Phenotypic diversity and genotypic flexibility of Burkholderia cenocepacia during long-term chronic infection of cystic fibrosis lungs.

Genome Res. 2017 Mar 21;:

Authors: Lee AH, Flibotte S, Sinha S, Paiero A, Ehrlich RL, Balashov S, Ehrlich GD, Zlosnik JE, Mell JC, Nislow C

Abstract
Chronic bacterial infections of the lung are the leading cause of morbidity and mortality in cystic fibrosis patients. Tracking bacterial evolution during chronic infections can provide insights into how host selection pressures-including immune responses and therapeutic interventions-shape bacterial genomes. We carried out genomic and phenotypic analyses of 215 serially collected Burkholderia cenocepacia isolates from 16 cystic fibrosis patients, spanning a period of 2-20 yr and a broad range of epidemic lineages. Systematic phenotypic tests identified longitudinal bacterial series that manifested progressive changes in liquid media growth, motility, biofilm formation, and acute insect virulence, but not in mucoidy. The results suggest that distinct lineages follow distinct evolutionary trajectories during lung infection. Pan-genome analysis identified 10,110 homologous gene clusters present only in a subset of strains, including genes restricted to different molecular types. Our phylogenetic analysis based on 2148 orthologous gene clusters from all isolates is consistent with patient-specific clades. This suggests that initial colonization of patients was likely by individual strains, followed by subsequent diversification. Evidence of clonal lineages shared by some patients was observed, suggesting inter-patient transmission. We observed recurrent gene losses in multiple independent longitudinal series, including complete loss of Chromosome III and deletions on other chromosomes. Recurrently observed loss-of-function mutations were associated with decreases in motility and biofilm formation. Together, our study provides the first comprehensive genome-phenome analyses of B. cenocepacia infection in cystic fibrosis lungs and serves as a valuable resource for understanding the genomic and phenotypic underpinnings of bacterial evolution.

PMID: 28325850 [PubMed - as supplied by publisher]

Impaired defenses of neonatal mouse alveolar macrophage with cftr deletion are modulated by glutathione and TGFβ1.

Physiol Rep. 2017 Mar;5(6):

Authors: Gauthier TW, Grunwell JR, Ping XD, Harris FL, Brown LA

Abstract
Our understanding of the intrinsic effects of cystic fibrosis (CF) transmembrane conductance regulator (cftr) deletion on resident neonatal alveolar macrophage (AM) remains limited. We previously demonstrated that diminished glutathione (GSH) or excessive AM transforming growth factor beta one (TGFβ1) contributes to AM dysfunction in a variety of disease states. In this study, using a gut-corrected cftr neonatal knockout (KO) mouse model and a siRNA-manipulated macrophage-like cell line (THP-1 cell), we hypothesized (1) that cftr mutation alone increases neonatal AM oxidant stress and cellular TGFβ1 signaling via altered GSH, thereby impairing cellular function, and (2) that exogenous GSH attenuates AM alterations and dysfunction in the KO AM In neonatal KO mice, the baseline bronchoalveolar lavage fluid demonstrated a near doubling in mixed disulfides (P ≤ 0.05) and oxidized GSSG (P ≤ 0.05) without concurrent inflammation compared to WT littermates. KO AM demonstrated diminished AM thiols (P ≤ 0.05), increased AM mitochondrial ROS (P ≤ 0.05), increased AM TGFβ1 (P ≤ 0.05) with increased TGFβ1 signaling (P ≤ 0.05), and impaired phagocytosis (P ≤ 0.05). KO AM mitochondrial ROS was modulated by exogenous GSH (P ≤ 0.05). Conversely, TGFβ1 was reduced (P ≤ 0.05) and impaired phagocytosis was rescued (P ≤ 0.05) by exogenous GSH in the KO AM These results suggest that an altered neonatal AM phenotype may contribute to the initiation of lung inflammation/infection in the CF lung. Modulation of the AM in the neonatal CF lung may potentially alter progression of disease.

PMID: 28325787 [PubMed - in process]

Pseudomonas aeruginosa Alginate Overproduction Promotes Coexistence with Staphylococcus aureus in a Model of Cystic Fibrosis Respiratory Infection.

MBio. 2017 Mar 21;8(2):

Authors: Limoli DH, Whitfield GB, Kitao T, Ivey ML, Davis MR, Grahl N, Hogan DA, Rahme LG, Howell PL, O'Toole GA, Goldberg JB

Abstract
While complex intra- and interspecies microbial community dynamics are apparent during chronic infections and likely alter patient health outcomes, our understanding of these interactions is currently limited. For example, Pseudomonas aeruginosa and Staphylococcus aureus are often found to coinfect the lungs of patients with cystic fibrosis (CF), yet these organisms compete under laboratory conditions. Recent observations that coinfection correlates with decreased health outcomes necessitate we develop a greater understanding of these interbacterial interactions. In this study, we tested the hypothesis that P. aeruginosa and/or S. aureus adopts phenotypes that allow coexistence during infection. We compared competitive interactions of P. aeruginosa and S. aureus isolates from mono- or coinfected CF patients employing in vitro coculture models. P. aeruginosa isolates from monoinfected patients were more competitive toward S. aureus than P. aeruginosa isolates from coinfected patients. We also observed that the least competitive P. aeruginosa isolates possessed a mucoid phenotype. Mucoidy occurs upon constitutive activation of the sigma factor AlgT/U, which regulates synthesis of the polysaccharide alginate and dozens of other secreted factors, including some previously described to kill S. aureus Here, we show that production of alginate in mucoid strains is sufficient to inhibit anti-S. aureus activity independent of activation of the AlgT regulon. Alginate reduces production of siderophores, 2-heptyl-4-hydroxyquinolone-N-oxide (HQNO), and rhamnolipids-each required for efficient killing of S. aureus These studies demonstrate alginate overproduction may be an important factor driving P. aeruginosa coinfection with S. aureusIMPORTANCE Numerous deep-sequencing studies have revealed the microbial communities present during respiratory infections in cystic fibrosis (CF) patients are diverse, complex, and dynamic. We now face the challenge of determining the influence of these community dynamics on patient health outcomes and identifying candidate targets to modulate these interactions. We make progress toward this goal by determining that the polysaccharide alginate produced by mucoid strains of P. aeruginosa is sufficient to inhibit multiple secreted antimicrobial agents produced by this organism. Importantly, these secreted factors are required to outcompete S. aureus, when the microbes are grown in coculture; thus we propose a mechanism whereby mucoid P. aeruginosa can coexist with S. aureus Finally, the approach used here can serve as a platform to investigate the interactions among other CF pathogens.

PMID: 28325763 [PubMed - in process]

Effect of allergic bronchopulmonary aspergillosis on FEV1 in children and adolescents with cystic fibrosis: a European Cystic Fibrosis Society Patient Registry analysis.

Arch Dis Child. 2017 Mar 21;:

Authors: Kaditis AG, Miligkos M, Bossi A, Colombo C, Hatziagorou E, Kashirskaya N, de Monestrol I, Thomas M, Mei-Zahav M, Chrousos G, Zolin A

Abstract
OBJECTIVE: To evaluate the effect of allergic bronchopulmonary aspergillosis (ABPA) on FEV1 percent predicted in children and adolescents with cystic fibrosis.
DESIGN: Longitudinal data analysis (2008-2010).
SETTING: Patients participating in the European Cystic Fibrosis Society Patient Registry.
PARTICIPANTS: 3350 patients aged 6-17 years.
MAIN OUTCOME MEASURE: FEV1percent predicted was the main outcome measure (one measurement per year per child). To describe the effect of ABPA (main explanatory variable) on FEV1 while controlling for other prognostic factors, a linear mixed effects regression model was applied.
RESULTS: In 2008, the mean (±SD) FEV1 percent predicted was 78.6 (±20.6) in patients with ABPA (n=346) and 88 (±19.8) in those without ABPA (n=2806). After considering other variables, FEV1 in subjects with ABPA on entry to the study was 1.47 percentage points lower than FEV1 in patients of similar age without ABPA (p=0.003). There was no FEV1 decline associated with ABPA over the subsequent study years as the interaction of ABPA with age was not significant (p>0.05). For patients aged 11.82 years (population mean age), poor body mass index had the greatest impact on FEV1 in 2008, followed by high-risk genotype (two severe mutations), female gender, diabetes mellitus, chronic Pseudomonas aeruginosa infection and ABPA in descending order of effect size.
CONCLUSIONS: In contrast to the common clinical belief of ABPA having a serious impact on lung function, the difference in FEV1 between young patients with and without the complication was found to be modest when the effect of other prognostic factors was considered.

PMID: 28325727 [PubMed - as supplied by publisher]

Real-life initiation of lumacaftor/ivacaftor combination in adults with cystic fibrosis homozygous for the Phe508del CFTR mutation and severe lung disease.

J Cyst Fibros. 2017 Mar 15;:

Authors: Hubert D, Chiron R, Camara B, Grenet D, Prévotat A, Bassinet L, Dominique S, Rault G, Macey J, Honoré I, Kanaan R, Leroy S, Desmazes Dufeu N, Burgel PR

Abstract
OBJECTIVE: To investigate the short-term adverse events and effectiveness of lumacaftor/ivacaftor combination treatment in adults with cystic fibrosis (CF) and severe lung disease in a real life setting.
METHODS: A multicentre observational study investigated adverse events, treatment discontinuation, FEV1 and body mass index (BMI) one month and three months after lumacaftor/ivacaftor initiation in adults with CF and FEV1 below 40% predicted.
RESULTS: Respiratory adverse events (AEs) were reported by 27 of 53 subjects (51%) and 16 (30%) discontinued treatment. The mean absolute change in FEV1 was +2.06% after one month of treatment (P=0.086) and +3.19% after 3 months (P=0.009). BMI was unchanged.
CONCLUSIONS: Treatment with lumacaftor/ivacaftor in patients with CF and severe lung disease was discontinued more frequently than reported in clinical trials, due to respiratory AEs. Nevertheless, the patients who continued treatment had an increase in lung function comparable to what was observed in pivotal trials.

PMID: 28325531 [PubMed - as supplied by publisher]

Relationship of Antibiotic Treatment to Recovery after Acute FEV1 Decline in Children with Cystic Fibrosis.

Ann Am Thorac Soc. 2017 Mar 21;:

Authors: Morgan WJ, Wagener JS, Pasta DJ, Millar SJ, VanDevanter DR, Konstan MW, Scientific Advisory Group, Investigators, and Coordinators of the Epidemiologic Study of Cystic Fibrosis

Abstract
RATIONALE: Children with cystic fibrosis often suffer acute declines in lung function. We previously showed that such declines are not always treated with antibiotics, but did not assess whether treatment improves the likelihood of recovery.
OBJECTIVES: To determine whether new antibiotic treatment was associated with recovery from acute FEV1 decline.
METHODS: We studied episodes of FEV1 decline (≥10% from baseline) from the Epidemiologic Study of Cystic Fibrosis. Treatments were hospitalization, home IV antibiotic, or new inhaled, oral quinolone, or other oral antibiotic. We used logistic regression to evaluate whether treatment was associated with recovery to baseline, or near baseline.
RESULTS: Logistic regression of 9,875 patients showed that new antibiotic treatment was associated with an increased likelihood of recovery to 90% of baseline (P<0.001), especially for hospitalization compared to no new antibiotic (OR 2.79, 95%CI 2.41-3.23). All four outpatient treatments were associated with greater likelihood of recovery compared to no treatment (OR 1.27 to 1.64). Inpatient treatment was better than outpatient treatments (OR 1.94, 95%CI 1.68-2.23). Treatment type odds ratios were similar across recovery criteria and levels of baseline lung function.
CONCLUSIONS: New antibiotic therapy and especially inpatient treatment is associated with greater likelihood of recovery after acute decline in FEV1. Benefits extend across all disease stages and are especially important in patients with high lung function, who are at greatest risk for FEV1 decline.

PMID: 28324670 [PubMed - as supplied by publisher]

ACCRUAL OF BONE MASS IN CHILDREN AND ADOLESCENTS WITH CYSTIC FIBROSIS.

J Clin Endocrinol Metab. 2017 Feb 22;:

Authors: Sharma S, Jaksic M, Fenwick S, Byrnes C, Cundy T

Abstract
Context: Low bone density is a recognised complication of cystic fibrosis (CF).
Hypothesis: Accrual of bone mass is most impaired in the sickest children, as judged by nutritional status and pulmonary function.
Design: Retrospective analysis of correlation between lumbar spine BMD, BMI and FEV1 z-score in children and adolescents with CF.
Setting: Specialist CF service at a pediatric hospital.
Patients: 60 participants aged 5.9 - 18.8 years (24 female), with confirmed CF.
Interventions: Lumbar spine BMD, BMI and FEV1 z-score measured at time of first BMD scan. 40 participants had sequential scans. Change in L1-L4 z-score over time was used as a measure of bone accrual, and BMI as a measure of nutritional status.
Outcome measures: Correlations between lumbar spine BMD, BMI and FEV1 z-scores.
Results: Mean BMI and BMD z-score were strongly correlated at the initial scan (p <0.0001) suggesting that nutritional status is a major determinant of BMD. In the sequential scan at a mean age of 16.1 years, height centile was maintained, indicating normal linear growth. Changes in BMI and BMD z-score were positively correlated (p= 0.001), indicating that patients failing to gain weight appropriately with growth were also failing to acquire bone normally. Change in FEV1 z-score was correlated with both change in BMD z-score (p <0.0001), and change in BMI z-score (p= 0.02).
Conclusion: Although they may be maintaining normal growth in height, bone accrual is impaired in those young people with CF who have the poorest nutritional status and lung function.

PMID: 28323913 [PubMed - as supplied by publisher]

Boosting joint models for longitudinal and time-to-event data.

Biom J. 2017 Mar 21;:

Authors: Waldmann E, Taylor-Robinson D, Klein N, Kneib T, Pressler T, Schmid M, Mayr A

Abstract
Joint models for longitudinal and time-to-event data have gained a lot of attention in the last few years as they are a helpful technique clinical studies where longitudinal outcomes are recorded alongside event times. Those two processes are often linked and the two outcomes should thus be modeled jointly in order to prevent the potential bias introduced by independent modeling. Commonly, joint models are estimated in likelihood-based expectation maximization or Bayesian approaches using frameworks where variable selection is problematic and that do not immediately work for high-dimensional data. In this paper, we propose a boosting algorithm tackling these challenges by being able to simultaneously estimate predictors for joint models and automatically select the most influential variables even in high-dimensional data situations. We analyze the performance of the new algorithm in a simulation study and apply it to the Danish cystic fibrosis registry that collects longitudinal lung function data on patients with cystic fibrosis together with data regarding the onset of pulmonary infections. This is the first approach to combine state-of-the art algorithms from the field of machine-learning with the model class of joint models, providing a fully data-driven mechanism to select variables and predictor effects in a unified framework of boosting joint models.

PMID: 28321912 [PubMed - as supplied by publisher]

The Pseudomonas aeruginosa two-component regulator AlgR directly activates rsmA expression in a phosphorylation independent manner.

J Bacteriol. 2017 Mar 20;:

Authors: Stacey SD, Williams DA, Pritchett CL

Abstract
Pseudomonas aeruginosa is an important pathogen of the immunocompromised, causing both acute and chronic infections. In cystic fibrosis (CF) patients, P. aeruginosa causes chronic disease. The impressive sensory network of P. aeruginosa allows the bacteria to sense and respond to a variety of stimuli found in diverse environments. Transcriptional regulators, including alternative sigma factors and response regulators, integrate signals changing gene expression allowing P. aeruginosa to cause infection. The two-component transcriptional regulator AlgR is important in P. aeruginosa pathogenesis in both acute and chronic infections. In chronic infections, AlgR and the alternative sigma factor AlgU activate the genes responsible for alginate production. Previous work demonstrated that AlgU controlled rsmA expression. RsmA is a post-transcriptional regulator that is antagonized by two small RNAs, RsmY and RsmZ. In this work, we demonstrate that AlgR directly activates rsmA expression from the same promoter as AlgU. In addition, phosphorylation was not necessary for AlgR activation of rsmA using algR and algZ mutant strains. AlgU and AlgR appear to affect the antagonizing small RNAs, rsmY and rsmZ, indirectly. RsmA was active in a mucA22 strain using leader fusions of two RsmA targets, tssA1 and hcnA AlgU and AlgR were necessary for posttranscriptional regulation of tssA1 and hcnA Altogether, our work demonstrates that the alginate regulators, AlgU and AlgR, are important in the control of the RsmA posttranscriptional regulatory system. These findings suggest that RsmA plays an unknown role in mucoid strains due to AlgU and AlgR activity.IMPORTANCEP. aeruginosa infections are difficult to treat and frequently cause significant mortality in CF patients. Understanding the mechanisms of persistence is important. Our work has demonstrated that the alginate regulatory system also significantly impacts the posttranscriptional regulator system RsmA/Y/Z. We demonstrate AlgR directly activates rsmA expression and this impacts the RsmA regulon. This leads to the possibility that the RsmA/Y/Z system plays a role in helping P. aeruginosa persist during chronic infection. In addition, this furthers our understanding of the reach of the alginate regulators AlgU and AlgR.

PMID: 28320883 [PubMed - as supplied by publisher]

Nutritional status, nutrient intake and use of enzyme supplements in paediatric patients with Cystic Fibrosis; a European multicentre study with reference to current guidelines.

J Cyst Fibros. 2017 Mar 17;:

Authors: Calvo-Lerma J, Hulst JM, Asseiceira I, Claes I, Garriga M, Colombo C, Fornés V, Woodcock S, Martins T, Boon M, Ruperto M, Walet S, Speziali C, Witters P, Masip E, Barreto C, de Boeck K, Ribes-Koninckx C, MyCyFAPP Project

Abstract
BACKGROUND: The New European guidelines have established the most updated recommendations on nutrition and pancreatic enzyme replacement therapy (PERT) in CF. In the context of MyCyFAPP project - a European study in children with CF aimed at developing specific tools for improvement of self-management - the objective of the current study was to assess nutritional status, daily energy and macronutrient intake, and PERT dosing with reference to these new guidelines.
METHODS: Cross sectional study in paediatric patients with CF from 6 European centres. SD-scores for weight-for-age (WFA), height-for-age (HFA) and body mass index-for-age (BMI) were obtained. Through a specific 4-day food and enzyme-dose record, energy and macronutrients intake and PERT-use (LU/g lipids) were automatically calculated by the MyCyFAPP system. Comparisons were made using linear regression models.
RESULTS: The lowest quartiles for BMI and HFA were between 0 and -1SD in all the centres with no significant differences, and 33.5% of the patients had a SD-score <0 for all three parameters. The minimum energy intake recommendation was not reached by 40% of the children and mean nutrients intake values were 14%, 51% and 34% of the total energy for protein, carbohydrates and lipids respectively. When assessed per centre, reported PERT doses were in the recommended range in only 13.8% to 46.6% of the patients; from 5.6% up to 82.7% of children were above the recommended doses and 3.3% to 75% were below.
CONCLUSION: Among the 6 centres, a large variability and inconsistency with new guidelines on nutrition and PERT-use was found. Our findings document the lack of a general criterion to adjust PERT and suggest the potential benefit of educational and self-managerial tools to ensure adherence to therapies, both for clinical staff and families. They will be taken into account when developing these new tools during the next stages of MyCyFAPP Project.

PMID: 28320633 [PubMed - as supplied by publisher]

Is acute recurrent pancreatitis in children a precursor of chronic pancreatitis? A long-term follow-up study of 93 cases.

Dig Liver Dis. 2017 Mar 06;:

Authors: Poddar U, Yachha SK, Borkar V, Srivastava A

Abstract
BACKGROUND/AIMS: In view of paucity of literature we analyzed our experience of acute recurrent pancreatitis (ARP) to study clinical profile and long-term outcome.
METHODS: Over 13 years, 93 consecutive children (≤18 years) diagnosed to have ARP were included in this study. Magnetic resonance cholangiopancreatography was done at baseline and on follow-up. Common mutations for serine-protease-inhibitor (SPINK1 N34S), protease inhibitor (PRSS1 R122S) and cystic fibrosis transmembrane conductance regulator (CFTR deltaF508, 5T) were studied in 22 idiopathic cases.
RESULTS: The median age of the children with ARP was 13 (10-14.5) years, 53 were males. Etiology included biliary in 14 (15%), pancreas divisum in 6 (7%), others in 3 (3.5%) and idiopathic in the remaining 70 (75%). SPINK1 mutation was found in 10/22 (45%) cases. Over a median follow-up of 25.5 (8.25-48) months, 37 (42%) of 88 (5 lost to follow-up) developed chronic pancreatitis (CP). On multivariate analysis idiopathic etiology (p<0.03), presence of SPINK1 mutation (p=0.01), longer follow-up (p<0.001) were associated with progression to CP.
CONCLUSIONS: Biliopancreatic structural/obstructive causes should always be looked for. It seems ARP is a precursor of CP and progression is associated with idiopathic etiology and presence of genetic mutations. Hence, patients with ARP should be kept on regular follow-up to detect CP.

PMID: 28320629 [PubMed - as supplied by publisher]

Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

PLoS Genet. 2016 07;12(7):e1006220

Authors: Lewis WR, Malarkey EB, Tritschler D, Bower R, Pasek RC, Porath JD, Birket SE, Saunier S, Antignac C, Knowles MR, Leigh MW, Zariwala MA, Challa AK, Kesterson RA, Rowe SM, Drummond IA, Parant JM, Hildebrandt F, Porter ME, Yoder BK, Berbari NF

Abstract
Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.

PMID: 27472056 [PubMed - indexed for MEDLINE]

Genomic characterisation of HER2-positive breast cancer and response to neoadjuvant trastuzumab and chemotherapy - results from the ACOSOG Z1041 (Alliance) trial.

Ann Oncol. 2017 Feb 21;:

Authors: Lesurf R, Griffith OL, Griffith M, Hundal J, Trani L, Watson MA, Aft R, Ellis MJ, Ota D, Suman VJ, Meric-Bernstam F, Leitch AM, Boughey JC, Unzeitig G, Buzdar AU, Hunt KK, Mardis ER

Abstract
Background: HER2 ( ERBB2 ) gene amplification and its corresponding overexpression are present in 15-30% of invasive breast cancers. While HER2-targeted agents are effective treatments, resistance remains a major cause of death. The American College of Surgeons Oncology Group Z1041 trial (NCT00513292) was designed to compare the pathologic complete response (pCR) rate of distinct regimens of neoadjuvant chemotherapy and trastuzumab, but ultimately identified no difference [1].
Patients and methods: In supplement to tissues from 37 Z1041 cases, 11 similarly treated cases were obtained from a single institution study (NCT00353483). We have extracted genomic DNA from both pre-treatment tumor biopsies and blood of these 48 cases, and performed whole genome (WGS) and exome sequencing. Coincident with these efforts, we have generated RNA-seq profiles from 42 of the tumor biopsies. Among patients in this cohort, 24 (50%) achieved a pCR.
Results: Here we characterized the genomic landscape of HER2-positive breast cancer and investigated associations between genomic features and pCR. Cases assigned to the HER2-enriched subtype by RNA-seq analysis were more likely to achieve a pCR compared to the luminal, basal-like, or normal-like subtypes (19/27 versus 3/15; P =0.0032). Mutational events led to the generation of putatively active neoantigens, but were overall not associated with pCR. ERBB2 and GRB7 were the genes most commonly observed in fusion events, and genomic copy number analysis of the ERBB2 locus indicated that cases with either no observable or low-level ERBB2 amplification were less likely to achieve a pCR (7/8 versus 17/40; P =0.048). Moreover, among cases that achieved a pCR, tumors consistently expressed immune signatures that may contribute to therapeutic response.
Conclusion: The identification of these features suggests that it may be possible to predict, at the time of diagnosis, those HER2-positive breast cancer patients who will not respond to treatment with chemotherapy and trastuzumab.

PMID: 28327926 [PubMed - as supplied by publisher]

Analysis of exome data for 4293 trios suggests GPI-anchor biogenesis defects are a rare cause of developmental disorders.

Eur J Hum Genet. 2017 Mar 22;:

Authors: Pagnamenta AT, Murakami Y, Taylor JM, Anzilotti C, Howard MF, Miller V, Johnson DS, Tadros S, Mansour S, Temple IK, Firth R, Rosser E, Harrison RE, Kerr B, Popitsch N, DDD Study, Kinoshita T, Taylor JC, Kini U

Abstract
Over 150 different proteins attach to the plasma membrane using glycosylphosphatidylinositol (GPI) anchors. Mutations in 18 genes that encode components of GPI-anchor biogenesis result in a phenotypic spectrum that includes learning disability, epilepsy, microcephaly, congenital malformations and mild dysmorphic features. To determine the incidence of GPI-anchor defects, we analysed the exome data from 4293 parent-child trios recruited to the Deciphering Developmental Disorders (DDD) study. All probands recruited had a neurodevelopmental disorder. We searched for variants in 31 genes linked to GPI-anchor biogenesis and detected rare biallelic variants in PGAP3, PIGN, PIGT (n=2), PIGO and PIGL, providing a likely diagnosis for six families. In five families, the variants were in a compound heterozygous configuration while in a consanguineous Afghani kindred, a homozygous c.709G>C; p.(E237Q) variant in PIGT was identified within 10-12 Mb of autozygosity. Validation and segregation analysis was performed using Sanger sequencing. Across the six families, five siblings were available for testing and in all cases variants co-segregated consistent with them being causative. In four families, abnormal alkaline phosphatase results were observed in the direction expected. FACS analysis of knockout HEK293 cells that had been transfected with wild-type or mutant cDNA constructs demonstrated that the variants in PIGN, PIGT and PIGO all led to reduced activity. Splicing assays, performed using leucocyte RNA, showed that a c.336-2A>G variant in PIGL resulted in exon skipping and p.D113fs*2. Our results strengthen recently reported disease associations, suggest that defective GPI-anchor biogenesis may explain ~0.15% of individuals with developmental disorders and highlight the benefits of data sharing.European Journal of Human Genetics advance online publication, 22 March 2017; doi:10.1038/ejhg.2017.32.

PMID: 28327575 [PubMed - as supplied by publisher]

Insights from early experience of a Rare Disease Genomic Medicine Multidisciplinary Team: a qualitative study.

Eur J Hum Genet. 2017 Mar 22;:

Authors: Ormondroyd E, Mackley MP, Blair E, Craft J, Knight JC, Taylor J, Taylor JC, Wilkie AO, Watkins H

Abstract
Whole-exome/whole-genome sequencing (WES/WGS) has the potential to enhance genetic diagnosis of rare disease, and is increasingly becoming part of routine clinical care in mainstream medicine. Effective translation will require ongoing efforts in a number of areas including: selection of appropriate patients, provision of effective consent, pre- and post-test genetic counselling, improving variant interpretation algorithms and practices, and management of secondary findings including those found incidentally and those actively sought. Allied to this is the need for an effective education programme for all members of clinical teams involved in care of patients with rare disease, as well as to maintain public confidence in the use of these technologies. We established a Genomic Medicine Multidisciplinary Team (GM-MDT) in 2014 to build on the experiences of earlier successful research-based WES/WGS studies, to address these needs and to review results including pertinent and secondary findings. Here we report on a qualitative study of decision-making in the GM-MDT combined with analysis of semi-structured interviews with GM-MDT members. Study findings show that members appreciate the clinical and scientific diversity of the GM-MDT and value it for education and oversight. To date, discussions have focussed on case selection including the extent and interpretation of clinical and family history information required to establish likely monogenic aetiology and inheritance model. Achieving a balance between effective use of WES/WGS - prioritising cases in a diverse and highly complex patient population where WES/WGS will be tractable - and meeting the recruitment targets of a large project is considered challenging.European Journal of Human Genetics advance online publication, 22 March 2017; doi:10.1038/ejhg.2017.37.

PMID: 28327571 [PubMed - as supplied by publisher]

PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features.

Eur J Hum Genet. 2017 Mar 22;:

Authors: Low KJ, Ansari M, Abou Jamra R, Clarke A, El Chehadeh S, FitzPatrick DR, Greenslade M, Henderson A, Hurst J, Keller K, Kuentz P, Prescott T, Roessler F, Selmer KK, Schneider MC, Stewart F, Tatton-Brown K, Thevenon J, Vigeland MD, Vogt J, Willems M, Zonana J, Study DD, Smithson SF

Abstract
PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through similar projects. Detailed phenotypic analysis of all patients was undertaken. All 12 patients had de novo heterozygous PUF60 variants on exome analysis, each confirmed by Sanger sequencing: four frameshift variants resulting in premature stop codons, three missense variants that clustered within the RNA recognition motif of PUF60 and five essential splice-site (ESS) variant. Analysis of cDNA from a fibroblast cell line derived from one of the patients with an ESS variants revealed aberrant splicing. The consistent feature was developmental delay and most patients had short stature. The phenotypic variability was striking; however, we observed similarities including spinal segmentation anomalies, congenital heart disease, ocular colobomata, hand anomalies and (in two patients) unilateral renal agenesis/horseshoe kidney. Characteristic facial features included micrognathia, a thin upper lip and long philtrum, narrow almond-shaped palpebral fissures, synophrys, flared eyebrows and facial hypertrichosis. Heterozygote loss-of-function variants in PUF60 cause a phenotype comprising growth/developmental delay and craniofacial, cardiac, renal, ocular and spinal anomalies, adding to disorders of human development resulting from aberrant RNA processing/spliceosomal function.European Journal of Human Genetics advance online publication, 22 March 2017; doi:10.1038/ejhg.2017.27.

PMID: 28327570 [PubMed - as supplied by publisher]

Lessons learned from additional research analyses of unsolved clinical exome cases.

Genome Med. 2017 Mar 21;9(1):26

Authors: Eldomery MK, Coban-Akdemir Z, Harel T, Rosenfeld JA, Gambin T, Stray-Pedersen A, Küry S, Mercier S, Lessel D, Denecke J, Wiszniewski W, Penney S, Liu P, Bi W, Lalani SR, Schaaf CP, Wangler MF, Bacino CA, Lewis RA, Potocki L, Graham BH, Belmont JW, Scaglia F, Orange JS, Jhangiani SN, Chiang T, Doddapaneni H, Hu J, Muzny DM, Xia F, Beaudet AL, Boerwinkle E, Eng CM, Plon SE, Sutton VR, Gibbs RA, Posey JE, Yang Y, Lupski JR

Abstract
BACKGROUND: Given the rarity of most single-gene Mendelian disorders, concerted efforts of data exchange between clinical and scientific communities are critical to optimize molecular diagnosis and novel disease gene discovery.
METHODS: We designed and implemented protocols for the study of cases for which a plausible molecular diagnosis was not achieved in a clinical genomics diagnostic laboratory (i.e. unsolved clinical exomes). Such cases were recruited to a research laboratory for further analyses, in order to potentially: (1) accelerate novel disease gene discovery; (2) increase the molecular diagnostic yield of whole exome sequencing (WES); and (3) gain insight into the genetic mechanisms of disease. Pilot project data included 74 families, consisting mostly of parent-offspring trios. Analyses performed on a research basis employed both WES from additional family members and complementary bioinformatics approaches and protocols.
RESULTS: Analysis of all possible modes of Mendelian inheritance, focusing on both single nucleotide variants (SNV) and copy number variant (CNV) alleles, yielded a likely contributory variant in 36% (27/74) of cases. If one includes candidate genes with variants identified within a single family, a potential contributory variant was identified in a total of ~51% (38/74) of cases enrolled in this pilot study. The molecular diagnosis was achieved in 30/63 trios (47.6%). Besides this, the analysis workflow yielded evidence for pathogenic variants in disease-associated genes in 4/6 singleton cases (66.6%), 1/1 multiplex family involving three affected siblings, and 3/4 (75%) quartet families. Both the analytical pipeline and the collaborative efforts between the diagnostic and research laboratories provided insights that allowed recent disease gene discoveries (PURA, TANGO2, EMC1, GNB5, ATAD3A, and MIPEP) and increased the number of novel genes, defined in this study as genes identified in more than one family (DHX30 and EBF3).
CONCLUSION: An efficient genomics pipeline in which clinical sequencing in a diagnostic laboratory is followed by the detailed reanalysis of unsolved cases in a research environment, supplemented with WES data from additional family members, and subject to adjuvant bioinformatics analyses including relaxed variant filtering parameters in informatics pipelines, can enhance the molecular diagnostic yield and provide mechanistic insights into Mendelian disorders. Implementing these approaches requires collaborative clinical molecular diagnostic and research efforts.

PMID: 28327206 [PubMed - in process]

Whole exome sequencing with genomic triangulation implicates CDH2-encoded N-cadherin as a novel pathogenic substrate for arrhythmogenic cardiomyopathy.

Congenit Heart Dis. 2017 Mar 21;:

Authors: Turkowski KL, Tester DJ, Bos JM, Haugaa KH, Ackerman MJ

Abstract
BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is a heritable disease characterized by fibrofatty replacement of cardiomyocytes, has a prevalence of approximately 1 in 5000 individuals, and accounts for approximately 20% of sudden cardiac death in the young (≤35 years). ACM is most often inherited as an autosomal dominant trait with incomplete penetrance and variable expression. While mutations in several genes that encode key desmosomal proteins underlie about half of all ACM, the remainder is elusive genetically.
OBJECTIVE: Here, whole exome sequencing (WES) was performed with genomic triangulation in an effort to identify a novel explanation for a phenotype-positive, genotype-negative multi-generational pedigree with a presumed autosomal dominant, maternal inheritance of ACM.
METHODS: WES and genomic triangulation was performed on a symptomatic 14-year-old female proband, her affected mother and affected sister, and her unaffected father to elucidate a novel ACM-susceptibility gene for this pedigree. Following variant filtering using Ingenuity® Variant Analysis, gene priority ranking was performed on the candidate genes using ToppGene and Endeavour. The phylogenetic and physiochemical properties of candidate mutations were assessed further by 6 in silico prediction tools. Species alignment and amino acid conservation analysis was performed using the Uniprot Consortium. Tissue expression data was abstracted from Expression Atlas.
RESULTS: Following WES and genomic triangulation, CDH2 emerged as a novel, autosomal dominant, ACM-susceptibility gene. The CDH2-encoded N-cadherin is a cell-cell adhesion protein predominately expressed in the heart. Cardiac dysfunction has been demonstrated in prior CDH2 knockout and over-expression animal studies. Further in silico mutation prediction, species conservation, and protein expression analysis supported the ultra-rare (minor allele frequency <0.005%) p.Asp407Asn-CDH2 variant as a likely pathogenic variant.
CONCLUSIONS: Herein, it is demonstrated that genetic mutations in CDH2-encoded N-cadherin may represent a novel pathogenetic basis for ACM in humans. The prevalence of CDH2-mediated ACM in heretofore genetically elusive ACM remains to be determined.

PMID: 28326674 [PubMed - as supplied by publisher]

Exome Analysis of the Evolutionary Path of Hepatocellular Adenoma-Carcinoma Transition, Vascular Invasion and Brain Dissemination.

J Hepatol. 2017 Mar 17;:

Authors: Vilarinho S, Erson-Omay Z, Mitchell-Richards K, Cha C, Nelson-Williams C, Harmancı AS, Yasuno K, Günel M, Taddei TH

Abstract
Hepatocellular adenoma (HCA) is a rare benign liver tumor, predominantly seen in young women. Its major complications are malignant transformation, spontaneous hemorrhage, and rupture. We describe a case of a young female with no underlying liver disease who presented with acute abdominal pain and was found to have a 17 cm heterogeneous mass in the left lobe of the liver. She underwent left hepatectomy and pathology revealed a 14 cm moderately differentiated hepatocellular carcinoma (HCC) arising in a shell of a HCA. At that time, vascular invasion was already present. She rapidly developed recurrent multifocal hepatic lesions and subsequent spread to the brain, leading to her death 18 months after surgery. To investigate the underlying genetic events occurring during hepatocellular adenoma-carcinoma transition and extra-hepatic dissemination, we performed whole-exome sequencing of DNA isolated from peripheral blood leucocytes, HCA, HCC, tumor thrombus and brain metastasis. Our data show a step-wise addition of somatic mutations and copy number variations with disease progression, suggesting a linear tumor evolution, which is supported by clonality analysis. Specifically, using a model based clustering of somatic mutations, one single founding clone arising in the HCA, which included CTNNB1 and IL6ST driver mutations, was identified and displayed an increasing clonality rate in HCC, tumor thrombus and brain metastasis. Our data highlight the feasibility of performing whole-exome capture, sequencing and analysis using FFPE samples, and we describe the first genomic longitudinal study of hepatocellular adenoma-carcinoma transition, vascular invasion and brain metastasis with detailed clinicopathologic annotation.

PMID: 28323122 [PubMed - as supplied by publisher]

A novel aberrant splice site mutation in COL27A1 is responsible for Steel syndrome and extension of the phenotype to include hearing loss.

Am J Med Genet A. 2017 Mar 21;:

Authors: Gariballa N, Ben-Mahmoud A, Komara M, Al-Shamsi AM, John A, Ali BR, Al-Gazali L

Abstract
Steel syndrome is an autosomal recessive disease characterized by skeletal abnormalities and dysmorphic features. The first mutation associated with this syndrome was reported in Puerto Rican children. In this study, we identified a novel homozygous splice site variant in COL27A1 (c.3556-2A>G) in a consanguineous Emirati family with a child affected by Steel syndrome. In addition, the affected child had severe non-progressive sensorineural hearing loss not reported previously. The variant segregated in the family in an autosomal recessive manner and we show that the variant alters mRNA splicing. Furthermore, relative quantitative analysis revealed a marked reduction in gene expression in the proposita compared to healthy controls. Segregation analysis of heterozygous variants, related to hearing loss, identified by whole exome sequencing in the child (ILDR1: c.1159T>C, SYNE4: c.313G>C, and GPR98: c.18746T>G) excluded them from being responsible for the hearing loss in the proposita. In addition, the products of these genes are not interacting in the same pathway and have only been reported to cause deafness in an autosomal recessive manner. Therefore, we conclude that the novel splice-site variant identified in COL27A1 is the most likely cause for Steel syndrome in this family and that the hearing loss is part of this syndrome's phenotype.

PMID: 28322503 [PubMed - as supplied by publisher]