A Genotypic-oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macro-categories of Cystic Fibrosis.

Cystic Fibrosis (CF) is a monogenic disease caused by mutations of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The genotype-phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, 125 different mutated alleles (11 of which with novel mutations and 10 of which complex) and 225 genotypes were found. A strong correlation between mutational patterns at the genotypic level and phenotypic macro-categories emerged. This specificity appears to be largely dependent on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macro-categories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype - phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype - phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway

Cystic fibrosis transmembrane conductance regulator (CFTR) allelic variants relate to shifts in faecal microbiota of cystic fibrosis patients.

IntroductionIn this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age.MethodsThirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure.ResultsPatients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced.ConclusionsThis is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a 'systemic disease', linking the lung and the gut in a joined axis

Il pattern mutazionale del gene CFTR in diverse forme di fibrosi cistica influenza le caratteristiche operative del test genetico

La Fibrosi Cistica ( FC; OMIM 219700) può originare da oltre 1800 diverse variazioni di sequenza del gene CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Le manifestazioni cliniche sono estremamente variabili, con una relazione tra genotipo e fenotipo spesso poco chiara. L a ricerca mutazionale non è inoltre sempre in grado di rilevare tutte le mutazioni presenti. L'obiettivo specifico di questo lavoro è valutare se le caratteristiche operative del test genetico per FC siano influenzate dalla metodologia di indagine mutazionale eventualmente applicata a diverse forme cliniche di FC. Abbiamo studiato 610 pazienti classificati in 4 popolazioni: 1) FC con insufficienza pancreatica (FC-PI, 354 pazienti); 2) FC con sufficienza pancreatica (FC-PS, 138 pazienti); 3) forme atipiche e/o mono- od oligo-sintomatiche di FC (nel complesso identificate come CFTR-RD, 71 pazienti); 4) assenza bilaterale congenita dei vasi deferenti, quale unica manifestazione clinica monosintomatica (CBAVD, 47 pazienti). La ricerca mutazionale nel CFTR è stata condotta con un approccio multistep volto all'analisi: a) delle 32 mutazioni più frequenti al mondo (saggio CF-OLA, Abbott); b) delle 14 mutazioni più frequenti nell'area geografica specifica (mediante un nostro saggio di primer extension); c) del tratto variante (TG)mTn (mediante un nostro saggio di sequenziamento); d) della regione 5'-prossimale, di tutti gli esoni e delle zone introniche adiacenti (mediante un nostro saggio di sequenziamento); e) delle 7 macro-delezioni più frequenti al mondo (saggio FC-DEL, Nuclear Laser Medicine). Abbiamo evidenziato 125 diversi alleli mutati (tra i quali 11 nuovi e 10 complessi con più di una mutazione in cis), suddivisi in 225 diversi genotipi. Sono state riscontrate differenze significative (chi-quadrato, p<0.0001) tra i pattem mutazionali delle 4 popolazioni analizzate. Questa eterogeneità influenza la detection rate (DR) sia allelica (DRa = proporzione di alleli mutati identificati) che genotipica (DRe = proporzione di genotipi completamente caratterizzati con 2 alleli mutati) dei singoli step della ricerca mutazionale. Ad esempio, gli step di ricerca di pannelli mutazionali (a+b) risultano avere, da soli, un'elevata DRa in FC-PI (0.890), un valore intermedio in FC-PS (0.725), ma valori piuttosto bassi di DRa in CFTR-RD (0.535) e CBAVD (0.255). Aggiungendo gli altri step di ricerca mutazionale (c+d+e) si ottiene un notevole incremento nella DRa in FC-PI (0.993), FC-PS (0.967) e CFTR-RD (0.965), ma un valore di DRa totale comunque limitato in CBAVD (0.564). Non risulta possibile individuare un unico pannello mutazionale che massimizzi la DRa in tutte le popolazioni. Inoltre, solo per la popolazione CF-PI appare possibile individuare un pannello con un numero limitato di mutazioni ed elevata DR. Per ottenere un'elevata DR nelle altre popolazioni è necessaria l'indagine di un elevato numero di mutazioni, anche rare e/o individuali. Oltre a ciò, 19 diversi genotipi mutati sono stati trovati in almeno 2 diverse popolazioni, evidenziando la complessità del rapporto tra genotipo e fenotipo. Questi risultati hanno importanti ricadute sull'organizzazione e interpretazione del test genetico in FC, in particolare per il test del portatore, nonché per la comprensione della relazione tra genotipo e fenotipo

OPLS-DA analysis of TTGE profiles, according to disease severity.

<p>Panel A) On the left are reported representative TTGE profiles from severe and mild CF patients. Simca-P+ software was used to compute weight coefficients for each TTGE band (87 <i>x</i> variables) on the two subgroups of CF patients (2 <i>y</i> variables) harbouring at least one F508del mutation in one allele: severe (S) and mild (M), with a scaled a centred data set. A clustered image heatmap was generated with CIMminer online software (panel A, right). As depicted in the color-coded legend, the higher that the coefficient value is, the higher the weight (red), while the lower that the value is, the lower the weight (turquoise). Panel B) Faecal TTGE profiles of all 24 CF patients harbouring at least one F508del allele were analysed by OPLS-DA, and the resulting 3D score plot model gave a significant separation among the two sub-groups: severe (S, black diamonds) and mild (M, red diamonds).</p

Ecological parameters.

a<p>Hm, homozygous F508del; Ht, heterozygous F508del; N, non-F508del. The lowest <i>P</i> value was reported for all comparisons (within round brackets is reported the corresponding match). In bold are the significant <i>P</i> values.</p

PLS-DA analysis of TTGE profiles, according to F508del mutation.

<p>Panel A) On the left are reported representative TTGE profiles from homozygous-F508del (Hm), heterozygous-F508del (Ht) and non-F508del (N) patients. Simca-P+ software was used to compute weight coefficients for each TTGE band (87 <i>x</i> variables) on the three different sub-groups of CF patients (3 <i>y</i> variables, Hm-Ht-N), with a scaled and centred data set. These coefficients were useful to interpret the influence of the x variables on the y ones. A clustered image heatmap was generated with CIMminer online software (panel A, right). As depicted in the color-coded legend, the higher that the coefficient value is, the higher the weight (red), while the lower that the value is, the lower the weight (turquoise). Panel B) Faecal TTGE profiles of all 36 CF patients were analysed by PLS-DA, and the resulting 3D score plot model gave a significant separation between the three sub-groups: homozygous-F508del (Hm, red circles); heterozygous-F508del (Ht, black circles), and non-F508del (N, blue circles).</p

Species-specific PCR, according to F508del mutation.

<p>Species-specific PCR were performed on 16 bacterial species or groups, and a Mann-Whitney U test was employed to assess putative differences in their relative abundances (expressed as ng/µL) among homozygous-F508del (Hm, black bars), heterozygous-F508del (Ht, grey bars) and non-F508del (N, white bars) patients. In figure were reported the five bacterial species (or groups) with significant <i>P</i> values (in bold) among sub-groups.</p

Patients’ genetics and demographics.

a<p>(Hm = 1; Ht = 2; N = 3).</p>b<p>when the value is indicated in parenthesis tentative data are available.</p>c<p>s1, s2 a s3 denote a sibling relationship for these three couples of patients.</p>d<p>1 = pancreas insufficiency, 0 = pancreas sufficiency.</p