Vol 1 - No. 1 - February 2012
Bi-monthly Journal of Pediatrics - ISSN 2240-791X
Cystic Fibrosis a prism with thousand sides: a genotype-phenotype analysis
Vitaliti G, Leonardi S, Lionetti E, Salpietro C, La Rosa M
Department of Pediatrics, University of Catania and Messina, Italy
Abstract
Genetic, environmental, and stochastic factors contribute to phenotype variation of diseases in childhood.
Disease variability in patients with Cystic Fibrosis (CF) bearing the same combination of mutations emphasizes the role of genetic background and environment, which appears to
be the rule rather than the exception.
Cystic fibrosis (CF), a single-gene-recessive disorder, is an ideal model for the identification and characterization of factors that cause disease variation.
As a matter of fact its monogenic etiology allow a better and schematic study of the possible gene mutations that characterize the disease variability and severity.
The dysfunctional protein Cystic Fibrosis transmembrane regulator (CFTR) in CF patients conducts chloride across the apical membranes of polarized epithelia.
Loss of CFTR function affects the transport of chloride, sodium, and water across epithelial tissues, leading to inadequate hydration of mucous secretions in CF patients.
Patients with CF manifest disease in the lungs, pancreas, intestine, liver, male reproductive tract, and sweat gland.
The amount of CFTR required by each organ involved in the disease to remain phenotypically “normal” varies, and, similarly, the extent to which each organ contributes to the CF
phenotypes varies in each individual.
Moreover the complex genetic expression is further modified by both environmental factors and other genetic influences such as those coming from modifier genes, that also
contribute to the disease severity.
Thus, these influences increase the number of phenotypic expressions of the same disease.
The aim of this review is to analyse the multiple phenotypic aspects of Cystic Fibrosis related to the genotypic mutations that determine this high variability of the same disease and
to describe our personal clinical experience.
Introduction
Genetic, environmental and stochastic factors contribute to phenotype variation.
However, the relative effect of each component is difficult to assess, especially for common diseases where a myriad of environmental factors may play a role.
In many cases, some portion of phenotype variability can be associated with the nature of the mutations in the disease-causing gene.
However, disease variability in patients bearing the same combination of mutations emphasizes the role of genetic background and environment, which appears to be the rule rather
than the exception (1).
Thus, the current challenge form any studying monogenic disorders is to assess the relative contribution of genetic factors distinct from the disease causing gene and to identify
those genes that modify outcome (2).
Identification of such modifier genes increases our understanding of the elements that affect disease variability and thereby identifies new targets for therapy.
Cystic fibrosis (CF), a single-gene-recessive disorder that affects 60.
000 individuals worldwide, is an ideal model for the identification and characterization of factors that cause disease variation (3-4).
As a matter of fact its monogenic etiology allow a better and schematic study of the possible gene mutations that characterize the disease variability and severity.
The aim of this review is to analyse the multiple phenotypic aspects of Cystic Fibrosis related to the genotypic mutations that determine this high variability of the same disease and
to describe our personal clinical experience.
The genetic background of Cystic Fibrosis
Patients with CF manifest disease in the lungs, pancreas, intestine, liver, male reproductive tract, and sweat gland (5).
Cystic Fibrosis transmembrane regulator (CFTR), the dysfunctional protein in CF patients, conducts chloride across the apical membranes of polarized epithelia (6).
Loss of CFTR function affects the transport of chloride, sodium, and water across epithelial tissues, leading to inadequate hydration of mucous secretions in CF patients.
Obstruction of luminal space follows, and recurrent cycles of inflammation and fibrosis ultimately destroys affected organs (5, 7).
Obstruction of the exocrine pancreas causes intestinal malabsorption and an abnormal nutritional status in almost all CF patients.
Obstructive lung disease is the cause of death in almost 90% of patients (8).
The large number of families with multiple affected offspring facilitated identification of the CF transmembrane conductance regulator (CFTR) gene, an early and remarkable success
in positional cloning, and actually a network of research labs that joined to form the CF Genetic Analysis Consortium has identified over 1, 800 mutations in CFTR
(http://www.genet.sickkids.on.ca/cftr/app).
One mutation, a deletion of three nucleotides causing the loss of a phenylalanine at codon 508 (p.Phe508del or î​F508), accounts for approximately 70% of CF alleles in Caucasian
patients (9).
CF patients homozygous for î​F508 constitute the most common CFTR genotype (about 50% of patients, designated as î​F508 homozygotes from here on), and, as such, have
served as a reference population for genotype/phenotype correlations (10).
In Europe there is a clear gradient in the frequency of ​"F508 mutation from the highest in the northwest (88% in Denmark) to the lowest in the southeast (25% in Turkey) (9).
The remaining mutations are highly heterogeneous, with the vast majority being private or limited to a small number of individuals (9), while de novo CFTR mutations are
exceptionally rare (11).
In literature it is described that CFTR genotype is highly correlated with preservation of some function of the exocrine pancreas (termed ​ pancreatic sufficiency​ ), as that sweat
chloride levels tend to be less elevated in patients with CFTR genotypes associated with preserved pancreatic function (10, 12), while minimal correlation has been found between
CFTR genotype and severity of lung disease, the major cause of mortality for CF patients (4, 13-14).
These observations prompted a search for the causes of disease variation either linked to CFTR genotype and independent to the same genotype pattern.
Classification of CFTR mutations and clinical consequences
Mutations in the CFTR gene have been classified based on the qualitative and/or quantitative impact the have at a cellular level (table 1 and table 2).
- Class I mutations introduce a stop codon prematurely (15-16).
- Class II mutations block the processing of the protein (15).
- Class III mutations produce a CFTR protein that does not function as a chloride channel due to a block in regulation, despite being fully processed and currently located (15-16).
- Class IV mutations also produce a fully processed and correctly located CFTR protein (15-16).
- Class V mutations result in a reduction in the synthesis of CFTR (15).
Consensus by Castellani and colleagues (17) also agrees that a CFTR mutation can be classified as disease causing if it has been shown to:
- cause a change in the amino-acid sequence that severely affects CFTR synthesis and or function
- introduces a premature termination signal (insertion, deletion or nonsense mutation)
- alters the invariant nucleotide of intron splice sites (the first two or last two nucleotides)
- cause a deletion of one or more exons.
Based on their molecular mechanisms, insertions, deletions and non-sense mutations are likely to belong to the CF disease causing group (17-18).
Genotype-phenotype correlation in Cystic Fibrosis and the role of modifiers genes
Cystic fibrosis is a highly heterogeneous condition, with considerable variability in the severity and rate of disease progression in different organs (17).
The greatest variability is observed for the respiratory system, followed by pancreatic function with the lowest being for sweat gland involvement and male infertility (14, 19).
The heterogeneity of the CF phenotype suggests that there are more factors involved in the determination of this variable phenotype, for example environmental factors and other
genetic determinants, such as gene modifiers (19-21) and every organ affected by the disease is controlled by these mechanisms in its phenotypic expression.
Lung disease
The lung function, measured as forced expiratory volume in one second (FEV1), is highly variable among cystic fibrosis patients with identical CFTR genotypes (e.g. !F508
homozygotes) (22).
With the exception of a few mutations that confer a milder pancreatic phenotype (e.g., p.Arg455Glu (23-24), correlation between different CFTR genotypes associated with
pancreatic insufficiency and lung function measures is minimal. Although a portion of the wide variation in pulmonary severity is attributable to genotype, other significant influences
on this phenotype include environmental factors and genetic modifiers that are not related or linked to CFTR (4, 25-29). For example, polymorphisms in the gene encoding
transforming growth factor beta are associated with severity of CF lung disease (26, 30). As a matter of fact, Transforming growth factor beta 1 (TGFb1) has been investigated
numerous times for potential modifier effect on CF.
It is a compelling candidate as TGFb1 genotypes have been shown to alter risk for other lung diseases such as asthma and chronic obstructive pulmonary disease (32-34).
It is a compelling candidate as TGFb1 genotypes have been shown to alter risk for other lung diseases such as asthma and chronic obstructive pulmonary disease (32-34).
Furthermore, TGFb1 plays a key role in processes central to CF lung pathophysiology, such as regulation of inflammation and tissue remodelling (35). One of the largest CF genetic
modifier studies to date, the Genetic Modifier Study (GMS) analyzed 808 !F508 homozygotes drawn from the extreme of lung function (highest 30 percentile and lowest 30
percentile) and reported that alleles in the promoter ("509) and first exon (codon 10) of TGFb1 are associated with worse lung function (26).
This finding was replicated in 498 patients with various CFTR genotypes and was independently confirmed when a haplotype composed of the opposite alleles at –509 and codon
10 were associated with improved lung function (35) . It also appears that gene and environmental interactions are important to consider in evaluating TGFb1, as variation in CFTR
and in MBL2 as well as exposure to second-hand smoke affect association with CF lung function (30, 36-37).
Six studies encompassing over 2.500 CF patients report association between TGFb1 and CF lung function while one study composed of 118 patients did not (38), and another
involving 171 patients (39) found association with worse lung function and the opposite alleles than reported by Drumm and colleagues (26) and Bremer and colleagues (30). In
aggregate, these studies indicate that alleles that increase TGFb1 expression cause worse lung function in CF, although definitive experiments in patients remain to be performed.
Despite the very limited predictive value of CFTR genotype with respect to pulmonary phenotype, certain patterns have been noted. For example, compound heterozygotes with
#F508/A455E have better pulmonary function than individuals who are homozygous for #F508 (24). In individuals with one or two R117H mutations, the severity of lung disease
depends on the presence of a variation in the poly T tract of intron 8 (40-41). Individuals with the 5T variant in cis configuration with the R117H mutation plus a second CFTR
disease-causing mutation usually develop the lung disease of CF, but those individuals with the 7T variant in cis configuration with the R117H mutation plus a second CFTR
disease-causing mutation have a highly variable phenotype, which can range from no symptoms to mild lung disease (42-43).
Because the A455E and R117H mutations are associated with pancreatic sufficiency, the less severe lung disease seen in individuals with these mutations could be the
consequence of better nutritional status.
Disseminated bronchiectasis
Mutations in the CFTR gene are also thought to be involved in other obstructive pulmonary diseases besides CF. In a study of 16 patients with disseminated bronchectasis (an
obstructive pulmonary disorder involving morphological abnormalities that is associated with childhood lung infections and with some genetic disorders and immunodeficiencies), the
frequency of the intron 8 T5 allele was significantly increased when compared to a control population (44).
Among them, 56% of patients analysed carried the T5 allele or an alternative CFTR mutation. In a similar study 32 disseminated bronchiectasis patients were analysed and 13
CFTR mutations were detected (45).
Exocrine pancreatic function
Pancreatic function is abnormal in almost all patients with CF.
Most patients have severe dysfunction (pancreatic insufficiency) leading to steatorrhea and malabsorption that manifests in the first year of life.
A fraction of CF patients (about 10-20%) do not develop steatorrhea and are termed “pancreatic sufficient”.
CFTR genotype is highly predictive of pancreatic exocrine status (46).
Patients with pancreatic sufficiency have at least one “mild” allele (e.g. class IV, partial misplicing mutations of class I or protein stability mutations) whereas pancreatic insufficient
patients are homozygous or compound heterozygous for two “severe” mutations (e.g.
Class I, II or III).
Mutations R117H, R334W and R347P are usually associated with less impaired pancreatic function (47).
There are patients who appears to have delayed transition to pancreatic insufficiency suggesting a possible role for genetic modifiers.
Serum immunoreactive trypsinogen (IRT) is a pancreatic enzyme precursor that is elevated in neonates with CF and, as such, serves as a marker for newborn screening for CF.
In young CF patients that develop exocrine pancreatic insufficiency, IRT levels decline rapidly (48).
Heritability for variation in IRT levels was estimated in 23 sibling pairs with CFTR genotypes associated with pancreatic insufficiency using variance analysis.
Evidence of genetic control was noted in patients at 2 months of age (h2 = 0.51) and at 6 months of age (0.45) (49).
Finally, the link between pancreatic disorders and genotype features in cystic fibrosis patients is also demonstrated by the increased incidence of CFTR mutations in patients with
pancreatitis.
In a study of 134 patients with chronic pancreatitis, about 14% carried CFTR mutations on one allele and 10% had the T5 allele (twice the expected frequency) (50).
In the same year (51) showed that of 27 patients suffering from idiopathic chronic pancreatitis (ICP), 37% carried at least one abnormal CFTR allele.
In two other further studies, of 39 and 20 ICP patients respectively (52-53) at least 30% of patients carried CFTR mutations, demonstrating that these mutations are associated with
idiopathic chronic pancreatitis.
Diabetes
CF-related diabetes mellitus (CFRDM) may present in adolescence.
It is diagnosed in 7% of those aged 11–17 years (54).
The prevalence increases in adulthood as a consequence of the successful management of CF in childhood.
Diabetes in CF patients typically occurs in the absence of obesity and is associated with a significantly worse prognosis.
While CFRDM has been viewed as a condition distinct from diabetes seen in the general population, there are number of clinical and pathologic similarities with type 1 and 2
diabetes mellitus.
Both type 1 and 2 diabetes mellitus show evidence of strong genetic control.
Twin study has used to assess genetic modifier contribution to CFRDM.
Particularly, candidate gene studies for diabetes were informed by the observation that diabetes in CF may share genetic origins with type 2 diabetes (55).
Single nucleotide polymorphisms (SNPs) near the transcription factor 7-like 2 (TCF7L2) gene (56) demonstrate the most consistent association with type 2 diabetes with an
estimated odds ratio of about 1.5 per allele (57).
Treatment with systemic glucocorticoids, an independent environmental risk factor for diabetes in CF (58), obscured the association with TCF7L2.
However, risk for diabetes associated with TCF7L2 was substantially increased (HR 2.9 per allele; P = 0.00011) when patients who had not been treated with glucocorticoids in the
past year were analyzed (59).
Together, these observations indicate that TCF7L2 modifies risk for diabetes in CF patients who have not had recent or prolonged exposure to systemic steroids.
TCF7L2 is postulated to play a role in proliferation and function of the beta cells of pancreatic islets (60-61).
Thus, implication of TCF7L2 as a modifier of risk in CF-related diabetes suggests that factors intrinsic to the beta cells of the pancreas contribute to the development of this common
complication of CF.
Intestinal obstruction. Obstruction of the intestine by abnormal meconium in the neonatal period is a characteristic sign of CF. The condition, termed “meconium ileus” (MI), affects
about 15% of newborns with CF (5). While formally lethal, modern treatments consisting of enemas and/or surgery have reduced mortality to less than 10% (62). CFTR genotype
appears to make a sizable contribution to risk as patients bearing the missense mutation p.Gly551Asp are at considerably lower risk (6.4) than p.Phe508del homozygotes (19.5%)
(63), while patients carrying the non-sense mutation p.Gly542X may be at higher risk (46).
However, genetic modifiers make an important contribution as noted by two observations: 1- recurrence risk for MI in affected siblings (25%) is significantly higher than in unrelated
CF patients (15%) (25), and 2- rates of intestinal obstruction in mouse models of CF differ by strain (i.e., genetic background) (64).
Intriguingly, neonatal intestinal obstruction that is anatomically and temporally similar to humans occur with a prevalence of 100% in a porcine model of CF (65) and 75% in a ferret
model of CF (66). Thus, it appears that humans may have genetic modifiers that protect from this complication.Twins and sibling analyses have been used to estimate heritability of
MI, and a similar phenotype observed in older CF patients termed “distal intestinal obstruction syndrome” (DIOS).
Blackman and colleagues noted that concordance for MI in monozygotic twin pairs (82%) was significantly higher than in dizygotic twin and siblings of similar age and sex (22%; P =
0.009) suggesting that heritability for MI approaches 1.0 (25) . Non-genetic factors must play some role as concordance is not 100% in monozygotic twins. On the other hand, DIOS
showed no differences in concordance rates among monozygotic and dizygotic twins and siblings, indicating little, if any, genetic modifier effect (25). The latter observation is notable
knowing that DIOS was formally termed MI equivalent due to anatomic and pathologic similarities to the neonatal condition.
The intestinal and pancreatic disease in CF leads to substantial disruptions in growth manifesting as short stature and low weight. Aggressive nutritional supplementation has
improved both parameters, but chronic illness also affects patients’ entry into puberty. Thus, the use of growth metrics such as BMI or percent of predicted weight for height (WfH%)
that have been standardized in healthy individuals presents a challenge for assessing nutritional status in CF. Preliminary analysis of “average” BMI displays moderate heritability
(about 0.6) (59). Genetic control is also noted when BMI measures from select ages are used to avoid confounding by effects of the underlying disease process. Initial analysis of
WfH% by the European CF Twin and Sibling Study demonstrated that intra-pair discordance for this measure was similar between 29 monozygotic and 12 dizygotic twin pairs (67).
However, this issue was recently revisited, and a significant trend was noted when comparing intrapair differences in WfH% in 38 monozygotic, 24 dizygotic and 396 sibling pairs.
Modelling of these three classes of CF siblings estimated that genetic factors account for about 80% of variation in WfH% in 466 related patients (about 60% in #F508 homozygotes)
(68). Thus, anthropometric measures also appear to be under genetic control in CF patients.
Further study is needed to assess the degree to which height and weight independently contribute to the heritability of these composite measures. In cystic fibrosis there is also a
liver involvement and liver expression phenotype seems to be also modulated by genetic factors, even if these factors are different from the cystic fibrosis transmembrane regulator
genotype. As a matter of fact Castaldo G et al. studied five sets of cystic fibrosis siblings bearing a strongly discordant liver phenotype, three with genotype #F508/unknown, and
one with genotype unknown/unknown. The siblings of each set were raised in the same family environment. All siblings had pancreatic insufficiency and moderate respiratory
expression.
One sibling of each of the five sets was free of liver involvement, and the other had severe liver expression. Other causes of liver disease (viral, metabolic and genetic other than
cystic fibrosis) were excluded. Authors concluded that liver expression in cystic fibrosis in the five unrelated siblings was determined by modifier genes, inherited independently of
the cystic fibrosis transmembrane regulator gene (69).
Male reproductive tract: congenital absence of the vase deferens (CAVD) and obstructive azoospermia
Congenital absence of the vase deferens (CAVD) usually results from the combination of one severe CFTR mutation on one chromosome with either a mild CFTR mutation or the
5T allele (even in the absence of R117H) on the other chromosome.
However, some overlap exists between the CAVD phenotype and a very mild CF phenotype, with some fraction of individuals with CAVD also reporting respiratory or pancreatic
problems (70-71).
Moreover, the 5T allele may be associated with lung disease in adult females with CF-like symptoms (72).
Moreover, the 5T allele may be associated with lung disease in adult females with CF-like symptoms (72).
Thus, caution must be exercised in attempting to use genotype to predict the future course of individuals initially diagnosed with CAVD only.
Genotype-phenotype correlations are most relevant for genetic counselling of two carriers who have not had an affected child but who have been detected either through evaluation
of at-risk family members or screening programs.
The considerations in predicting the phenotype of potential offspring are the same as described above for CF and CAVD probands.
Prediction of the risk of CAVD from genotype is reasonably reliable, but couples should be aware that mild respiratory and/or pancreatic disease is sometimes seen in individuals
with genotypes usually associated with CAVD.
The mechanism of partial penetrance of the 5T allele for CAVD is due to variation in the length of the adjacent TG tract (estimated at 60% in one study) (73-74).
Thus, interpretation of the disease implications of the 5T variant requires assessment of the number of TG repeats adjacent to the polythymidine tract (74).
Besides CAVD, a higher than expected frequency of CF mutations/variants has been observed in other more common forms of infertility, including obstructive azoospermia and
oligospermia (75).
Unlike the observations in patients with CAVD, men with obstructive azoospermia rarely harboured two distinct CF mutations.
It has been proposed that in absence of a well-defined CFTR mutation, frequent polymorphic CFTR variants may propose to obstructive azoospermia and perhaps other CFTRassociated diseases (73).
Two such variants, the variable (TG) m repeat in front of the polythymidine tract in intron 8 and the frequent M470V variant, producing less functional CFTR, were found to be a risk
for developing obstructive azoospermia, oligospermia and testicular failure (76).
Although the CFTR defect has not been generally implicated in spermatogenesis, a report by Larriba and colleagues (77) suggested that the sperm maturation process can be
delayed in CAVD patients.
Overall, it seems that the male reproductive tract is the most sensitive system of all CFTR-affected tissues to even very minor defects in CFTR function.
Personal studies and clinical experience
Although the correlation of genotype-phenotype in CF has been extensively studied, it is difficult to predict for rare mutations and, given the increasing number of mutations that
have been discovered in these last years, understanding the role of genes on clinical outcomes is becoming more challenging. In our Bronchopneumology and Cystic Fibrosis Unit,
we follow about 105 patients affected by CF. The most of them carries class I-II or III mutations in homozygosis, and these patients show a worse quality of life and long-term
prognosis. When #F508 mutation is implicated in homozygosis, the classic CF phenotype manifests with predominant severe respiratory disease, pancreatic insufficiency,
endocrinologic disorders and male infertility. The estimated life of these patients is about 30-40 years of age, even if actually the better understanding of the disease and the
progress of target therapies allow to these patients a longer estimated life. Nevertheless, other different relationships between genotype and phenotype produce a wild range of
clinical situations.
Among these patients we actually follow a 7 year-old child affected by CF, carrier of the mutation N1303K in homozygosis (N1303K/N1303K) . This kind of mutation belongs to class
II mutations and it is considered severe because it can affect predominantly the pancreas function. As a matter of fact this patient show all the clinical features of classic CF
phenotype (respiratory failure, pancreas insufficiency, growth retardation), moreover since he was one year old, he has already suffered of bronchiectasis, precociously diagnosed
by HRCT scans.
His estimated life is low, his nutritional status is bad and he frequently is admitted to our department for respiratory failure. In recent years it has been acknowledge that there is a
wide clinical spectrum of disease associated with the CFTR mutations. About 10% of the patients present a mild form of CF often involving a single organ dysfunction, in many cases
with mild respiratory symptoms, pancreatic sufficiency and normal or borderline sweat test. The diagnosis of these mild forms of CF has been possible thanks to the development of
II level DNA molecular analysis, that allow a deeper analysis of the CFTR gene, evidencing new mutations in non codifying alleles. These often are mutations in heterozygosis with
class I or II mutations, and these patients in the past were considered as “CF healthy carriers”.
Because of the unusual presentation, the diagnosis of these “non classic” CF forms can be difficult. The disease severity, to some extent, correlates with organ’s sensitivity to CFTR
dysfunction and with the amount of functional protein which is influenced by the type of mutation. In 2001, Leonardi S and colleagues leaded a study on 35 infertile male adults,
whose infertility was caused by congenital absence of vas deferens.
They found that about half of the individuals studied presented a mutation of the CFTR gene, in 35% of cases represented by the #F508 associated to a second mutation, and in
those years the incapacity to identify this second mutation, despite the analysis of the entire codifying sequence, was explained by the presence of the second mutation in the region
of the non codifying gene, with a consequent expression of a “mild” phenotype, involving only the deferens, saving the other organs (lungs, pancreas, liver, sweat glands) (78). In
2005, Leonardi S and colleagues published a case of azoospermia as clinical manifestation of CF in a 46-year-old man, whose diagnosis was missed in childhood. The man was
admitted to our Cystic Fibrosis Unit for respiratory failure, always diagnosed as Obstructive Bronchopneumopathy as he was a smoker and liver cirrhosis associated with
azoospermia. When we analysed his DNA, we found that the patient was carrier of the homozygous 3849+10kbC>T mutation, and this kind of mutation has been frequently reported
in literature in adult patients with normal sweat tests, subsequently leading to a late diagnosis of CF (79). Recently, in 2011, Spicuzza L and colleagues (18) studied the role of the
rare missense N-terminus CFTR P5L mutation in patients affected by Cystic Fibrosis. They studied a cohort of 7 patients carriers of the P5L mutation in association with a severe
class II mutation, evaluating the clinical phenotype of these patients. They found that these patients were affected by a mild CF phenotype, with normal respiratory function and
pancreatic sufficiency. Their quality of life and long-term outcome was surely better than patients with the #F508 in homozygosis. As a matter of fact all patients were in good
nutritional status with two patients slightly overweight. All had pancreatic sufficiency and no hepatobiliary disease was recorded. Both adults and children had no chronic respiratory
symptoms.
Respiratory exacerbations requiring oral antibiotics prescription were uncommon in all patients over the time. Chest HRCT showed the absence of bronchiectasis in all patients
except in one of them. Sputum or throat swab cultures revealed episodic presence of Staphylococcus Aureus, while the occurrence of Pseudomonas Aeruginosa in the sputum was
reported in some occasions only in two patients. Among these patients, 5 had recurrent episodes of dehydration and hypochloronatremia, curiously only during the summer, when
the environmental temperature was high (July-August), sometimes so severe to require hospitalisation. For the two adult males spermiograms were performed and both revealed
normal values.
Clinical features of patients carriers the P5L mutation in heterozygosis are shown in Table 3. Actually in our Department we follow some atypical cases of cystic fibrosis, and most of
them show a mild phenotype of the disease. In December 2011 we admitted to our Cystic Fibrosis day hospital e 52-year-old man, affected by obstructive bronchopneumopathy,
always treated like asthma, and bronchiectasis. He suffered of chronic cough resistant to common antibiotics and his sputum culture was firstly positive for Pseudomonas
Aeruginosa and then positive for Stenotrophomonas Maltophila. In our day hospital he underwent a sweat chloride test that was negative and a venous withdraw for DNA genotype
and the first level analysis revealed the presence of a !F508 mutation in heterozygosis with a second mutation, that we are actually studying with a second level DNA genotype
analysis. We moreover follow two adult brothers with signs of pancreatitis and suffering of infertility have been admitted in our Department. They are respectively 37- and 35- year
old men and they have not suffered of any other relevant diseases until 2005, when they were admitted in another nearby hospital for abdominal pain.
During the hospitalization a pancreatic insufficiency was diagnosed by increased levels of amilasis and lipasis, and spermiograms diagnosed the presence of male infertility. Thus, a
genotype analysis for CF was performed and it revealed in both patients the presence of a N1303K mutation in heterozygosis with a D110 M mutation. Another delayed diagnosis
was made in a 37-year old female that suffered of recurrent bronchitis always treated as asthmatic episodes. The diagnosis of Cystic Fibrosis was made in adulthood when these
respiratory episodes were associated with female infertility. The genetic analysis of the patient revealed the presence of the #F508 in heterozygosis with a 2789+5 G>A mutation.
Another case of CF delayed diagnosis was made in a 21 year-old female with a mild phenotype of CF, characterized by recurrent respiratory infections, without pancreatic
insufficiency and/or other clinical typical signs of the disease. The patients carried a mutation #F508/E193 K. Another mild form of CF was diagnosed in a 5-month-child, whose
neonatal screening was positive and the genetic analysis showed a rare pattern characterized by a severe mutation in one allele (#F508) and two mutations in the other allele
(R1438W and Y1032 C). Actually this child is in good health conditions, her auxologic parameters are in the normal range and her nutritional status is good. She has not yet suffered
of respiratory symptoms and it seems that her long-term prognosis will be good. Table 4 summarize the clinical and genotypic features of the above described patients. The
knowledge of these mild form of cystic fibrosis is important for physicians in order to recognise the disease and to start treatment in an early époque of life. As a matter of fact most
of physicians suggest a CF diagnosis when the patient is affected by the classical clinical signs of the disease (growth retardation, worsening of the nutritional status, recurrent
respiratory symptoms, pancreatic insufficiency).
The improvement of genetic diagnostic analysis has allowed to the scientific community to discover new mild forms of CF, that perhaps in the past were considered as “healthy
carrier forms” and were undervalued, thus that these patients were not identified with a clinical diagnosis.
From our above described personal experience we can speculate that a severe mutation such as class I-II or III mutation (#F508, N1303K, W12182X), when associated with a class
IV or V mutation in the other allele, cause a milder form of the disease, as that the second mutation attenuate the expression of the first mutation, even if this theory should be better
clarified by further studies on these atypical forms of CF.
Concluding remarks
Is it is well known CF is a disorder that results in a complex spectrum of disease phenotypes.
The amount of CFTR required by each organ involved in the disease to remain phenotypically “normal” varies, and, similarly, the extent to which each organ contributes to the CF
phenotypes varies in each individual. Moreover the complex genetic expression is further modified by both environmental factors and other genetic influences such as those coming
from modifier genes, that also contribute to the disease severity.
Thus, these influences increase the number of phenotypic expressions of the same disease.
The reproductive, pancreatic, and nutritional manifestations of CF are somewhat predictable in terms of genotype-phenotype correlation, and also relatively straightforward to treat.
The respiratory manifestations of CF are much less predictable, and despite marked improvements in antimicrobial and anti-inflammatory therapy over the past 40 years, may
inexorably progress to end-stage lung disease. In many cases, nutritional decline presages progression of CF lung disease.
Surely modifying genes have a more important role in the expression and severity of other phenotypic aspects of CF, such as diabetes and intestinal obstruction.
Indeed, although the correlation of genotype–phenotype in CF has been extensively studied, it is difficult to predict for rare mutations and, given the increasing number of mutations
discovered, understanding the role of genes on clinical outcomes is becoming more challenging.
On this regard the analyses of the genetic role in cystic fibrosis phenotype finds an important role in the target for treatments, in order to individualize therapeutic protocols for a
better management of the disease.
References
1- Scriver CR, Waters PJ. Monogenic traits are not simple: lessons from phenylketonuria. Trends Genet 1999;15: 267–272.
1- Scriver CR, Waters PJ. Monogenic traits are not simple: lessons from phenylketonuria. Trends Genet 1999;15: 267–272.
2- Nadeau JH. Modifier genes in mice and humans. Nat Rev Genet 2001; 2:165–174.
3- Drumm M. Modifier genes and variation in cystic fibrosis. Respir Res 2001; 2: 125–128.
4- Cutting GR. Modifier genetics: cystic fibrosis. Ann Rev Genomics Hum Genet 2005; 6: 237–260.
5- Welsh MJ, Ramsey BW, Accurso FJ, Cutting GR. Cystic Fibrosis. In The Metabolic and MolecularBases of Inherited Disease, Vol III 2001. Scriver CR, Beaudet AL, Valle D & Sly
WS, Eds.:5121-5188, Mc Graw-Hill, Inc. New York.
6- Anderson MP, Gregory RJ, Thompson S, et al. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 1991;253: 202–205.
7- Cutting GR. Cystic fibrosis. In Emery and Rimoin’s Principles and Practice of Medical Genetics, 2007, Vol. 2. Rimoin DL, Connor JM, Pyeritz RE and Korf BR, Eds.: 1354– 1394.
Churchill Livingstone Elsevier. Philadelphia.
8- Cystic Fibrosis Foundation. Cystic Fibrosis Foundation Patient Registry Annual Data Report 2005. Cystic Fibrosis Foundation. Bethesda, MD.
9- Bobadilla JL, Macek M, Fine JP, et al. Cystic fibrosis: a worldwide analysis of CFTR mutations— correlation with incidence data and application to screening. Hum Mutat 2002;19:
575–606.
10- The Cystic Fibrosis Genotype-Phenotype Consortium. Correlation between genotype and phenotype in patients with cystic fibrosis. N. Engl. J. Med. 1993;329: 1308–1313.
11- Cremonesi L, Cainarca S, Rossi A, et al. Detection of a de novo R1066H mutation in an Italian patient affected by cystic fibrosis. Hum Genet. 1996;98:119-21.
12- Wilschanski M. Dupuis A, Ellis L, et al. Mutations in cystic fibrosis transmembrane regulator gene and in vivo transepithelial potentials. Am J Respir Crit Care Med 2006; 78:787–
794.
13- Mickle JE, Cutting GR. Genotype-phenotype relationships in cystic fibrosis. Med Clin North Am 2000; 84:597–607.
14- Zielenski J. Genotype and phenotype in cystic fibrosis. Respiration 2000; 67: 117–133.
15- Zielenski J, Tsui LC. Cystic fibrosis: genotypic and phenotypic variations. Annu Rev Genet. 1995;29:777-807.
16- Welsh MJ, Smith AE. Molecular mechanism of CFTR chloride channel dysfunction in cystic fibrosis. Cell 1993;73:1251-4
17- Castellani C, Cuppens H, Macek M Jr, et al. Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice. J Cyst Fibros. 2008;7:179-96.
18- Spicuzza L, Sciuto C, Di Dio L, Mattina T, Leonarsi S, Miraglia del Giudice M, La Rosa M. mild cystic fibrosis in patients with the rare P5L CFTR mutation. Journal of Cystic
Fibrosis. Article in Press.
19- Kerem E, Kerem B. Genotype-phenotype correlations in cystic fibrosis. Pediatr Pulmonol. 1996;22:387-95.
20- McKone EF, Emerson SS, Edwards KL, et al. Effect of genotype on phenotype and mortality in cystic fibrosis: a retrospective cohort study. Lancet. 2003;17;361:1671-6.
21- McKone EF, Goss CH, Aitken ML. CFTR genotype as a predictor of prognosis in cystic fibrosis. Chest. 2006;130:1441-7.
22- Kerem E, Corey M, Kerem BS, et al. The relation between genotype and phenotype in cystic fibrosis— analysis of the most common mutation (deltaF508) . N Engl J Med
1990;323: 1517–1522.
23- Gan KH, Veeze HJ, Van Den Ouweland AMW, et al. A cystic fibrosis mutation associated with mild lung disease. N. Engl. J. Med. 1995; 333: 95–99.
24- De Braekeleer M, Allard C, Leblanc JP, et al. Genotype-phenotype correlation in cystic fibrosis patients compound heterozygous for the A455E mutation. Hum Genet
1997;101:208–211.
25- Blackman SM, Deering-Brose R, McWilliams R, et al. Relative contribution of genetic and nongenetic modifiers to intestinal obstruction in cystic fibrosis. Gastroenterology
2006;131:1030–1039.
26- Drumm ML, Konstan MW, Schluchter MD, et al. Genetic modifiers of lung disease in cystic fibrosis. N Engl J Med 2005;353:1443–1453.
27- Vanscoy LL, Blackman SM, Collaco JM, et al. Heritability of lung disease severity in cystic fibrosis. Am J Respir Crit Care Med 2007;175:1036–1043.
28- Braun AT, Farrell PM, Ferec C, et al. Cystic fibrosis mutations and genotype-pulmonary phenotype analysis. J Cyst Fibros 2006;5:33–41.
29- Goss CH, Newsom SA, Schildcrout JS, et al. Effect of ambient air pollution on pulmonary exacerbations and lung function in cystic fibrosis. Am J Respir Crit Care Med
2004;169:816–821.
30- Bremer LA, Blackman SM, Vanscoy LL, et al. Interaction between a novel TGFB1 haplotype and CFTR genotype is associated with improved lung function in cystic fibrosis.
Hum Mol Genet. 2008;17:2228-37.
31- CFTR genotype is associated with improved lung function in cystic fibrosis. Hum Mol Genet 2008;17:2228–2237.
32- Pulleyn LJ, Newton R, Adcock, et al. TGFbeta1 allele association with asthma severity. Hum Genet 2001;109: 623–627.
33- Wu L, Chau J, Young RP, et al. Transforming growth factor-beta1 genotype and susceptibility to chronic obstructive pulmonary disease. Thorax 2004 ;59: 126–129.
34- Silverman ES, Palmer LJ, Subramaniam V, et al. Transforming growth factor-beta1 promoter polymorphism C-509T is associated with asthma. Am J Respir Crit Care Med
2004;169: 214–219.
35- Akhurst RJ. TGFbeta signaling in health and disease. Nat. Genet. 2004; 36: 790–792.
36- Dorfman R, Sandford A, Taylor C, et al. Complex two-gene modulation of lung disease severity in children with cystic fibrosis. J Clin Invest 2008; 118: 1040–1049.
37- Collaco JM, Vanscoy L, Bremer L, et al. Interactions between second hand smoke and genes that affect cystic fibrosis lung disease. JAMA 2008; 299: 417–424.
38- Brazova J, Sismova K, Vavrova V, et al. Polymorphismsof TGF-beta1 in cystic fibrosis patients. Clin. Immunol 2006;121: 350–357.
39- Arkwright PD, Laurie S, Super M, et al. TGFbeta (1) genotype and accelerated decline in lung function of patients with cystic fibrosis. Thorax 2000; 55: 459–462.
40- Alper OM, Wong LJ, Young S, et al. Identification of novel and rare mutations in California Hispanic and African American cystic fibrosis patients. Hum Mutat 2004;24:353.
41- Massie RJ, Poplawski N, Wilcken B, et al. Intron-8 polythymidine sequence in Australasian individuals with CF mutations R117H and R117C. Eur Respir J 2001;17:1195–1200.
42- Kiesewetter S, Macek M Jr, Davis C, et al. A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet 1993;5:274–278.
43- Chmiel JF, Drumm ML, Konstan MW, et al. Pitfall in the use of genotype analysis as the sole diagnostic criterion for cystic fibrosis. Pediatrics 1999;103:823–826.
44- Pignatti PF, Bombieri C, Benetazzo M, et al. CFTR gene variant IVS8-5T in disseminated bronchiectasis. Am J Hum Genet 1996; 58:889-892.
45- Giordon E, Cazenueve C, Lebargy F, et al. CFTR mutations in adults with disseminated bronchiectasies. Eur J Hum Genet 1997; 5:149-55.
46- Kristidis P, Bozon D, Corey M, et al. Genetic determination of exocrine pancreatic function in cystic fibrosis. Am J Hum Genet. 1992;50:1178-84.
47- Tsui LC. The spectrum of cystic fibrosis mutations. Trends Genet. 1992;8:392-8.
48- Durie P, Forstner GG, Gaskin KJ, et al.Age-related alteration of immunoreactive pancreatic cationic trypsinogen in sera from cystic fibrosis patients with and without pancreatic
insufficiency. Pediatr Res 1986;20: 209–213.
49- Sontag MK, Corey M, Hokanson JE, et al. Genetic and physiologic correlates of longitudinal immunoreactive trypsinogen decline in infants with cystic fibrosis identified through
newborn screening. J Pediatr 2006 ;149: 650– 65.
50- Sharer N, Schwarz M, Malone G, et al. Mutations of the cystic fibrosis gene in patients with chronic pancreatitis. N Engl J Med. 1998;339:645-52.
51- Cohn JA, Friedman KJ, Noone PG, et al. Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis. N Engl J Med. 1998;339:653-8.
52- Audrézet MP, Chen JM, Le Maréchal C, et al.. Determination of the relative contribution of three genes-the cystic fibrosis transmembrane conductance regulator gene, the
cationic trypsinogen gene, and the pancreatic secretory trypsin inhibitor gene-to the etiology of idiopathic chronic pancreatitis. Eur J Hum Genet. 2002;10:100-6.
53- Ockenga J, Stuhrmann M, Ballmann M, et al. Mutations of the cystic fibrosis gene, but not cationic trypsinogen gene, are associated with recurrent or chronic idiopathic
pancreatitis. Am J Gastroenterol. 2000;9:2061-7.
54- Cystic Fibrosis Foundation. CFF Patient Registry. Bethesda, Maryland: 2006.
55- Blackman SM, Hsu S, Ritter SE, et al. A susceptibility gene for type 2 diabetes confers substantial risk for diabetes complicating cystic fibrosis. Diabetologia 2009; 52: 1858–
1865
56- Grant SF, Thorleifsson G, Reynisdottir I, et al. Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes. Nat Genet 2006;38: 320–323.
57- Cauchi, S., Y. El Achhab, H. Choquet, et al. TCF7L2 is reproducibly associated with type 2 diabetes in various ethnic groups: a global meta-analysis. J Mol Med 2007; 85: 777–
782.
58- Marshall BC, Butler SM, Stoddard M, et al. Epidemiology of cystic fibrosis-related diabetes. J Pediatr 2005; 146: 681–687.
59- Blackman SM, Vanscoy LL, Collaco JM, et al. Variability in body mass index in cystic fibrosis is determined partly by a genetic locus on chromosome 5. Pediatr Pulmonol 2008;
Supp. 31: 271.
60- Liu Z, Habener JF. Glucagon-like peptide-1 activation of TCF7L2-dependentWnt signalling enhances pancreatic beta-cell proliferation. J Biol Chem 2008; 283: 8723– 8735.
61- Wegner L, Hussain MS, Pilgaard K, et al. Impact of TCF7L2 rs7903146 on insulin secretion and action in young and elderly Danish twins. J Clin Endocrinol Metab 2008 ; 93:
4013–4019.
62- Escobar MA, Grosfeld JL, Burdick JJ, et al. Surgical considerations in cystic fibrosis: a 32-year evaluation of outcomes. Surgery 2005; 138: 560–571.
63- Hamosh A, King TM, Rosenstein BJ, et al. Cystic fibrosis patients bearing the common missense mutation Gly->Asp at codon 551 and the deltaF508 are indistinguishable from
deltaF508 homozygotes except for decreased risk of meconium ileus. Am J Hum Genet 1992;51: 245–250.
64- Rozmahel R, Wilschanski M, Matin A, et al. Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor.
Nat. Genet. 1996; 12: 280–287.
65- Stoltz DA, Meyerholz DK, Pezzulo AA, et al. Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth. Sci Transl Me. 2010;2: 29-31.
66- Sun X, Sui H, Fisher JT, et al. Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis. J Clin Invest 2010; 120: 3149–3160.
67- Mekus F, Ballmann M, Bronsveld I, et al. Categories of deltaF508 homozygous cystic fibrosis twin and sibling pairs with distinct phenotypic characteristics. Twin. Res.
2000;3:277–293.
68- Stanke F, Becker T, Kumar V, et al. Genes that determine immunology and inflammation modify the basic defect of impaired ion conductance in cystic fibrosis epithelia. J Med
Genet. 2011;4:24-31.
69- Castaldo G, Fuccio A, Salvatore D, et al. Liver expression in cystic fibrosis could be modulated by genetic factors different from the cystic fibrosis trans membrane regulator
genotype. Am J Med Genet 2001;98:294-7.
70- Dork T, Dworniczak B, Aulehla-Scholz C, et al. Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens. Hum Genet 1997;100:365–377.
71- Gilljam M, Moltyaner Y, Downey GP, et al. Airway inflammation and infection in congenital bilateral absence of the vas deferens. Am J Respir Crit Care Med 2004;169:174–179.
72- Noone PG, Pue CA, Zhou Z, et al. Lung disease associated with the IVS8 5T allele of the CFTR gene. Am J Respir Crit Care Med 2000;162:1919–1924.
73- Cuppens H, Lin W, Jaspers M, et al. Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg) m locus explains the partial
penetrance of the T5 polymorphism as a disease mutation. J Clin Invest 1998;101:487–496.
74- Groman JD, Hefferon TW, Casals T, et al. Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator
gene is pathogenic or benign. Am J Hum Genet 2004;74:176–179
75- Jarvi K, Zielenski J, Wilschanski M, et al. Cystic fibrosis transmembrane conductance regulator and obstructive azoospermia. Lancet 1995M345:1578.
76- Zielenski J, Jarvi K, Ray P, et al. Increased risk of infertility associated with specific DNA marker haplotypes in the CFTR locus. Pediatr Pulmonol 1998; (suppl 17) :297.
77- Larriba S, Bassas, L, Gimenez J, et al. testicular CFTR splice variants in patients with congenital absence of the vas deferens. Hum Mol Genet 1998;7:1739-1743.
78- Leonardi S, Bombace V, Rotolo N, Sciuto C, La Rosa M. Congenital absence of vas deferens and cystic fibrosis. Minerva Pediatrica 2003;55:43-50
79- Leonardi S, Sciuto C, La Rosa M. A missed cystic fibrosis diagnosis in childhood. Allergy asthma Proc 2005;26:487-8.
Legend for tables
Table 1: classification scheme for CFTR mutations (15)
Table 2: phenotypes of 10 most common CFTR alleles in whites with CF (20)
Table 3: clinical features of CF patients carriers of the missense P5L mutation (18).
Table 4: clinical and genotypic features of the patients followed by our Cystic Fibrosis Unit
Table 1: classification scheme for CFTR mutations (15)
Table 2: phenotypes of 10 most common CFTR alleles in whites with CF (20)
Table 3: clinical features of CF patients carriers of the missense P5L mutation (18)
Table 4: clinical and genotypic features of the patients followed by our Cystic Fibrosis Unit
www.theChild.it bimestrale di divulgazione scientifica dell'Associazione Pediatrica di Immunologia e Genetica
Legge 7 marzo 2001, n. 62 - Registro della Stampa Tribunale di Messina n. 3/09 - 11 maggio 2009
Direttore scientifico Carmelo Salpietro - Direttore responsabile Giuseppe Micali - Segreteria redazione Basilia Piraino - Piera Vicchio
Direzione-redazione: UOC Genetica e Immunologia Pediatrica - AOU Policlicnico Messina