Ferdinando Bonfiglio

Dynamics of the human gut microbiome in inflammatory bowel disease

Inflammatory bowel disease (IBD) is characterized by flares of inflammation with a periodic need for increased medication and sometimes even surgery. The aetiology of IBD is partly attributed to a deregulated immune response to gut microbiome dysbiosis. Cross-sectional studies have revealed microbial signatures for different IBD subtypes, including ulcerative colitis, colonic Crohns disease and ileal Crohns disease. Although IBD is dynamic, microbiome studies have primarily focused on single time points or a few individuals. Here, we dissect the long-term dynamic behaviour of the gut microbiome in IBD and differentiate this from normal variation. Microbiomes of IBD subjects fluctuate more than those of healthy individuals, based on deviation from a newly defined healthy plane (HP). Ileal Crohns disease subjects deviated most from the HP, especially subjects with surgical resection. Intriguingly, the microbiomes of some IBD subjects periodically visited the HP then deviated away from it. Inflammation was not directly correlated with distance to the healthy plane, but there was some correlation between observed dramatic fluctuations in the gut microbiome and intensified medication due to a flare of the disease. These results will help guide therapies that will redirect the gut microbiome towards a healthy state and maintain remission in IBD.

Chronic Pro-oxidative State and Mitochondrial Dysfunctions are more Pronounced in Fibroblasts from Down Syndrome Foeti with Congenital Heart Defects

Trisomy of chromosome 21 is associated to congenital heart defects in ∼50% of affected newborns. Transcriptome analysis of hearts from trisomic human foeti demonstrated that genes involved in mitochondrial function are globally downregulated with respect to controls, suggesting an impairment of mitochondrial function. We investigated here the properties of mitochondria in fibroblasts from trisomic foeti with and without cardiac defects. Together with the upregulation of Hsa21 genes and the downregulation of nuclear encoded mitochondrial genes, an abnormal mitochondrial cristae morphology was observed in trisomic samples. Furthermore, impairment of mitochondrial respiratory activity, specific inhibition of complex I, enhanced reactive oxygen species production and increased levels of intra-mitochondrial calcium were demonstrated. Seemingly, mitochondrial dysfunction was more severe in fibroblasts from cardiopathic trisomic foeti that presented a more pronounced pro-oxidative state. The data suggest that an altered bioenergetic background in trisomy 21 foeti might be among the factors responsible for a more severe phenotype. Since the mitochondrial functional alterations might be rescued following pharmacological treatments, these results are of interest in the light of potential therapeutic interventions.

Functional variants in the sucrase-isomaltase gene associate with increased risk of irritable bowel syndrome

Objective IBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase–isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase–isomaltase (SI) gene variants for their potential relevance in IBS. Design We sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population. Results CSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case–control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05). Conclusions SI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients.

Stool frequency is associated with gut microbiota composition

We read with great interest the two recent communications by Vandeputte et al 1 and Tigchelaar et al ,2 which reported an association between stool consistency and gut microbiota composition. Both studies focused on stool consistency, based on the Bristol Stool Scale (BSS) as the main trait, due to its reported correlation with colonic transit time (CTT) that is of clinical relevance for several GI conditions. However, stool frequency, another feature of human bowel function that is easy to measure and record, has also been shown to correlate with CTT,3 ,4 although to a lesser extent than stool form, and has not yet been investigated in relation to microbiota composition. We examined the association between gut microbiota and stool frequency in the Population-based Colonoscopy study, a general population-based cohort from Stockholm, Sweden, previously described in detail.5 Sixty-nine individuals (21 males and 48 females, aged 55.6±10.33) with available frozen faecal samples and daily recordings of defaecation …

Dense genotyping of immune-related loci identifies HLA variants associated with increased risk of collagenous colitis

Objective Collagenous colitis (CC) is a major cause of chronic non-bloody diarrhoea, particularly in the elderly female population. The aetiology of CC is unknown, and still poor is the understanding of its pathogenesis. This possibly involves dysregulated inflammation and immune-mediated reactions in genetically predisposed individuals, but the contribution of genetic factors to CC is underinvestigated. We systematically tested immune-related genes known to impact the risk of several autoimmune diseases for their potential CC-predisposing role. Design Three independent cohorts of histologically confirmed CC cases (N=314) and controls (N=4299) from Sweden and Germany were included in a 2-step association analysis. Immunochip and targeted single nucleotide polymorphism (SNP) genotype data were produced, respectively, for discovery and replication purposes. Classical human leucocyte antigen (HLA) variants at 2-digit and 4-digit resolution were obtained via imputation from single marker genotypes. SNPs and HLA variants passing quality control filters were tested for association with CC with logistic regression adjusting for age, sex and country of origin. Results Forty-two markers gave rise to genome-wide significant association signals, all contained within the HLA region on chromosome 6 (best p=4.2×10−10 for SNP rs4143332). Among the HLA variants, most pronounced risk effects were observed for 8.1 haplotype alleles including DQ2.5, which was targeted and confirmed in the replication data set (p=2.3×10−11; OR=2.06; 95% CI (1.67 to 2.55) in the combined analysis). Conclusions HLA genotype associates with CC, thus implicating HLA-related immune mechanisms in its pathogenesis.

Metformin restores the mitochondrial network and reverses mitochondrial dysfunction in Down syndrome cells.

Alterations in mitochondrial activity and morphology have been demonstrated in human cells and tissues from individuals with Down syndrome (DS), as well as in DS mouse models. An impaired activity of the transcriptional coactivator PGC-1α/PPARGC1A due to the overexpression of chromosome 21 genes, such as NRIP1/RIP140, has emerged as an underlying cause of mitochondrial dysfunction in DS. We tested the hypothesis that the activation of the PGC-1α pathway might indeed reverse this mitochondrial dysfunction. To this end, we investigated the effects of metformin, a PGC-1α-activating drug, on mitochondrial morphology and function in DS foetal fibroblasts. Metformin induced both the expression of PGC-1α and an augmentation of its activity, as demonstrated by the increased expression of target genes, strongly promoting mitochondrial biogenesis. Furthermore, metformin enhanced oxygen consumption, ATP production, and overall mitochondrial activity. Most interestingly, this treatment reversed the fragmentation of mitochondria observed in DS and induced the formation of a mitochondrial network with a branched and elongated tubular morphology. Concomitantly, cristae remodelling occurred and the alterations observed by electron microscopy were significantly reduced. We finally demonstrated that the expression of genes of the fission/fusion machinery, namely OPA1 and MFN2, was reduced in trisomic cells and increased by metformin treatment. These results indicate that metformin promotes the formation of a mitochondrial network and corrects the mitochondrial dysfunction in DS cells. We speculate that alterations in the mitochondrial dynamics can be relevant in the pathogenesis of DS and that metformin can efficiently counteract these alterations, thus exerting protective effects against DS-associated pathologies.

TRPM8 polymorphisms associated with increased risk of IBS-C and IBS-M

Recently in Gut , genetic variation affecting ion channels activity has been highlighted in relation to bowel function and the biology of stool frequency.1 It is also known that 2% of patients with IBS carry functional missense mutations in the voltage-gated channel NaV1.5 ( SCN5A gene).2 Hence, channelopathies represent potential abnormalities underlying GI dysfunction and IBS. We inspected data from our previous genome-wide association study (GWAS) of IBS,3 in relation to 27 genes whose ion channel products contribute to GI sensorimotor development and function, visceral sensation and GI motility (see online supplementary table S1). Significant (uncorrected) results were detected for four genes (calcium voltage-gated channels CACNA1A and CACNA1E , and transient receptor potential channels TRPV3 and TRPM8 ; see online supplementary figure S1), which were selected for replication analyses in an independent set of IBS cases (N=386) and controls (N=357) (see online supplementary material methods). A sex-adjusted logistic regression analysis of genotype data from this cohort (see online supplementary material methods) detected significant associations for …

LACC1 polymorphisms in inflammatory bowel disease and juvenile idiopathic arthritis

The function of the Laccase domain-containing 1 (LACC1) gene is unknown, but genetic variation at this locus has been reported to consistently affect the risk of Crohns disease (CD) and leprosy. Recently, a LACC1 missense mutation was found in patients suffering from monogenic forms of CD, but also systemic juvenile idiopathic arthritis. We tested the hypothesis that LACC1 single nucleotide polymorphisms (SNPs), in addition to CD, are associated with juvenile idiopathic arthritis (JIA, non-systemic), and another major form of inflammatory bowel disease, ulcerative colitis (UC). We selected 11 LACC1 tagging SNPs, and tested their effect on disease risk in 3855 Swedish individuals from three case–control cohorts of CD, UC and JIA. We detected false discovery rate corrected significant associations with individual markers in all three cohorts, thereby expanding previous results for CD also to UC and JIA. LACC1’s link to several inflammatory diseases suggests a key role in the human immune system and justifies further characterization of its function(s).

A meta-analysis of reflux genome-wide association studies in 6750 Northern Europeans from the general population.

Gastroesophageal reflux disease (GERD), the regurgitation of gastric acids often accompanied by heartburn, affects up to 20% of the general population. Genetic predisposition is suspected from twin and family studies but gene‐hunting efforts have so far been scarce and no conclusive genome‐wide study has been reported. We exploited data available from general population samples, and studied self‐reported reflux symptoms in relation to genome‐wide single nucleotide polymorphism (SNP) genotypes.

Overexpression of Chromosome 21 miRNAs May Affect Mitochondrial Function in the Hearts of Down Syndrome Fetuses

Dosage-dependent upregulation of most of chromosome 21 (Hsa21) genes has been demonstrated in heart tissues of fetuses with Down syndrome (DS). Also miRNAs might play important roles in the cardiac phenotype as they are highly expressed in the heart and regulate cardiac development. Five Hsa21 miRNAs have been well studied in the past: miR-99a-5p, miR-125b-2-5p, let-7c-5p, miR-155-5p, and miR-802-5p but few information is available about their expression in trisomic tissues. In this study, we evaluated the expression of these miRNAs in heart tissues from DS fetuses, showing that miR-99a-5p, miR-155-5p, and let-7c-5p were overexpressed in trisomic hearts. To investigate their role, predicted targets were obtained from different databases and cross-validated using the gene expression profiling dataset we previously generated for fetal hearts. Eighty-five targets of let-7c-5p, 33 of miR-155-5p, and 10 of miR-99a-5p were expressed in fetal heart and downregulated in trisomic hearts. As nuclear encoded mitochondrial genes were found downregulated in trisomic hearts and mitochondrial dysfunction is a hallmark of DS phenotypes, we put special attention to let-7c-5p and miR-155-5p targets downregulated in DS fetal hearts and involved in mitochondrial function. The let-7c-5p predicted target SLC25A4/ANT1 was identified as a possible candidate for both mitochondrial and cardiac anomalies.