- Email: [email protected]
Mild cystic fibrosis in patients with the rare P5L CFTR mutation
Journal of Cystic Fibrosis 11 (2012) 30 – 33 www.elsevier.com/locate/jcf
Original Article
Mild cystic fibrosis in patients with the rare P5L CFTR mutation Lucia Spicuzza a,⁎, Concetta Sciuto a , Lucia Di Dio b , Teresa Mattina b , Salvatore Leonardi a , Michele Miraglia del Giudice c , Mario La Rosa a a
Department of Pediatrics, Bronchopneumology Unit, University of Catania, Italy b Department of Pediatrics, Genetics Unit, University of Catania, Italy c Department of Pediatrics, University of Napoli, Napoli, Italy
Received 19 July 2011; received in revised form 26 August 2011; accepted 29 August 2011 Available online 7 October 2011
Abstract Over 1800 Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) mutations have been identified so far, determining different degrees of CFTR dysfunction and a range of different cystic fibrosis phenotypes. The P5L CFTR mutation is a recently described N-terminus missense variant which may cause defect of protein folding and processing/trafficking, but the functional classification is still unclear. Given the rarity of the mutation, the associated clinical phenotype is still unknown. The aim of our study was to describe the clinical phenotypes in a group of 7 patients with the P5L mutation including 2 adults, 2 adolescents and 3 children. The P5L variant was associated with ΔF508 in 5 patients and with W1282X in two patients. All patients had positive or borderline sweat test values. All had pancreatic sufficiency, no hepatobiliary disease, no or mild respiratory symptoms and normal lung function. The two adult males were fertile. Most of the patients presented recurrent episodes of dehydration and hypochloronatremia. We conclude that, although it has been speculated that the N-terminus CFTR missense variants may severely affect the behaviour of the CFTR chloride channel, patients with the P5L CFTR mutation, in association with a severe class II mutation, may be asymptomatic or may be affected by mild disease. © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; CFTR; P5L
1. Introduction Cystic fibrosis (CF) is caused by mutations of the CF Transmembrane Conductance Regulator gene (CFTR). The classic CF phenotype includes multi-organ involvement with predominant severe respiratory disease, pancreatic insufficiency and male infertility [1,2]. In recent years it has been acknowledged 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 ⁎ Corresponding author at: Department of Pediatrics, Bronchopneumology Unit, Via Santa Sofia 78, 95123 Catania, Italy. Tel.: +39 095 3782764; fax: +39 095 222532. E-mail address: [email protected] (L. Spicuzza).
mild respiratory symptoms, pancreatic sufficiency and normal or borderline sweat test [3–5]. Because of the unusual presentation, the diagnosis of these “non classic” cystic fibrosis can be difficult to make. 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. More than 1800 CF mutations have been described since the cloning of the gene in 1989, even though the association of genotype–phenotype has been made only for a few common mutations [6]. The P5L mutation consists an N-terminus CFTR missense variant which has been recently described in Spain [7]. Eight different N-terminus missense natural variants have been detected which cause a defect of CFTR folding and or/trafficking, thus severely affecting the chloride channel activity. Although a severe impact of this CFTR variant has been predicted on the CF
1569-1993/$ - see front matter © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2011.08.009
L. Spicuzza et al. / Journal of Cystic Fibrosis 11 (2012) 30–33
phenotype, so far no clinical data have been produced on patients with this mutation to support this hypothesis, perhaps due to its rarity. The aim of this study was therefore to describe clinical features in patients bearing the rare P5L CFTR mutation. 2. Methods 2.1. Patients population We reviewed data from a cohort of 105 patients with a diagnosis of cystic fibrosis followed in the Cystic Fibrosis Centre of the Catania University Hospital (Southern Italy). All patients were followed in the Centre since the time of the diagnosis. Sweat test, clinical data and genotypes were available for all the patients. Seven patients (5 males, age range 15 months– 30 years) who were heterozygous for the P5L mutation were included in the study. The study was approved by our local Committee for Clinical Investigation and informed consent was obtained by each patient or parent. 2.2. Diagnostic tests and clinical data Sweat chloride concentrations were obtained according to guidelines by the Gibson and Cooke method [8]. Three tests were performed in each patient and the mean value is reported. Blood immunoreactive trypsinogen (IRT) was measured in patients who underwent neonatal screening for CF (in Italy newborn screening is mandatory since 1998) one and three weeks after birth. Pancreas sufficiency was defined as a faecal elastase N 200 mg/g faeces. A quarterly clinical evaluation was performed in our Centre after the diagnosis. During each visit lung function (in patients N 6 yrs old) was measured according to the American Thoracic Standard criteria [9]. Values of forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were expressed as % predicted of normal values adjusted for age, gender, sex height and weight. Sputum samples for microbiology were obtained for all adults and adolescents. In younger children who could not expectorate sputum, oropharyngeal swabs were collected. Chest High Resolution Computed Tomography scan (HRCT) was performed in all patients. Demographic data included in the study were derived from the last routine evaluation in the Centre. The age at the diagnosis was derived from the date of the first sweat test performed. 2.3. Genetic analysis Genotyping of the CFTR was carried out on DNA extracted from blood leucocytes. In our laboratory basic genetic test was performed by reverse dot blot hybridisation by using INNO-LiPA CFTR19 and CFTR17+Tn assays (Innogenetics, Zwiljnaarde, Belgium). The test, performed according to the recommendation of the supplier, allows the detection of the 36 more common mutations in Europe [10]. In patients suspected of CF and a single allele mutation shown by basic genotyping, advanced genetic testing is routinely performed in our laboratory. The P5L mutation was identified by denaturing high-performance liquid chromatography (d-HPLC) and confirmed by gene sequencing analysis with an
31
automated procedure (Genetic Analyzer, Applied Biosystems). Methods and procedures for dHPLC and DNA sequencing were reported previously [11–13]. 3. Results 3.1. Genotype Advanced genetic analysis showed that 7, out of 105 patients followed in our Centre, were heterozygous for the P5L CFTR mutation. Five patients also carried the ΔF508 mutation in the second allele while two carried the W1282X mutation. The group included two adults, two adolescents and three children. Demographic data of the patients are shown in Table 1. 3.2. Diagnosis Only three children, who were born after 1998, were screened for CF. These children had abnormal IRT values and for this reason the CF diagnosis was made soon after birth (Table 1). For patient n. 6 hypertrophic pyloric stenosis was reported after birth. In the other patients the diagnosis was made during childhood: recurrent episodes of dehydration (in some cases severe) were the main reason for addressing the child to the Centre for sweat test evaluation. Four patients had clearly abnormal sweat chloride values (N 60 mmol/l), while three patients were in the intermediate range (30–60 mmol/l). 3.3. Clinical data and clinical course All patients were in good nutritional status with two patients slightly overweight (Table 1). All had pancreatic sufficiency and no hepatobiliary disease was recorded (Table 2). Both adults and children had no chronic respiratory symptoms. Respiratory exacerbations, requiring oral antibiotics prescription were uncommon in all patients over the time. Patient n. 3 required i.v. antibiotic once. Chest HRCT showed the absence of bronchiectasis in all patients except in n. 1 (the older of the group), in which mild bilateral basal bronchiectasis were present. In all cooperating patients we found a normal lung function with all FEV1 values well above 80% of predicted. Sputum or throat swab cultures revealed episodic presence of Staphylococcus aureus in some patients, while the occurrence of Pseudomonas aeruginosa in the sputum was reported in one occasion in patient n. 1 and in three occasions (through the course of life) in n. 3 (Table 2). All patients except n. 4 and n. 7, had recurrent episodes of dehydration and hypochloronatremia, particularly during the summer, sometimes so severe to require hospitalisation. For the two adult males spermiograms were performed and both revealed normal values. Patient n. 2 also had a healthy child. 4. Discussion The present study provides evidence that the P5L CFTR mutation is associated with a mild CF phenotype. In fact, all patients had normal respiratory function and pancreatic sufficiency.
32
L. Spicuzza et al. / Journal of Cystic Fibrosis 11 (2012) 30–33
Table 1 Demographic data of the patients. Patient
Sex
Age
BMI
1 2 3 4 5 6 7
M M F M F M M
30 yrs 24 15 13 6 6 15 months
25.5 30.1 29.1 25.2
Height (centiles)
50° N97° 50°
Weight (centiles)
75° 97° 50°
Age of diagnosis
Genotype
Sweat Chloride (mmol/L)
IRT (ng/ml)
8 yrs 3 yrs 4 months 8 yrs Birth Birth Birth
ΔF508/P5L ΔF508/P5L ΔF508/P5L ΔF508/P5L ΔF508/P5L W1282X/P5L W1282X/P5L
87 120 46 79 57 62 57
NA NA NA NA 60/40 106/90 94/42
BMI: body mass index. IRT: immunoreactive trypsinogen, first week/third week after birth. Normal range values b60 ng/ml first week and b40 ng/ml third week.
Table 2 Clinical data of the patients. Patient
Fev1 (%)
SaO2 (%)
Bronchiectasis
Sputum pathogens
Rhino/sinus disease
Pancreatic sufficiency
Dehydration
1 2 3 4 5 6 7
101 90 95 86 NA NA NA
97 97 98 97 98 98 98
Mild No No No No No No
S. aureus, P. aeruginosa No S. aureus, P. aeruginosa No No S. aureus S. aureus
Yes No No No No No No
Yes Yes Yes Yes Yes Yes Yes
Yes (severe) Yes Yes No Yes Yes No
The P5L mutation is one of the N-terminus CFTR missense variants, first studied in the Spanish population [14]. Functional classification of this mutation still remains controversial [10]. Some Spanish authors suggested that this mutation, along with other N-terminus CFTR missense variants, may alter the protein folding and/or trafficking/processing. However, these authors found that although the expression of the P5L CFTR mutant was reduced in the plasmatic membrane, still a small but significant amount of mature protein was present [7]. Electrophysiological analysis of the mature P5L CFRT fraction revealed an altered channel activity and it has been speculated that the N-terminus alteration may inhibit the channel's gating processes [15]. Other authors included the P5L mutation in the functional class II of the CFRT mutations, similar to the common severe ΔF508 [16]. Although the different methodologies might explain these discrepancies, it seems unlikely that the P5L variant might be included among deleterious mutations. If it is true that the degree of organ involvement and the severity of the disease are linked to the amount of functional CFTR (and to organ's sensitivity to CFTR dysfunction) it is likely that some functional protein must be present in our patients. It is noteworthy that all patients with the P5L variant were bearing a severe mutation, causing loss of CFTR function on the associated allele, thus confirming a protective role of this variant on the phenotype. Most of the missense N-terminus mutations have been described in Spanish patients, however details on the clinical phenotype are lacking. The only patient reported so far is a 9-yr-old Spanish boy, with normal lung function and pancreatic sufficiency [14]. The mild course the disease has been confirmed in the present study as the two older patients maintained normal lung
function and stable clinical condition over the time. It is also intriguing that the two male patients were fertile, since most of the N-terminus missense variants have been associated with congenital bilateral absence of vas deferens [7]. 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. In our patients we found abnormal/borderline sweat test values along with one single allele mutation at basic genetic test and this warranted further genetic search for the detection of a second CFTR mutation. As the P5L is a rare and still unclassified mutation, and given the association with a severe mutation such as ΔF508, it is clear that only a follow-up of the patients could allow definitive conclusion on the course of the disease. Indeed our data confirm previous observations that the identification of two CFTR mutations does not necessary settle the diagnostic issues, and most of the consensus statements agree on the importance of the clinical condition, organ's involvement and symptoms to make diagnosis of cystic fibrosis [1,17]. In conclusion, although it has been speculated that the Nterminus CFTR missense variants may severely affect the behaviour of the CFTR chloride channel, patients with the P5L CFTR mutation, in association with a severe class II mutation, may be asymptomatic or affected by mild disease. References [1] Rosenstein BJ, Cutting GR. The diagnosis of cystic fibrosis: a consensus statement. Cystic Fibrosis Foundation Consensus Panel. J Pediatr 1998;132:589–95.
L. Spicuzza et al. / Journal of Cystic Fibrosis 11 (2012) 30–33 [2] Knowles MR, Durie PR. What is cystic fibrosis? N Engl J Med 2002;347: 439–42. [3] De Boeck K, Wilschanski M, Castellani C, Taylor C, Cuppens H, Dodge J, et al. Diagnostic working group. Cystic fibrosis: terminology and diagnostic algorithms. Thorax 2006;61:627–35. [4] Kerem E. Atypical CF and CF related diseases. Paediatr Respir Rev 2006;7(Suppl 1):S144–6. [5] Wallis C. Atypical cystic fibrosis: diagnostic and management dilemmas. J R Soc Med 2003;96(Suppl 43):2–10. [6] Cystic Fibrosis Genetic Database. www.genet.sickkids.on.ca/. [7] Gené GG, Llobet A, Larriba S, de Semir D, Martínez I, Escalada A, et al. N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel. Hum Mutat 2008;29(5):738–49. [8] LeGrys VA, Yankaskas JR, Quittell LM, Marshall BC, Mogayzel Jr PJ. Diagnostic sweat testing: the Cystic Fibrosis Foundation guidelines. J Pediatr 2007;151:85–9. [9] American Thoracic Society. Standardization of spirometry, 1994 update. Am J Respir Crit Care Med 1995;152:1107–36. [10] Chehab FF, Wall J. Detection of multiple cystic fibrosis mutations by reverse dot blot hybridization: technology for carrier screening. Hum Genet 1992;89:163–8. [11] Le Marechal C, Audrezet M, Quere I, Raguenes O, Langonne S, Ferec C. Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid
[12]
[13]
[14]
[15]
[16]
[17]
33
chromatography (d-HPLC): major implications for genetic counselling. Hum Genet 2001;108:290–8. Strom CM, Huang D, Chen C, Buller A, Peng M, Quan F, et al. Extensive sequencing of the cystic fibrosis transmembrane regulator gene: assay validation and unexpected benefits of developing a comprehensive test. Genet Med 2003;5:9–14. McGinniss MJ, Chen C, Redman JB, Buller A, Quan F, Peng M, et al. Extensive sequencing of the CFTR gene: lessons learned from the first 157 patient samples. Hum Genet 2005;118:331–8. Chillón M, Casals T, Giménez J, Nunes V, Estivill X. Analysis of the CFTR gene in the Spanish population: SSCP-screening for 60 known mutations and identification of four new mutations (Q30X, A120T, 18121G→A, and 3667del4). Hum Mutat 1994;3:223–30. Fu J, Ji HL, Naren AP, Kirk KL. A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating. J Physiol 2001;536:459–70. Thelin WR, Chen Y, Gentzsch N, Kreda SM, Sallee JL, Scarlett CO, et al. Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR. J Clin Invest Feb. 2007;117:364–74. Goubau C, Wilschanski M, Skalická V, Lebecque P, Southern KW, Sermet I, et al. Phenotypic characterisation of patients with intermediate sweat chloride values: towards validation of the European diagnostic algorithm for cystic fibrosis. Thorax 2009;64:683–91.
Original Article
Mild cystic fibrosis in patients with the rare P5L CFTR mutation Lucia Spicuzza a,⁎, Concetta Sciuto a , Lucia Di Dio b , Teresa Mattina b , Salvatore Leonardi a , Michele Miraglia del Giudice c , Mario La Rosa a a
Department of Pediatrics, Bronchopneumology Unit, University of Catania, Italy b Department of Pediatrics, Genetics Unit, University of Catania, Italy c Department of Pediatrics, University of Napoli, Napoli, Italy
Received 19 July 2011; received in revised form 26 August 2011; accepted 29 August 2011 Available online 7 October 2011
Abstract Over 1800 Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) mutations have been identified so far, determining different degrees of CFTR dysfunction and a range of different cystic fibrosis phenotypes. The P5L CFTR mutation is a recently described N-terminus missense variant which may cause defect of protein folding and processing/trafficking, but the functional classification is still unclear. Given the rarity of the mutation, the associated clinical phenotype is still unknown. The aim of our study was to describe the clinical phenotypes in a group of 7 patients with the P5L mutation including 2 adults, 2 adolescents and 3 children. The P5L variant was associated with ΔF508 in 5 patients and with W1282X in two patients. All patients had positive or borderline sweat test values. All had pancreatic sufficiency, no hepatobiliary disease, no or mild respiratory symptoms and normal lung function. The two adult males were fertile. Most of the patients presented recurrent episodes of dehydration and hypochloronatremia. We conclude that, although it has been speculated that the N-terminus CFTR missense variants may severely affect the behaviour of the CFTR chloride channel, patients with the P5L CFTR mutation, in association with a severe class II mutation, may be asymptomatic or may be affected by mild disease. © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; CFTR; P5L
1. Introduction Cystic fibrosis (CF) is caused by mutations of the CF Transmembrane Conductance Regulator gene (CFTR). The classic CF phenotype includes multi-organ involvement with predominant severe respiratory disease, pancreatic insufficiency and male infertility [1,2]. In recent years it has been acknowledged 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 ⁎ Corresponding author at: Department of Pediatrics, Bronchopneumology Unit, Via Santa Sofia 78, 95123 Catania, Italy. Tel.: +39 095 3782764; fax: +39 095 222532. E-mail address: [email protected] (L. Spicuzza).
mild respiratory symptoms, pancreatic sufficiency and normal or borderline sweat test [3–5]. Because of the unusual presentation, the diagnosis of these “non classic” cystic fibrosis can be difficult to make. 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. More than 1800 CF mutations have been described since the cloning of the gene in 1989, even though the association of genotype–phenotype has been made only for a few common mutations [6]. The P5L mutation consists an N-terminus CFTR missense variant which has been recently described in Spain [7]. Eight different N-terminus missense natural variants have been detected which cause a defect of CFTR folding and or/trafficking, thus severely affecting the chloride channel activity. Although a severe impact of this CFTR variant has been predicted on the CF
1569-1993/$ - see front matter © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2011.08.009
L. Spicuzza et al. / Journal of Cystic Fibrosis 11 (2012) 30–33
phenotype, so far no clinical data have been produced on patients with this mutation to support this hypothesis, perhaps due to its rarity. The aim of this study was therefore to describe clinical features in patients bearing the rare P5L CFTR mutation. 2. Methods 2.1. Patients population We reviewed data from a cohort of 105 patients with a diagnosis of cystic fibrosis followed in the Cystic Fibrosis Centre of the Catania University Hospital (Southern Italy). All patients were followed in the Centre since the time of the diagnosis. Sweat test, clinical data and genotypes were available for all the patients. Seven patients (5 males, age range 15 months– 30 years) who were heterozygous for the P5L mutation were included in the study. The study was approved by our local Committee for Clinical Investigation and informed consent was obtained by each patient or parent. 2.2. Diagnostic tests and clinical data Sweat chloride concentrations were obtained according to guidelines by the Gibson and Cooke method [8]. Three tests were performed in each patient and the mean value is reported. Blood immunoreactive trypsinogen (IRT) was measured in patients who underwent neonatal screening for CF (in Italy newborn screening is mandatory since 1998) one and three weeks after birth. Pancreas sufficiency was defined as a faecal elastase N 200 mg/g faeces. A quarterly clinical evaluation was performed in our Centre after the diagnosis. During each visit lung function (in patients N 6 yrs old) was measured according to the American Thoracic Standard criteria [9]. Values of forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were expressed as % predicted of normal values adjusted for age, gender, sex height and weight. Sputum samples for microbiology were obtained for all adults and adolescents. In younger children who could not expectorate sputum, oropharyngeal swabs were collected. Chest High Resolution Computed Tomography scan (HRCT) was performed in all patients. Demographic data included in the study were derived from the last routine evaluation in the Centre. The age at the diagnosis was derived from the date of the first sweat test performed. 2.3. Genetic analysis Genotyping of the CFTR was carried out on DNA extracted from blood leucocytes. In our laboratory basic genetic test was performed by reverse dot blot hybridisation by using INNO-LiPA CFTR19 and CFTR17+Tn assays (Innogenetics, Zwiljnaarde, Belgium). The test, performed according to the recommendation of the supplier, allows the detection of the 36 more common mutations in Europe [10]. In patients suspected of CF and a single allele mutation shown by basic genotyping, advanced genetic testing is routinely performed in our laboratory. The P5L mutation was identified by denaturing high-performance liquid chromatography (d-HPLC) and confirmed by gene sequencing analysis with an
31
automated procedure (Genetic Analyzer, Applied Biosystems). Methods and procedures for dHPLC and DNA sequencing were reported previously [11–13]. 3. Results 3.1. Genotype Advanced genetic analysis showed that 7, out of 105 patients followed in our Centre, were heterozygous for the P5L CFTR mutation. Five patients also carried the ΔF508 mutation in the second allele while two carried the W1282X mutation. The group included two adults, two adolescents and three children. Demographic data of the patients are shown in Table 1. 3.2. Diagnosis Only three children, who were born after 1998, were screened for CF. These children had abnormal IRT values and for this reason the CF diagnosis was made soon after birth (Table 1). For patient n. 6 hypertrophic pyloric stenosis was reported after birth. In the other patients the diagnosis was made during childhood: recurrent episodes of dehydration (in some cases severe) were the main reason for addressing the child to the Centre for sweat test evaluation. Four patients had clearly abnormal sweat chloride values (N 60 mmol/l), while three patients were in the intermediate range (30–60 mmol/l). 3.3. Clinical data and clinical course All patients were in good nutritional status with two patients slightly overweight (Table 1). All had pancreatic sufficiency and no hepatobiliary disease was recorded (Table 2). Both adults and children had no chronic respiratory symptoms. Respiratory exacerbations, requiring oral antibiotics prescription were uncommon in all patients over the time. Patient n. 3 required i.v. antibiotic once. Chest HRCT showed the absence of bronchiectasis in all patients except in n. 1 (the older of the group), in which mild bilateral basal bronchiectasis were present. In all cooperating patients we found a normal lung function with all FEV1 values well above 80% of predicted. Sputum or throat swab cultures revealed episodic presence of Staphylococcus aureus in some patients, while the occurrence of Pseudomonas aeruginosa in the sputum was reported in one occasion in patient n. 1 and in three occasions (through the course of life) in n. 3 (Table 2). All patients except n. 4 and n. 7, had recurrent episodes of dehydration and hypochloronatremia, particularly during the summer, sometimes so severe to require hospitalisation. For the two adult males spermiograms were performed and both revealed normal values. Patient n. 2 also had a healthy child. 4. Discussion The present study provides evidence that the P5L CFTR mutation is associated with a mild CF phenotype. In fact, all patients had normal respiratory function and pancreatic sufficiency.
32
L. Spicuzza et al. / Journal of Cystic Fibrosis 11 (2012) 30–33
Table 1 Demographic data of the patients. Patient
Sex
Age
BMI
1 2 3 4 5 6 7
M M F M F M M
30 yrs 24 15 13 6 6 15 months
25.5 30.1 29.1 25.2
Height (centiles)
50° N97° 50°
Weight (centiles)
75° 97° 50°
Age of diagnosis
Genotype
Sweat Chloride (mmol/L)
IRT (ng/ml)
8 yrs 3 yrs 4 months 8 yrs Birth Birth Birth
ΔF508/P5L ΔF508/P5L ΔF508/P5L ΔF508/P5L ΔF508/P5L W1282X/P5L W1282X/P5L
87 120 46 79 57 62 57
NA NA NA NA 60/40 106/90 94/42
BMI: body mass index. IRT: immunoreactive trypsinogen, first week/third week after birth. Normal range values b60 ng/ml first week and b40 ng/ml third week.
Table 2 Clinical data of the patients. Patient
Fev1 (%)
SaO2 (%)
Bronchiectasis
Sputum pathogens
Rhino/sinus disease
Pancreatic sufficiency
Dehydration
1 2 3 4 5 6 7
101 90 95 86 NA NA NA
97 97 98 97 98 98 98
Mild No No No No No No
S. aureus, P. aeruginosa No S. aureus, P. aeruginosa No No S. aureus S. aureus
Yes No No No No No No
Yes Yes Yes Yes Yes Yes Yes
Yes (severe) Yes Yes No Yes Yes No
The P5L mutation is one of the N-terminus CFTR missense variants, first studied in the Spanish population [14]. Functional classification of this mutation still remains controversial [10]. Some Spanish authors suggested that this mutation, along with other N-terminus CFTR missense variants, may alter the protein folding and/or trafficking/processing. However, these authors found that although the expression of the P5L CFTR mutant was reduced in the plasmatic membrane, still a small but significant amount of mature protein was present [7]. Electrophysiological analysis of the mature P5L CFRT fraction revealed an altered channel activity and it has been speculated that the N-terminus alteration may inhibit the channel's gating processes [15]. Other authors included the P5L mutation in the functional class II of the CFRT mutations, similar to the common severe ΔF508 [16]. Although the different methodologies might explain these discrepancies, it seems unlikely that the P5L variant might be included among deleterious mutations. If it is true that the degree of organ involvement and the severity of the disease are linked to the amount of functional CFTR (and to organ's sensitivity to CFTR dysfunction) it is likely that some functional protein must be present in our patients. It is noteworthy that all patients with the P5L variant were bearing a severe mutation, causing loss of CFTR function on the associated allele, thus confirming a protective role of this variant on the phenotype. Most of the missense N-terminus mutations have been described in Spanish patients, however details on the clinical phenotype are lacking. The only patient reported so far is a 9-yr-old Spanish boy, with normal lung function and pancreatic sufficiency [14]. The mild course the disease has been confirmed in the present study as the two older patients maintained normal lung
function and stable clinical condition over the time. It is also intriguing that the two male patients were fertile, since most of the N-terminus missense variants have been associated with congenital bilateral absence of vas deferens [7]. 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. In our patients we found abnormal/borderline sweat test values along with one single allele mutation at basic genetic test and this warranted further genetic search for the detection of a second CFTR mutation. As the P5L is a rare and still unclassified mutation, and given the association with a severe mutation such as ΔF508, it is clear that only a follow-up of the patients could allow definitive conclusion on the course of the disease. Indeed our data confirm previous observations that the identification of two CFTR mutations does not necessary settle the diagnostic issues, and most of the consensus statements agree on the importance of the clinical condition, organ's involvement and symptoms to make diagnosis of cystic fibrosis [1,17]. In conclusion, although it has been speculated that the Nterminus CFTR missense variants may severely affect the behaviour of the CFTR chloride channel, patients with the P5L CFTR mutation, in association with a severe class II mutation, may be asymptomatic or affected by mild disease. References [1] Rosenstein BJ, Cutting GR. The diagnosis of cystic fibrosis: a consensus statement. Cystic Fibrosis Foundation Consensus Panel. J Pediatr 1998;132:589–95.
L. Spicuzza et al. / Journal of Cystic Fibrosis 11 (2012) 30–33 [2] Knowles MR, Durie PR. What is cystic fibrosis? N Engl J Med 2002;347: 439–42. [3] De Boeck K, Wilschanski M, Castellani C, Taylor C, Cuppens H, Dodge J, et al. Diagnostic working group. Cystic fibrosis: terminology and diagnostic algorithms. Thorax 2006;61:627–35. [4] Kerem E. Atypical CF and CF related diseases. Paediatr Respir Rev 2006;7(Suppl 1):S144–6. [5] Wallis C. Atypical cystic fibrosis: diagnostic and management dilemmas. J R Soc Med 2003;96(Suppl 43):2–10. [6] Cystic Fibrosis Genetic Database. www.genet.sickkids.on.ca/. [7] Gené GG, Llobet A, Larriba S, de Semir D, Martínez I, Escalada A, et al. N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel. Hum Mutat 2008;29(5):738–49. [8] LeGrys VA, Yankaskas JR, Quittell LM, Marshall BC, Mogayzel Jr PJ. Diagnostic sweat testing: the Cystic Fibrosis Foundation guidelines. J Pediatr 2007;151:85–9. [9] American Thoracic Society. Standardization of spirometry, 1994 update. Am J Respir Crit Care Med 1995;152:1107–36. [10] Chehab FF, Wall J. Detection of multiple cystic fibrosis mutations by reverse dot blot hybridization: technology for carrier screening. Hum Genet 1992;89:163–8. [11] Le Marechal C, Audrezet M, Quere I, Raguenes O, Langonne S, Ferec C. Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid
[12]
[13]
[14]
[15]
[16]
[17]
33
chromatography (d-HPLC): major implications for genetic counselling. Hum Genet 2001;108:290–8. Strom CM, Huang D, Chen C, Buller A, Peng M, Quan F, et al. Extensive sequencing of the cystic fibrosis transmembrane regulator gene: assay validation and unexpected benefits of developing a comprehensive test. Genet Med 2003;5:9–14. McGinniss MJ, Chen C, Redman JB, Buller A, Quan F, Peng M, et al. Extensive sequencing of the CFTR gene: lessons learned from the first 157 patient samples. Hum Genet 2005;118:331–8. Chillón M, Casals T, Giménez J, Nunes V, Estivill X. Analysis of the CFTR gene in the Spanish population: SSCP-screening for 60 known mutations and identification of four new mutations (Q30X, A120T, 18121G→A, and 3667del4). Hum Mutat 1994;3:223–30. Fu J, Ji HL, Naren AP, Kirk KL. A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating. J Physiol 2001;536:459–70. Thelin WR, Chen Y, Gentzsch N, Kreda SM, Sallee JL, Scarlett CO, et al. Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR. J Clin Invest Feb. 2007;117:364–74. Goubau C, Wilschanski M, Skalická V, Lebecque P, Southern KW, Sermet I, et al. Phenotypic characterisation of patients with intermediate sweat chloride values: towards validation of the European diagnostic algorithm for cystic fibrosis. Thorax 2009;64:683–91.