The Prevalence of Pseudomonas Aeruginosa Infection Over a Ten-Year Period in Children with Cystic Fibrosis

Background Pseudomonas aeruginosa (PA) infection is associated with an increased morbidity and adverse prognosis in children with Cystic Fibrosis(CF). The aim of the study was to evaluate the prevalence and characteristics of PA over a ten year period at a single paediatric tertiary referral centre in Ireland. Methods This was a retrospective cross-sectional study. Patient's case notes, microbiology laboratory results and CF Registry of Ireland(CFRI) data were used to collect the data. Results The overall chronic PA infection prevalence was 28.1%(45/160) in 2004 and 21.3%(35/164) in 2014. In 2004, 54/160(33.8%) patients were never infected with PA, 27/160(16.9%) were free for 12 months and 34/160(21.3%) were intermittently infected. In 2014; 80/164(49%) patients, 38/164(23.2%) and 11/164(6.7%) were never infected, free for 12 months and intermittently infected respectively. Conclusion There has been a decline in the overall prevalence of PA infection and a change in the pattern of prevalence over the last decade at our Centre.

Pulmonary aspiration in preschool children with cystic fibrosis.

Pulmonary aspiration of gastric refluxate (PAGR) has been demonstrated in association with pulmonary inflammation in school aged children with Cystic Fibrosis (CF). We sought to determine if similar findings were present in preschool children. Pepsin was measured in Broncho-alveolar lavage (BAL) fluid collected from clinically stable preschool children with CF and controls. Elevated pepsin levels were found in a subgroup of children with CF, but this was not found to be associated with pulmonary infection, pulmonary inflammation or respiratory or gastrointestinal symptoms.

Diagnosis and treatment of sleep related breathing disorders in children: 2007 to 2011.

Sleep related breathing disorders (SRBD) have historically been under-recognised and under-treated. Obstructive sleep apnoea (OSA) affects approximately 3% of children. In line with the increased recognition of SRBD there has been an increase in demand for diagnostic services. We determined the awareness of SRBD amongst Irish paediatricians, examined the provision of sleep services to children throughout the country between 2007 and 2011 and audited diagnostic sleep services in a tertiary centre in 2011. Amongst respondents there was an awareness of SRBD but a poor understanding of diagnostic evaluation with 31/46 (67) referring to inappropriate services. There has been a sharp increase in both diagnostic sleep tests (433-1793 [414]) and in the use of non-invasive ventilation (NIV) (31-186 [627]) for treatment of SRBD between 2007 and 2011. Paediatric sleep services are organized in an ad-hoc manner nationally with significant service variation. The use of domiciliary overnight oximetry reduced the requirement for more formal polysomnography by 70%.

Rhinovirus and the developing lung.

Human rhinovirus (HRV) infections are now widely accepted as the commonest cause of acute respiratory illnesses (ARIs) in children. Advanced PCR techniques have enabled HRV infections to be identified as causative agents in most common ARIs in childhood including bronchiolitis, acute asthma, pneumonia and croup. However, the long-term implications of rhinovirus infections are less clear. The aim of this review is to examine the relationship between rhinovirus infections and disorders of the lower airways in childhood.

The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene.

The Long-Evans Cinnamon (LEC) rat shows similarity to Wilson disease in many clinical and biochemical features. We have cloned cDNAs for the rat gene (Atp7b) homologous to the human Wilson disease gene (ATP7B) and have used them to identify a partial deletion in the Atp7b gene in the LEC rat. The deletion removes at least 900 bp of the coding region at the 3' end, includes the crucial ATP binding domain and extends downstream of the gene. Our results provide convincing evidence for defining the LEC rat as an animal model for Wilson disease. This model will be important for studying liver pathophysiology, for developing therapy for Wilson disease and for studying the pathway of copper transport and its possible interaction with other heavy metals.

The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene.

Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. The gene (WD) has been mapped to chromosome 13 q14.3. On yeast artificial chromosomes from this region we have identified a sequence, similar to that coding for the proposed copper binding regions of the putative ATPase gene (MNK) defective in Menkes disease. We show that this sequence forms part of a P-type ATPase gene (referred to here as Wc1) that is very similar to MNK, with six putative metal binding regions similar to those found in prokaryotic heavy metal transporters. The gene, expressed in liver and kidney, lies within a 300 kb region likely to include the WD locus. Two WD patients were found to be homozygous for a seven base deletion within the coding region of Wc1. Wc1 is proposed as the gene for WD.

The Wilson disease gene: spectrum of mutations and their consequences.

We have previously reported the cloning of a gene that encodes a copper transporting P-type ATPase (ATP7B) which is defective in Wilson disease. We have now identified in 58 WND patients, 20 new mutations as well as three of five previously published mutations: 11 small insertions and deletions, seven missense, two nonsense and three splice site mutations. Two of the mutations are relatively frequent, representing 38% of the mutations in patients of European origin. Our findings suggest a wider spectrum of age of onset than is considered typical of Wilson disease: mutations that completely disrupt the gene can produce liver disease in early childhood when Wilson disease may not typically considered in the differential diagnosis. The mutations identified provide an explanation for at least part of the wide phenotypic variation observed in Wilson disease.

Human microphthalmia associated with mutations in the retinal homeobox gene CHX10.

Isolated human microphthalmia/anophthalmia, a cause of congenital blindness, is a clinically and genetically heterogeneous developmental disorder characterized by a small eye and other ocular abnormalities. Three microphthalmia/anophthalmia loci have been identified, and two others have been inferred by the co-segregation of translocations with the phenotype. We previously found that mice with ocular retardation (the or-J allele), a microphthalmia phenotype, have a null mutation in the retinal homeobox gene Chx10 (refs 7,8). We report here the mapping of a human microphthalmia locus on chromosome 14q24.3, the cloning of CHX10 at this locus and the identification of recessive CHX10 mutations in two families with non-syndromic microphthalmia (MIM 251600), cataracts and severe abnormalities of the iris. In affected individuals, a highly conserved arginine residue in the DNA-recognition helix of the homeodomain is replaced by glutamine or proline (R200Q and R200P, respectively). Identification of the CHX10 consensus DNA-binding sequence (TAATTAGC) allowed us to demonstrate that both mutations severely disrupt CHX10 function. Human CHX10 is expressed in progenitor cells of the developing neuroretina and in the inner nuclear layer of the mature retina. The strong conservation in vertebrates of the CHX10 sequence, pattern of expression and loss-of-function phenotypes demonstrates the evolutionary importance of the genetic network through which this gene regulates eye development.

Association between human rhinovirus C and severity of acute asthma in children.

A new and potentially more pathogenic group of human rhinovirus (HRV), group C (HRVC), has recently been discovered. We hypothesised that HRVC would be present in children with acute asthma and cause more severe attacks than other viruses or HRV groups. Children with acute asthma (n = 128; age 2-16 yrs) were recruited on presentation to an emergency department. Asthma exacerbation severity was assessed, and respiratory viruses and HRV strains were identified in a nasal aspirate. The majority of the children studied had moderate-to-severe asthma (85.2%) and 98.9% were admitted to hospital. HRV was detected in 87.5% and other respiratory viruses in 14.8% of children, most of whom also had HRV. HRVC was present in the majority of children with acute asthma (59.4%) and associated with more severe asthma. Children with HRVC (n = 76) had higher asthma severity scores than children whose HRV infection was HRVA or HRVB only (n = 34; p = 0.018), and all other children (n = 50; p = 0.016). Of the 19 children with a non-HRV virus, 13 had HRV co-infections, seven of these being HRVC. HRVC accounts for the majority of asthma attacks in children presenting to hospital and causes more severe attacks than previously known HRV groups and other viruses.

The impact of MRSA infection in the airways of children with cystic fibrosis; a case-control study.

The prevalence of Methicillin Resistant Staphylococcus Aureus (MRSA) in patients with Cystic Fibrosis (CF) has risen dramatically over the past 10 years. The clinical significance of MRSA in CF patients remains undetermined. We conducted a review of patients with CF infected with MRSA over a 10 year period at Our Lady's Children's Hospital, Crumlin between 1999 and 2009. We collected data from 24 patients infected with MRSA and 24 control patients without MRSA There was a significant difference between the two groups in the rate of decline in percentage FEV1 two years after MRSA infection (Difference: -17.4, 95% CI: -30.48, -4.31, p = 0.01). A similar trend was seen for FVC% and FEF25-75% predicted. This study suggests that persistent MRSA infection in the airways of children with CF is associated with diminished lung function two years post acquisition, when compared to a matched control cohort without MRSA.

A deletion map of the human immunoglobulin heavy chain variable region.

Analysis of VH gene segments deleted in the process of immunoglobulin heavy chain (IGH) variable region assembly in three series of monoclonal B cell lines has been used to determine the human VH region organization. A deletion map of the relative positions of 21 different VH gene segments has been determined. The characterization of B cell lines from three unrelated adults of two racial groups yielded the same relative VH gene segment order, suggesting that the overall order of VH genes in the normal population is constant. This VH gene segment order was consistent with what we had previously generated from physical mapping techniques. DH segments from the second DH cluster, distinct from the major DH locus 3' of the VH region, were not observed to be used in 32 different rearrangements. Approximately 77% of the VH-(D)JH rearrangements involved VH gene segments within 500 kb of the JH region, indicating that human B cell lines preferentially rearrange JH-proximal VH gene segments. The switch, observed in mice, from the fetal use of JH-proximal VH gene segments to an adult VH use dependent upon VH family size may therefore not occur in humans. This detailed map of the VH gene segments is a necessary prerequisite for understanding VH usage in development and disease.

The role of rhinovirus infections in young children with cystic fibrosis.

Rhinovirus (RV) is an important virus in children with chronic respiratory conditions such as asthma; however, little is known about its role in CF. Our aim was to examine the prevalence and clinical impact of different RV species in young children with CF. We collected clinical data and nasal swabs on patients at home and in the hospital setting. Parents filled out symptom diaries and collected nasal swabs when their children were symptomatic and asymptomatic. A novel RV typing PCR assay was used to determine the RV species present. We collected 55 nasal swab samples from ten preschool CF patients over a six month period. The quality of parent collected samples at home was sufficient for PCR analysis. RV was the most common virus detected in young children with CF. There was no difference in the frequency of RV species between symptomatic and asymptomatic subjects. However, parental home-sampling is an acceptable and feasible approach to monitoring young children with CF.

Recommendations for locus-specific databases and their curation.

Expert curation and complete collection of mutations in genes that affect human health is essential for proper genetic healthcare and research. Expert curation is given by the curators of gene-specific mutation databases or locus-specific databases (LSDBs). While there are over 700 such databases, they vary in their content, completeness, time available for curation, and the expertise of the curator. Curation and LSDBs have been discussed, written about, and protocols have been provided for over 10 years, but there have been no formal recommendations for the ideal form of these entities. This work initiates a discussion on this topic to assist future efforts in human genetics. Further discussion is welcome.

A structured simple form for ordering genetic tests is needed to ensure coupling of clinical detail (phenotype) with DNA variants (genotype) to ensure utility in publication and databases.

Researchers and clinicians ideally need instant access to all the variation in their gene/locus of interest to efficiently conduct their research and genetic healthcare to the highest standards. Currently much key data resides in the laboratory books or patient records around the world, as there are many impediments to submitting this data. It would be ideal therefore if a semiautomated pathway was available, with a minimum of effort, to make the deidentified data publicly available for others to use. The Human Variome Project (HVP) meeting listed 96 recommendations to work toward this situation. This article is planned to initiate a strategy to enhance the collection of phenotype and genotype data from the clinician/diagnostic laboratory nexus. Thus, the aim is to develop universally applicable forms that people can use when investigating patients for each inherited disease, to assist in satisfying many of the recommendations of the HVP Meeting [Cotton et al., 2007]. We call for comment and collaboration in this article.

Wilson disease and Menkes disease: new handles on heavy-metal transport.

Little is known at the molecular level about the homeostatic control of heavy-metal concentrations in mammals. Recently, however, two human diseases that disrupt copper transport, Menkes disease and Wilson disease, were found to be caused by mutations in two closely related genes, MNK and WND, which encode proteins belonging to the P-type ATPase family of cation transporters. The MNK and WND proteins are unique in having at their amino termini six copies of a sequence that is remarkably similar to sequences previously found in bacterial heavy-metal-resistance proteins and in a P-type ATPase that appears to form part of a bacterial copper homeostatic system. These two human ATPases are the first putative heavy-metal transporters to be discovered in eukaryotes.

Susceptibility to multiple sclerosis is associated with the proximal immunoglobulin heavy chain variable region.

15 immunoglobulin heavy chain constant (CH) and variable region (VH) polymorphisms were selected to span the entire length of the heavy chain cluster. These polymorphisms were examined in 34 sib pairs concordant for multiple sclerosis (MS) and in 23 sporadic MS patients. Allele frequencies were calculated for the 2 MS patient groups and compared with those found in a control population from the same geographical location and of similar ethnic background. No significant association was found between MS and the 7 CH region polymorphisms examined. However, a significant correlation between the MS phenotype and a VH2 family polymorphism was observed in both MS patient populations (familial MS patients chi 2 = 8.16, P less than 0.005; sporadic MS patients chi 2 = 8.90, P less than 0.005). One allele of the VH2-5 gene segment was found to be over-represented in both MS groups. VH2-5 has recently been physically mapped close to the CH region, between 180 and 360 kb away. These results indicate that a locus near or within the CH-proximal VH region is associated with increased susceptibility to MS.

A null deficiency allele of alpha 1-antitrypsin, QOludwigshafen, with altered tertiary structure.

The most common deficiency allele of the plasma protease inhibitor alpha 1-antitrypsin (alpha 1AT) is PI*Z. Some rare deficiency alleles of alpha 1AT produce low but detectable amounts of plasma alpha 1AT (1-20% of normal), which can be differentiated by isoelectric focusing. Others, designated null (QO) alleles, produce no alpha 1AT detectable by routine quantitative methods. We have previously described a method using DNA polymorphisms, haplotypes, and polyacrylamide isoelectric focusing gels, to differentiate various deficiency alleles. Based on haplotypes, we previously identified, in eight patients, five different null alleles, four of which had been previously sequenced. We have now analyzed all 12 null alleles in these eight patients, using allele-specific oligonucleotide probes, and have identified six different null alleles. We have cloned and sequenced one of these, PI*QOludwigshafen, which has a base substitution in exon II, replacing isoleucine 92 in the normal sequence with an asparagine. This substitution of a polar for a nonpolar amino acid occurs in one of the alpha-helices and is predicted to disrupt the tertiary structure. A total of 13 different alpha 1AT deficiency alleles, 6 of them null alleles, have been sequenced to date.

Twenty-four novel mutations in Wilson disease patients of predominantly European ancestry.

Wilson disease (WND), an autosomal recessive disorder of copper transport, shows wide genotypic and phenotypic variability, with hepatic and/or neurological symptoms. The WND gene, ATP7B, encodes a copper transporting ATPase that is involved in the transport of copper into the plasma protein ceruloplasmin, and in the excretion of copper from the liver. ATP7B mutations result in copper storage in liver and brain. From 247 WND patients worldwide whose DNA has been sequenced in our laboratory, we have identified 24 new mutations. The origins of the patients were European white (one deletion, one nonsense, one splice site, and 18 missense), Chinese (one deletion, one missense) and Bangladeshi (one missense). Most of these had strong support as disease causing mutations, based on conservation between species, structural changes, and absence in controls. One missense mutation in a Chinese patient was considered uncertain because of its conservative nature and position in the protein. We also identified 15 nucleotide substitutions (11 of them new) causing silent or intronic changes, none of which produce an additional splice site that could lead to disease. Characterization of mutations, both disease-causing and normal variants, is essential for accurate molecular diagnosis of this condition.

Current status of human chromosome 14.

Over the past three decades, extensive genetic, physical, transcript, and sequence maps have assisted in the mapping of over 30 genetic diseases and in the identification of over 550 genes on human chromosome 14. Additional genetic disorders were assigned to chromosome 14 by studying either constitutional or acquired chromosome aberrations of affected subjects. Studies of benign and malignant tumours by karyotype analyses and by allelotyping with a panel of polymorphic genetic markers have further suggested the presence of several tumour suppressor loci on chromosome 14. The search for disease genes on human chromosome 14 has also been achieved by exploiting the human-mouse comparative maps. Research on uniparental disomy and on the search for imprinted genes has supported evidence of epigenetic inheritance as a result of imprinting on human chromosome 14. This review focuses on the current developments on human chromosome 14 with respect to genetic maps, physical maps, transcript maps, sequence maps, genes, diseases, mouse-human comparative maps, and imprinting.

Disruption of the neuronal PAS3 gene in a family affected with schizophrenia.

Schizophrenia and its subtypes are part of a complex brain disorder with multiple postulated aetiologies. There is evidence that this common disease is genetically heterogeneous, with many loci involved. In this report, we describe a mother and daughter affected with schizophrenia, who are carriers of a t(9;14)(q34;q13) chromosome. By mapping on flow sorted aberrant chromosomes isolated from lymphoblast cell lines, both subjects were found to have a translocation breakpoint junction between the markers D14S730 and D14S70, a 683 kb interval on chromosome 14q13. This interval was found to contain the neuronal PAS3 gene (NPAS3), by annotating the genomic sequence for ESTs and performing RACE and cDNA library screenings. The NPAS3 gene was characterised with respect to the genomic structure, human expression profile, and protein cellular localisation to gain insight into gene function. The translocation breakpoint junction lies within the third intron of NPAS3, resulting in the disruption of the coding potential. The fact that the bHLH and PAS domains are disrupted from the remaining parts of the encoded protein suggests that the DNA binding and dimerisation functions of this protein are destroyed. The daughter (proband), who is more severely affected, has an additional microdeletion in the second intron of NPAS3. On chromosome 9q34, the translocation breakpoint junction was defined between D9S752 and D9S972 and no genes were found to be disrupted. We propose that haploinsufficiency of NPAS3 contributes to the cause of mental illness in this family.