Cystic Fibrosis and Formes Frustes of CFTR-Related DiseaseSouthern K.W.
Royal Liverpool Children’s Hospital, Institute of Child Health, University of Liverpool, Liverpool, UK Corresponding Author
Cystic fibrosis (CF) is the commonest genetic cause of bronchiectasis in the Caucasian population. Since identification of the putative gene in 1989, the molecular basis of the condition has become clearer with characterisation of the unique pathophysiology. The small airways are the primary site of lung disease, with an intense but localised inflammatory picture, dominated by neutrophils. The clinical heterogeneity is explained to some degree by the distinct molecular consequences of the many mutations that have been recognised to affect the CF transmembrane conductance regulator (CFTR) gene; however other genes appear to modify the phenotype as well as environmental exposure. It has become increasingly apparent that certain conditions may result from CFTR dysfunction without fulfilling diagnostic criteria for CF. In some cases this may result in single organ disease for which the term CF (or CFTR)-related disease has been advocated. Congenital bilateral absence of the vas deferens is the most clearly characterised of these. In other cases where a mild CF phenotype is apparent, atypical CF is probably a better term. It remains unclear whether carrier status predisposes to certain conditions such as chronic rhinosinusitis or pancreatitis.
Copyright © 2007 S. Karger AG, Basel
Cystic fibrosis (CF) is a recessively inherited condition and the commonest genetic cause of bronchiectasis in the Caucasian population . The putative gene is the CF transmembrane conductance regulator (CFTR) . This paper will reflect on background to the condition, characterisation of the pathophysiology and consideration of atypical forms associated with CFTR dysfunction.
History of CF
Dorothy Andersen, a remarkable physician and paediatric pathologist, is credited with the first clear description of the CF phenotype in 1938 . At that point, prognosis was appalling and death from respiratory failure invariably occurred in the 1st decade of life. Shortly after Andersen’s description of the clinical condition, it was demonstrated that the disease followed an autosomal recessive inheritance . As well as a clear clinical characterisation, excess electrolytes were identified in sweat, and collection of sweat with pilocarpine iontophoresis and measurement of salt concentration became established as the diagnostic test . In the 1970s the transepithelial salt transport defect was characterised in the sweat gland and later in the airways [6, 7]. In the 1980s genetic linkage studies located the gene defect on the long arm of chromosome 7 and subsequent innovative techniques (chromosome walking and jumping) resulted in the identification and characterisation of the CFTR gene in 1989 . The gene spans over 250,000 base pairs and 27 exons code for a protein with 1,480 amino acids, which when fully processed is a glycosylated transmembrane channel with unique molecular features . Protein purification and gene transfer studies have demonstrated that CFTR can function as a chloride channel, as well as having interactions with other ion channels in epithelial cells, most notably the epithelial sodium channel (ENaC) in the airways [9, 10]. This paper will focus on the pathophysiology that results from disruption of CFTR function and will consider atypical conditions with CFTR dysfunction.
Genetics of CF
The high incidence of CF in the Caucasian population is the result of one mutation, a deletion of three base pairs (codon deletion) that results in the loss of phenylalanine at position 508 on the protein (in the first of two ATP-binding domains); termed ΔF508 or more recently phe508del . The molecular consequence of this and other mutations will be discussed later. Why phe508del is such a prevalent mutation remains unclear. The population incidence of CF directly correlates to the frequency of this mutation in that population (ranging from about 1 in 2,500 in the Northern European population to 1 in 32,000 in Eastern Asia) [12, 13]. The most plausible theory to explain a heterozygote advantage is the protection against severe diarrhoeal disease such as cholera; however it is not clear why this should be unique for phe508del [11, 14]. Codon deletions are unusual mutations and it is likely that all phe508del alleles derive from one genetic event, which occurred at an early point of human population drift (more than 10,000 years ago). The population frequency of phe508del varies considerably, even in Europe where incidence is highest (fig. 1). Outside Europe some continents (such as South East Asia) have a very low prevalence of phe508del and a low population incidence of CF. In Ashkenazi Jews, W1282X accounts for nearly 50% of mutated CFTR alleles and this is the only significant population in which phe508del is not the dominant CF-causing mutation .
|Fig. 1. Prevalence of the CFTR mutation, phe508del, as a percentage of all recognised CFTR mutations in the country (generated from data contained in the review of Bobadilla et al. ). The prevalence of phe508del is associated with the population incidence of CF (countries where phe508del is the most prevalent mutation have the greatest population incidence). A variation in phe508del prevalence is evident and there appears to be a northerly predisposition. Prevalence in Mediterranean countries is generally low, however there are contradictions to the North/South divide (Finland/Albania for example).|
Although phe508del is by far the most common CFTR mutation, there are over 1,500 reported mutations of the CFTR gene and an international database is a useful reference point for information on the phenotypic consequences . Interestingly, the most prevalent CFTR mutations aside from phe508del are thought to originate from ancient populations (G542X, Phoenicians ; G551D, Celtic , and 394delTT, Nordic ), though none approach the prevalence of phe508del. Most are point mutations and the molecular consequence depends on the site and nature of the gene change [18, 19]. A classification was developed to illustrate this (table 1). Distinct relationships exist between certain classes of CFTR mutations and phenotypes, however these are looser than expected and the phenotypic consequences of a significant number of CFTR gene changes remain unclear. Non-CFTR genes that modify the CF phenotype and environmental exposure both have a significant impact on clinical outcome.
|Table 1. Classes of CFTR mutations, with molecular and phenotypic consequences|
The impact of CFTR dysfunction on airway physiology is profound and results in a predisposition for chronic airway infection and inflammation . A key component of lung health is the constant clearance of surface liquid (removing particles from the distal airways every 10–15 min) . Airway surface liquid (ASL) is composed of two distinct parts, an upper mucus layer, termed the gel, and a periciliary liquid layer, termed the sol. Co-ordinated ciliary beating results in distal to proximal flow of ASL, with fluid absorption occurring in the larger airways. There is now compelling evidence that the clearance and composition of ASL is compromised in CF . A paradox in CF is that CFTR gene expression levels are low in the airways compared to other epithelia (table 2) [23, 24]. Despite this there appears to be a significant impact on transepithelial airway ion transport and this appears to be the fundamental defect in CF.
|Table 2. Six paradoxes associated with the CF condition|
In the airway epithelia, absence or dysfunction of CFTR results in over-absorption of water from ASL through increased sodium ion transport (CFTR normally downregulates ENaC) [22, 25]. In addition, the ability of the airways to respond to dehydrating stresses through fluid secretion is compromised as CFTR-mediated chloride secretion has a role in this function [26, 27]. Therefore in CF, ASL is prone to dehydration and this has an impact on mucus composition in the luminal gel layer. Ciliated cells from transplanted lungs grown in tissue culture with an air-liquid interface demonstrate that the CF ion transport defect results in reduced ASL volume, ciliary compression and disabled mucociliary transport .
Mucus is secreted by goblet cells in the proximal tubules of submucosal glands (and by goblet cells in the airways). The more distal serous cells in the submucosal glands express significant amounts of CFTR and appear to have a fluid secretory role (to eject the mucus secreted in more proximal tubules) . In CF, this serous cell function is absent, but mucus secretion is increased (possibly in response to dehydrated ASL and inflammation) [30, 31]. Inhibiting CFTR function in ex vivo pig submucosal glands results in reduced fluid secretion and a threefold increase in mucus viscosity, supporting the assertion that submucosal gland dysfunction is a key factor in CF airway disease [32, 33]. It remains unclear whether the abnormal composition of mucus in CF is an intrinsic defect (i.e. CFTR has a role in mucin gene function) or secondary as a result of the altered CF airway phenotype and ASL dehydration [34, 35]; whichever is the case, defects in mucus composition and hypersecretion significantly exacerbate the disabled airway clearance in CF.
A second paradox in CF is that, despite the profound defect in mucociliary transport that is evident in vitro, many people with CF maintain excellent respiratory function throughout their early years with standard therapies to aid airway clearance and prevent/treat infection . In addition, radioisotope studies show that people with CF are able to clear labelled particles from their distal airways and studies have demonstrated conserved mucociliary transport in CF [37,38,39]. Experiments on the in vitro tissue culture model may give some explanation to this paradox . It was noted that absent mucociliary transport was corrected if the CF culture dish was moved in a distinct manner. By applying phasic shear stress to the culture, fluid secretion through non-CFTR chloride transport was established. This enabled restoration of ASL volume and ciliary function. In vivo, shear stresses are applied to the airway epithelia by air movement, and the group designed their tissue culture movement to correspond to tidal breathing manoeuvres .
The Pathology of CF Lung Disease
The CFTR molecular defect results in an airway environment that is susceptible to unusual and chronic infection . The newborn infant with CF has macroscopically normal lungs and many maintain good respiratory condition throughout their childhood and even into early adult life, possibly as a result of the mechanisms described above. However the majority develop chronic airway infection. The trigger for chronic airway infection remains to be determined; however, in some cases viral infection of the airways may possibly disrupt the alternative fluid secretory mechanisms that maintain airway clearance . During this window of disabled airway clearance, bacterial infection may result in the cycle of inflammation, airway damage and further infection that characterises established CF lung disease. Patients with CF do not generally become systemically unwell with airway infection . A recent study of bronchoscopic lavage in screened infants with CF suggests that infection is required for lower airway inflammation, although previous studies have suggested contrasting results [43, 44].
Characteristic pathogens isolated from the CF airways include Staphylococcus aureus, Haemophilus influenzae and Moraxella catarrhalis, all common respiratory pathogens in childhood . Often these infections are transient, and in some countries prophylactic antibiotics are employed to prevent these infections, in particular S. aureus . A more entrenched infection that characterises CF lung disease is with Pseudomonas aeruginosa (PA) . Again it is not completely clear why this organism has a predilection for the CF airway, but, despite increasing use of eradication protocols, chronic PA airway infection remains common in people with CF . Once in the airways, PA undergoes a change in phenotype, possib- ly stimulated by an increasingly hypoxic environment, which is important in establishing chronic infection . A process of quorum sensing has been recognised and it is apparent that PA in CF airways express genes that activate this phenomenon . The PA sense each other and in response to a stressful hypoxic environment, secrete alginate gel and reduce their metabolism . The intra-luminal colonies of PA induce a powerful innate immune response with considerable neutrophil migration into the airways . A significant functional antibody response is mounted against the PA, however clearance is incompetent, possibly reflecting lack of avidity .
Inflammation and Lung Damage
The response of the CF airways to insult is unique. The inflammatory picture is one of an acute response, with neutrophils predominant and an intense but localised release of inflammatory mediators . Despite the chronic nature of the condition, the inflammatory response remains acute in nature with remarkable preservation of distal lung tissue (fig. 2) . Interleukin-8 is secreted in large amounts and all other key components of the inflammatory pathway are increased (Smad3, RhoA and NF-κB) [52, 53]. Inflammatory mediators have a profound impact on the surrounding airway architecture with cell infiltration and abnormal airway remodelling [54, 55]. Secretion of proteases (such as neutrophil elastase) results in structural damage with bronchiectasis of small and large airways, leading eventually to end-stage lung disease with abscess formation and pneumothorax, both prominent features .
|Fig. 2. Micrographs of lung tissue demonstrating pathological features associated with typical CF. Intense neutrophil-mediated inflammation localised to the airways (bold arrow) and relative sparing of surrounding distal lung tissue (thin arrow).|
For some time there has been debate as to whether the CF airway is intrinsically pro-inflammatory or whether the intense inflammatory response is a result of the unique infective environment. In favour of the first argument are studies demonstrating inflammatory mediators in the lavage fluid following bronchoscopy in well CF infants with no apparent lower airway infection  and an inflammatory profile identified in CF epithelial cells grown ex vivo [56,57,58]. Prolonged exposure of primary airway epithelial cells to a CFTR inhibitor did result in an increase in the inflammatory profile of those cells, despite minimal impact on sodium transport and no apparent infection . Recent work examining histone acetylation may have identified a mechanism for this pro-inflammatory profile, however further work is required to clarify this area [57, 58, 60]. Beyond debate is the fact that the inflammation present in CF airways is one of the most intense and sustained in human pathophysiology, making the localised nature of the process even more remarkable (table 2). The two hypotheses, dehydrated ASL through the CF ion transport defect and an intrinsic pro-inflammatory state are not incompatible.
Characteristic Clinical Picture
The respiratory phenotype is part of a distinct clinical picture that characterises typical CF. A challenge to the CF team is the increasingly heterogeneous clinical picture that is associated with CFTR dysfunction and the implication to individuals and families that such a diagnosis holds. The classic CF phenotype is well described and forms part of the diagnostic criteria . In addition to sino-pulmonary disease; pancreatic insufficiency resulting in malabsorption, liver disease, sweat salt loss and absence of the vas deferens are all characteristic. Whether people with CF have intrinsic renal or hearing impairment remains unclear. The heterogeneity of the typical CF phenotype can be explained, to some degree, by the distinct molecular consequences of the different classes of CFTR mutations (table 1). Certainly class IV and V mutations are more likely to be associated with pancreatic sufficiency (which is often associated with a milder respiratory phenotype) [19, 62]. Increasingly data from epidemiological studies suggest that there is a genetic predetermination as to whether a person with CF will develop significant CF-related liver disease .
Some Principles of Treatment
The expectations of CF patients and carers have changed significantly as we move into a new millennium. Recent data from the US Registry suggest median survival is moving toward 40 years, despite fears that conventional therapies had achieved their ceiling (http://www.cff.org/research/2006NACFC/Plenary III). The cornerstones of CF care remain excellent nutritional provision, pro-active treatment of chest infection and maintenance of an active healthy lifestyle. The multi-disciplinary team is critical in achieving these goals. Expansion of newborn screening for CF to more areas with high CF prevalence has provided CF carers with the opportunity to intervene at earlier points in the pathophysiological pathway . Together with this opportunity is the duty of the CF team to provide the family with a clear pathway, in order that they can clearly picture the progress their infant will make and the potential problems that they will face as a family. To have realistic hope of a more fundamental treatment intervention is necessary. The issue of sustainability of current treatment regimes becomes ever more pertinent. It is imperative that CF teams work with their patients and families in an open and frank manner to ensure that therapeutic interventions are introduced appropriately. In the progress of CF care, there have been some stumbles (the spread of epidemic bacterial strains  and identification of fibrosing colonopathy following the introduction of high-dose pancreatic enzyme supplements to name but two examples [65, 66]), however there is also good evidence that great strides have been taken toward better survival and, as importantly, quality of life. Two major advances have probably been most pertinent in achieving this – firstly the recognition of the importance of a multi-disciplinary team approach and secondly the assertion that high-calorie diets and attention to nutrition are essential  – supported by subsequent epidemiological studies that confirmed the importance of good nutrition in maintaining good respiratory health in CF . The challenge today for the CF team is to maintain good eating behaviour throughout childhood to achieve the maximum potential adult height. An illustration of the change in emphasis towards long-term nutritional well-being is the far more active nutritional intervention (often intravenous) that teams now employ for babies following neonatal surgery, in order to ensure that the early days and weeks of growth are not missed. Some indication of the importance of early nutrition has come from the work of the Wisconsin Newborn Screening project, data which demonstrated a close relationship between vitamin E levels at diagnosis and subsequent cognitive function entering school .
Most CF carers agree that the early and pro-active treatment of bacterial chest infection is an essential component of CF management, however many questions remain concerning the detail of care, for example:
Should we employ prophylactic antibiotics to reduce or prevent S. aureus infection ?
Should asymptomatic patients undertake regular chest physiotherapy for airway clearance ?
How should we treat a first (or recurrent) growth of PA?
What is an appropriate point to start nebulised dornase therapy (and same question for ibuprofen/azithromycin/hypertonic saline) ?
Should we immunise actively against PA and passively against respiratory syncytial virus ?
Despite systematic review of therapies  and an increasing number of well-designed clinical trials , a significant degree of variability in practice persists both across continents and regions. An increasing understanding of the pathophysiology of CF chest disease will help to determine more clearly treatment strategies and hopefully lead to novel therapeutic interventions.
Atypical Forms of CF
A key question is whether mild CFTR dysfunction can be associated with single organ disease. The following section will reflect on the evidence supporting formes frustes of CFTR-related disease.
Certain CFTR mutations are clearly associated with a milder CF phenotype. The most prevalent of these is the 5T variant of a poly T region of intron 8 [62, 75, 76]. This class V mutation can result in a mild CF phenotype when found as a compound heterozygote with a severe mutation and has been associated with atypical CF in homozygosity . Additionally 5T can occur in trans with R117H (a mild class IV mutation). This double hit on CFTR production and function invariably results in a significant disease phenotype. Given that men with CF frequently have absence of the vas deferens , a number of groups examined men attending fertility clinics for evidence of CFTR mutations [75, 79, 80]. A high prevalence of CFTR mutations was identified in men with congenital bilateral absence of the vas deferens (CBAVD), and the incidence of 5T was particularly high. In a small number diagnostic criteria for CF were fulfilled and it was evident that these men had CF, with lung and sweat gland involvement. However in a significant cohort the only phenotypic consequence was CBAVD and a strong argument can be raised that in these cases CBAVD represents a CFTR-related disease, rather than an atypical mild form of the condition.
Another condition that may constitute a CFTR-related disease is idiopathic pancreatitis [81, 82]. It is somewhat surprising, given the deranged anatomy of the CF pancreas, that pancreatitis is unusual in people with typical CF, however incidence is increased in people with CF who are pancreatic sufficient . Genetic studies of people with idiopathic pancreatitis have identified candidate genes (such as trypsinogen) associated with the condition . They have also demonstrated a greater than expected frequency of CFTR mutations, prompting the speculation that in certain circumstances, idiopathic pancreatitis represents a single organ pathology related to CFTR dysfunction [81, 82,85,86,87,88]. These studies identify a small number of individuals with two CFTR mutations that conform to a diagnosis of CF. The question arises as to whether idiopathic pancreatitis is a presentation of mild CF or represents a distinct CFTR-related entity, similar to CBAVD.
Another unusual condition, which occurs at increased frequency in people with CF, is allergic bronchopulmonary aspergillosis (ABPA) . Studies on people without CF who have ABPA have demonstrated increased frequency of CFTR mutations and again in some cases these studies have resulted in the recognition of mild cases of CF [90,91,92]. However, data on whether ABPA is associated with CFTR dysfunction are much less robust, given the small numbers examined, and further work is required in this area. A more common condition is chronic rhinosinusitis, which may affect 10–15% of certain populations . Two studies have found increased frequency of CFTR mutations in children and adults with chronic rhinosinusitis, suggesting that CF carriers may be susceptible to this condition [94, 95]. In the adult study, 9 of 10 men with a CFTR mutation were recognised to also have M470V. This gene change occurs at a high frequency in the general population and was considered a polymorphism; indeed M470V has increased chloride channel conductance, however, balancing against this M470V may affect protein maturation which despite its increased channel activity has a negative impact when in association with other mild class IV or V mutations . It has been demonstrated (in European populations) that the M470V allele associates more commonly with non-phe508del CFTR mutations than expected (in fact it is by far the major variant in these alleles) . The increased incidence of CFTR mutations in people with chronic rhinosinusitis prompted a prospective questionnaire study of parents of CF children . A higher than expected incidence of chronic rhinosinusitis was identified. These results raise the possibility that carriers may be predisposed to rhinosinusitis, however there are concerns about the specificity of the questionnaire and increased self reporting. Carrier status does have phenotypic consequences; for example, a higher than expected frequency of carrier infants are recognised by newborn screening because of persistent hypertrypsinaemia. This is supported by gene analysis of 10,000 infants born in Italy, which demonstrated that carrier infants had significantly increased immunoreactive trypsinogen levels compared to non-carriers . Carriers also have a small but significantly raised level of sweat salt . It does not seem unreasonable therefore that under certain circumstances carriers of a CFTR mutation may experience symptoms related to suboptimal CFTR function. Although rhinosinusitis is a common complaint in CF, the evidence would suggest that carrier status may predispose and that this is not a forme fruste of the condition.
The Diagnostic Challenge
Identification of the genetic basis of CF has improved diagnosis in most cases, however it still presents challenges in small but significant number and in some ways is more complex than ever . Improving technology means than sequencing the CFTR gene locus is increasingly straightforward and sometimes cheaper than specific mutation panels. There is a temptation to forgo physiological tests and rely on extensive gene analysis, however in patients with atypical presentation this may be disappointing and ultimately misleading. Extensive genotyping on a large cohort of patients with equivocal diagnosis from across the USA demonstrated that recognition of two CF-causing mutations was often achieved, however, in a significant number no mutation was recognised on either allele (this was particularly the case if no common mutation had been noted prior to referral) . The authors produced an intriguing argument (supported by two family studies and in vitro functional studies) that in some cases a mild CF phenotype can occur with normal CFTR gene expression and function. Further studies are needed to examine the possibility of a CF phenotype with normal CFTR function. Certainly mutations outside the CFTR locus may affect gene expression and this has to be taken into consideration. In addition, it is being increasingly recognised that large segment deletions or duplications are not infrequent causes of CF and are sometimes difficult to detect with standard techniques [103, 104].
Extensive DNA analysis is a key part of the assessment of an individual with an atypical presentation; however it is important that this is conducted together with tests that demonstrate a physiological abnormality consistent with CF. Measuring sweat salt levels remains a valuable procedure despite a lack of normal reference range data in adults. Sweat salt levels may be equivocal in atypical CF and this often prompts referral for further investigation . Some mutations (most commonly 3,849 + 10 kb C→T) result in normal sweat salt levels with characteristic pulmonary disease and should be considered in difficult cases . In contrast, a nonsense mutation (S1455X) results in truncation of the final 26 amino acids in CFTR and people with this mutation have a positive sweat test with raised sweat chloride levels, but no evidence of lung disease .
In the circumstance of clinical suspicion with incomplete gene analysis and equivocal sweat tests, it is appropriate to consider other electrophysiological tests to provide further information to help confirm or refute a diagnosis . A number of assays exist to identify a CF transepithelial ion transport defect, but most commonly this involves measurement of nasal potential difference or intestinal current measurement . Most intestinal current measurements are undertaken in the laboratory (ex vivo) on tissue obtained from rectal biopsy [108, 109]. Nasal potential difference, on the other hand, is measured in vivo and can be challenging in young patients . Both techniques require a significant degree of experience to undertake effectively and are not routinely available, however both can provide useful information to aid the clarification of a difficult diagnosis. Networks have been established in Europe and North America to provide access to such investigations.
Despite advances in DNA analysis and physiological tests, the most important element of the assessment of a patient with atypical presentation remains a full and thorough clinical evaluation with comprehensive investigation for evidence to support the multi-system nature of the condition.
The social and financial implications of a diagnosis of CF depend to some degree on health care provision and insurance in individual countries and states. In some cases, a patient can be significantly disadvantaged by a diagnosis of CF in a number of aspects of life, including obtaining life insurance and home mortgage. Balanced against this is the considerable advantage that a CF diagnosis can provide with respect to the initiation of appropriate care and advice. Most CF teams are adaptable in overcoming these issues but the situation is unlikely to improve. A clear classification is therefore desirable. The North American diagnostic criteria for classical CF are widely accepted and in the majority of cases the diagnosis is clear. Individuals who do not conform to these criteria but have a mild phenotype (although the term mild needs to be used with care as a significant number of these individuals can have quite marked lung disease albeit at a later stage) are often referred to as having atypical CF (table 3). Single organ disease that does not conform to a diagnosis of CF, but is associated with CFTR mutation, is increasingly being called CF (or CFTR)-related disease and this seems a sensible classification. Further studies are required to determine clearly whether carrier status predisposes to conditions such as chronic rhinosinusitis and idiopathic pancreatitis.
|Table 3. A proposed diagnostic classification|
The unique pathophysiological features and variable nature of the condition continues to provide a significant challenge to the CF physician. Technical advances have increased our understanding of the underlying disease process and have clarified diagnostic aspects, however difficulties remain. In some ways, the journey has been cyclical with a return to the appreciation that robust physiological testing is often necessary to confirm a diagnosis. An increased awareness and understanding of the pathophysiology of CF will lead to more consistent management strategies and hopefully therapeutic interventions that target the basic defect.
I would like to thank Dr. Michael Ashworth (Great Ormond Street Hospital, London, UK) for providing the photomicrographs for figure 2.
Kevin W. Southern, MBChB, PhD, FRCPCH
Institute of Child Health, University of Liverpool
Royal Liverpool Children’s Hospital, Eaton Road
Liverpool L12 2AP (UK)
Tel. +44 151 252 5396, Fax +44 151 252 5456, E-Mail email@example.com
Previous articles in this series: 1. Contopoulos-Ioannidis DG, Kouri IN, Ioannidis JPA: Genetic predisposition to asthma and atopy.
Number of Print Pages : 11
Number of Figures : 2, Number of Tables : 3, Number of References : 110
Respiration (International Journal of Thoracic Medicine)
Vol. 74, No. 3, Year 2007 (Cover Date: May 2007)
Journal Editor: Bolliger, C.T. (Cape Town)
ISSN: 0025–7931 (print), 1423–0356 (Online)
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