Marie Dorda

Extended spectrum of human glucose-6-phosphatase catalytic subunit 3 deficiency: Novel genotypes and phenotypic variability in severe congenital neutropenia

OBJECTIVE To delineate the phenotypic and molecular spectrum of patients with a syndromic variant of severe congenital neutropenia (SCN) due to mutations in the gene encoding glucose-6-phosphatase catalytic subunit 3 (G6PC3). STUDY DESIGN Patients with syndromic SCN were characterized for associated malformations and referred to us for G6PC3 mutational analysis. RESULTS In a cohort of 31 patients with syndromic SCN, we identified 16 patients with G6PC3 deficiency including 11 patients with novel biallelic mutations. We show that nonhematologic features of G6PC3 deficiency are good predictive indicators for mutations in G6PC3. Additionally, we demonstrate genetic variability in this disease and define novel features such as growth hormone deficiency, genital malformations, disrupted bone remodeling, and abnormalities of the integument. G6PC3 mutations may be associated with hydronephrosis or facial dysmorphism. The risk of transition to myelodysplastic syndrome/acute myeloid leukemia may be lower than in other genetically defined SCN subgroups. CONCLUSIONS The phenotypic and molecular spectrum in G6PC3 deficiency is wider than previously appreciated. The risk of transition to myelodysplastic syndrome or acute myeloid leukemia may be lower in G6PC3 deficiency compared with other subgroups of SCN.

The cystic fibrosis lower airways microbial metagenome

Chronic airway infections determine most morbidity in people with cystic fibrosis (CF). Herein, we present unbiased quantitative data about the frequency and abundance of DNA viruses, archaea, bacteria, moulds and fungi in CF lower airways. Induced sputa were collected on several occasions from children, adolescents and adults with CF. Deep sputum metagenome sequencing identified, on average, approximately 10 DNA viruses or fungi and several hundred bacterial taxa. The metagenome of a CF patient was typically found to be made up of an individual signature of multiple, lowly abundant species superimposed by few disease-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, as major components. The host-associated signatures ranged from inconspicuous polymicrobial communities in healthy subjects to low-complexity microbiomes dominated by the typical CF pathogens in patients with advanced lung disease. The DNA virus community in CF lungs mainly consisted of phages and occasionally of human pathogens, such as adeno- and herpesviruses. The S. aureus and P. aeruginosa populations were composed of one major and numerous minor clone types. The rare clones constitute a low copy genetic resource that could rapidly expand as a response to habitat alterations, such as antimicrobial chemotherapy or invasion of novel microbes. The CF lung metagenome is composed of few viruses and fungi and hundreds of bacterial species, clones and subclones http://ow.ly/ZiqUE

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients

ABSTRACT The chronic airway infections with Pseudomonas aeruginosa in people with cystic fibrosis (CF) are treated with aerosolized antibiotics, oral fluoroquinolones, and/or intravenous combination therapy with aminoglycosides and β-lactam antibiotics. An international strain collection of 361 P. aeruginosa isolates from 258 CF patients seen at 30 CF clinics was examined for mutations in 17 antimicrobial susceptibility and resistance loci that had been identified as hot spots of mutation by genome sequencing of serial isolates from a single CF clinic. Combinatorial amplicon sequencing of pooled PCR products identified 1,112 sequence variants that were not present in the genomes of representative strains of the 20 most common clones of the global P. aeruginosa population. A high frequency of singular coding variants was seen in spuE, mexA, gyrA, rpoB, fusA1, mexZ, mexY, oprD, ampD, parR, parS, and envZ (amgS), reflecting the pressure upon P. aeruginosa in lungs of CF patients to generate novel protein variants. The proportion of nonneutral amino acid exchanges was high. Of the 17 loci, mexA, mexZ, and pagL were most frequently affected by independent stop mutations. Private and de novo mutations seem to play a pivotal role in the response of P. aeruginosa populations to the antimicrobial load and the individual CF host.

Intraclonal genome diversity of the major Pseudomonas aeruginosa clones C and PA14

Summary Bacterial populations differentiate at the subspecies level into clonal complexes. Intraclonal genome diversity was studied in 100 isolates of the two dominant P seudomonas aeruginosa clones C and PA14 collected from the inanimate environment, acute and chronic infections. The core genome was highly conserved among clone members with a median pairwise within‐clone single nucleotide sequence diversity of 8 × 10−6 for clone C and 2 × 10−5 for clone PA14. The composition of the accessory genome was, on the other hand, as variable within the clone as between unrelated clones. Each strain carried a large cargo of unique genes. The two dominant worldwide distributed P. aeruginosa clones combine an almost invariant core with the flexible gain and loss of genetic elements that spread by horizontal transfer.

Effects of Lumacaftor–Ivacaftor Therapy on Cystic Fibrosis Transmembrane Conductance Regulator Function in Phe508del Homozygous Patients with Cystic Fibrosis

Rationale: The combination of the CFTR (cystic fibrosis transmembrane conductance regulator) corrector lumacaftor with the potentiator ivacaftor has been approved for the treatment of patients with cystic fibrosis homozygous for the Phe508del CFTR mutation. The phase 3 trials examined clinical outcomes but did not evaluate CFTR function in patients. Objectives: To examine the effect of lumacaftor‐ivacaftor on biomarkers of CFTR function in Phe508del homozygous patients with cystic fibrosis aged 12 years and older. Methods: This prospective observational study assessed clinical outcomes including FEV1% predicted and body mass index, and CFTR biomarkers including sweat chloride concentration, nasal potential difference, and intestinal current measurement before and 8‐16 weeks after initiation of lumacaftor‐ivacaftor. Measurements and Main Results: A total of 53 patients were enrolled in the study, and 52 patients had baseline and follow‐up measurements. After initiation of lumacaftor‐ivacaftor sweat chloride concentrations were reduced by 17.8 mmol/L (interquartile range [IQR], −25.9 to −6.1; P < 0.001), nasal potential difference showed partial rescue of CFTR function in nasal epithelia to a level of 10.2% (IQR, 0.0‐26.1; P < 0.011), and intestinal current measurement showed functional improvement in rectal epithelia to a level of 17.7% of normal (IQR, 10.8‐29.0; P < 0.001). All patients improved in at least one CFTR biomarker, but no correlations were found between CFTR biomarker responses and clinical outcomes. Conclusions: Lumacaftor‐ivacaftor results in partial rescue of Phe508del CFTR function to levels comparable to the lower range of CFTR activity found in patients with residual function mutations. Functional improvement was detected even in the absence of short‐term improvement of FEV1% predicted and body mass index. Clinical trial registered with www.clinicaltrials.gov (NCT02807415).

Multilocus amplicon sequencing of Pseudomonas aeruginosa cystic fibrosis airways isolates collected prior to and after early antipseudomonal chemotherapy

BACKGROUND Early antimicrobial chemotherapy can prevent or at least delay chronic cystic fibrosis (CF) airways infections with Pseudomonas aeruginosa. METHODS During a 10-year study period P. aeruginosa was detected for the first time in 54 CF patients regularly seen at the CF centre Hannover. Amplicon sequencing of 34 loci of the P. aeruginosa core genome was performed in baseline and post-treatment isolates of the 15 CF patients who had remained P. aeruginosa - positive after the first round of antipseudomonal chemotherapy. RESULTS Deep sequencing uncovered coexisting alternative nucleotides at in total 33 of 55,284 examined genome positions including six non-synonymous polymorphisms in the lasR gene, a key regulator of quorum sensing. After early treatment 42 of 50 novel nucleotide substitutions had emerged in exopolysaccharide biosynthesis, efflux pump and porin genes. CONCLUSIONS Early treatment selects pathoadaptive mutations in P. aeruginosa that are typical for chronic infections of CF lungs.

Impact of sample processing on human airways microbial metagenomes

Whole metagenome shotgun sequencing provides information about the gene content and the composition of microbial communities provided that the processing of the samples does not introduce a methodology-driven bias. We tested the impact of DNA isolation and storage period on the metagenome profile. Deep throat swabs were collected from healthy adults and an infected infant. DNA was isolated by sonification or enzymatic lysis either immediately or after 24h storage in agar gel Amies transport medium at room temperature. Disruption of cells and subsequent fragmentation of DNA by sonification was as suitable as the common enzymatic lysis to generate high-quality metagenomes particularly for low total DNA input of less than ten nanograms. Conversely, storage of samples for 24h produced severely distorted metagenomes. The majority of species became less abundant or even extinct, whereas a few Streptococcus, Neisseria and Haemophilus spp. proliferated so that the total number of bacterial reads increased at the expense of human reads. We recommend that samples for metagenome analysis should be immediately processed or frozen at -80°C.

98 The cystic fibrosis lower airways microbial metagenome

ABSTRACT Chronic airway infections determine most morbidity in people with cystic fibrosis (CF). Herein, we present unbiased quantitative data about the frequency and abundance of DNA viruses, archaea, bacteria, moulds and fungi in CF lower airways. Induced sputa were collected on several occasions from children, adolescents and adults with CF. Deep sputum metagenome sequencing identified, on average, approximately 10 DNA viruses or fungi and several hundred bacterial taxa. The metagenome of a CF patient was typically found to be made up of an individual signature of multiple, lowly abundant species superimposed by few disease-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, as major components. The host-associated signatures ranged from inconspicuous polymicrobial communities in healthy subjects to low-complexity microbiomes dominated by the typical CF pathogens in patients with advanced lung disease. The DNA virus community in CF lungs mainly consisted of phages and occasionally of human pathogens, such as adenoand herpesviruses. The S. aureus and P. aeruginosa populations were composed of one major and numerous minor clone types. The rare clones constitute a low copy genetic resource that could rapidly expand as a response to habitat alterations, such as antimicrobial chemotherapy or invasion of novel microbes.

37 The microbial metagenome of cystic fibrosis lower airways

The use of culture-independent microbiome analysis is expanding our view of respiratory tract infection in Cystic fibrosis (CF). The lower airways of people with CF are colonized with polymicrobial communities of bacteria, viruses, fungi and molds. We investigated the microbial metagenome in 20 exocrine pancreatic insufficient and 10 exocrine pancreatic sufficient CF patients. One to five sputum samples were collected from each patient over a 2-year period. The isolated DNA was sequenced by random whole genome sequencing. The reads were aligned to the human, bacterial, virus, fungi and molds reference genomes. Sequences were normalized by GC content and length reference genome. Analysis of temporal series of specimens indicate that each patient carries a specific signature of microbes in his airways unless drastic interventions were undertaken to eradicate unpleasant pathogens such as Burkholderia spp. The spectrum of bacterial species ranged from metagenomes indistinguishable from a healthy non-CF control to metagenomes dominated by the typical CF pathogens Staphylococcus aureus or Pseudomonas aeruginosa . During exacerbations the relative and absolute abundance of species changed, but not the overall signature. On the average several hundred bacterial and viral species, but just a few fungal species were detected by our pipeline. Five to forty species made up 95% of the bacterial metagenome. Our paradigmatic pilot study demonstrates that whole genome sequencing provides deep insight into the microbial CF lung metagenome. Supported by Mukoviszidose e.V. and the German Center for Lung Research (DZL).

WS19.4 Molecular epidemiology of hot-spots of mutation in antimicrobial resistance loci of Pseudomonas aeruginosa isolates from cystic fibrosis airways

High-throughput genome sequencing of serial isolates from cystic fibrosis (CF) airways collected over a 30-year period uncovered targets of antipseudomonal chemotherapy as hot-spots of mutation. Sporadic mutations were overrepresented in the loci gyrA, rpoB, ampD, mexA, mexS, mexY, mexZ, parS, parR, amrZ, envZ, oprD, oprM, pagL, spuE, spuF, fusA1 and fusA2. To reveal the frequency and spectrum of single nucleotide variations and frame-shift mutations in these loci in the contemporary P. aeruginosa population residing in CF airways, deep amplicon sequencing was performed with loci-spanning 405–1554 bp large PCR products amplified from DNA pooled from two strain collections. 305 isolates were obtained from 209 CF patients seen at 51 CF centers in Europe and USA and 56 isolates were retrieved from 38 CF patients after they had received early antipseudomonal chemotherapy of first lower airways colonization with P. aeruginosa. The impact of missense, stop and frame-shift mutations on bacterial antimicrobial susceptibility phenotype was assessed by determination of MIC values against amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, colistin, doripenem, fosfomycin, ghentamycin, imipenem, levofloxacin, meropenem, piperacillin, piperacillin/sulbactam, piperacillin/tazobactam and tobramycin. The spectrum of SNPs and indels and their association in the 18 susceptibility loci with the antipseudomonal resistance phenotype will be presented at the meeting. Supported by DFG (SFB 900, A2) and BMBF (DZL at BREATH).