooth neuropathy subtype in a family excluded for mutations in the common Charcot-Marie-Tooth genes, we used array-based sequence capture to simultaneously analyse the disease-linked protein coding exome at chromosome 14q32. A missense mutation in fibulin-5, encoding a widely expressed constituent of the extracellular matrix that has an essential role in elastic fibre assembly and has been shown to cause cutis laxa, was detected as the only novel non-synonymous sequence variant within the disease interval. Screening of 112 index probands with unclassified Charcot-Marie-Tooth neuropathies detected two further fibulin-5 missense mutations in two families with Charcot-Marie-Tooth disease and hyperextensible skin. Since fibulin-5 mutations have been described in patients with age-related macular degeneration, an additional 300 probands with exudative age-related macular degeneration were included in this study. Two further fibulin-5 missense mutations were identified in six patients. A mild to severe peripheral neuropathy was detected in the majority of patients with age-related macular degeneration carrying mutations in fibulin-5. This study identifies fibulin-5 as a gene involved in Charcot-Marie-Tooth neuropathies and reveals heterozygous fibulin-5 mutations in 2% of our patients with age-related macular degeneration. Furthermore, it adumbrates a new syndrome by linking concurrent pathologic alterations affecting peripheral nerves, eyes and skin to mutations in the fibulin-5 gene.

Whole-exome sequencing in patients with inherited neuropathies: outcome and challenges

Abstract Inherited peripheral neuropathies (IPN) are one of the most frequent inherited causes of neurological disability characterized by considerable phenotypic and genetic heterogeneity. Based on clinical and electrophysiological properties, they can be subdivided into three main groups: HMSN, dHMN, and HSN. At present, more than 50 IPN genes have been identified. Still, many patients and families with IPN have not yet received a molecular genetic diagnosis because clinical genetic testing usually only covers a subset of IPN genes. Moreover, a considerable proportion of IPN genes has to be identified. Here we present results of WES in 27 IPN patients excluded for mutations in many known IPN genes. Eight of the patients received a definite diagnosis. While six of these patients carried bona fide pathogenic mutations in known IPN genes, two patients had mutations in genes known to be involved in other types of neuromuscular disorders. A further group of eight patients carried sequence variations in IPN genes that could not unequivocally be classified as pathogenic. In addition, combining data of WES and linkage analysis identified SH3BP4, ITPR3, and KLHL13 as novel IPN candidate genes. Moreover, there was evidence that particular mutations in PEX12, a gene known to cause Zellweger syndrome, could also lead to an IPN phenotype. We show that WES is a useful tool for diagnosing IPN and we suggest an expanded phenotypic spectrum of some genes involved in other neuromuscular and neurodegenerative disorders. Nevertheless, interpretation of variants in known and potential novel disease genes has remained challenging.