Neonatal screening of cystic fibrosis: diagnostic problems with CFTR mild mutations

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1 JIMD Short Report #066 (2007) Online DOI /s NEWBORN SCREENING SHORT REPORT Neonatal screening of cystic fibrosis: diagnostic problems with CFTR mild mutations M. Roussey A. Le Bihannic V. Scotet M. P. Audrezet M. Blayau M. Dagorne V. David E. Deneuville J. L. Giniès M. Laurans V. Moisan-Petit G. Rault P. Vigneron C. Férec Received: 15 March 2007 /Submitted in revised form: 2 June 2007 /Accepted: 6 June 2007 / Published online: 12 July 2007 # SSIEM and Springer 2007 Summary Newborn screening (NBS) of cystic fibrosis (CF) was implemented throughout the whole of France in 2002, but it had been established earlier in three western French regions. It can reveal atypical CF with one or two known CFTR mild mutations, with an uncertain evolution. The sweat test can be normal or borderline. In Brittany, from 1989 to 2004, 196 CF cases were diagnosed (1/2885 births). The incidence of atypical CF diagnosed by NBS is 9.7% (19 from 196). The outcome of 17 (2 lost of view) has been studied, with 9 other atypical CF cases diagnosed by NBS in two other regions. The follow-up period extends from 0.25 to 19.8 years (NBS implemented in Normandy in 1980) with mean age 4.6 years. The most frequent mild mutation is R117H ISV8j7T (50%). At the time of the last visit, nutritional status is normal. All these CF patients are pancreatic sufficient. Only one patient exhibits respiratory infections, whereas 7 others have them intermittently. Two of them had intermittent Pseudomonas aeruginosa colonization at 2.8 and 6.5 years. Mean Shwachman score is 96.7, mean Brasfield score is Eight children have had lung function tests (mean follow-up of 10 years): mean FVC was 99% of predicted, mean FEV 1 101%, but one of them has FEV 1 of 48%. Predicting the phenotype of these atypical CF patients remains difficult, thus complicating any genetic counselling. A regular clinical evaluation is necessary, if possible by a CF unit, because CF symptoms may appear later. Communicating editor: Rodney Pollitt Competing interests: None declared M. Roussey : A. Le Bihannic : M. Dagorne : E. Deneuville Centre de Ressources et de compétences de la mucoviscidose (CRCM), Université de Rennes 1 et Association Régionale de Dépistage et de Prévention des Handicaps de l_enfant (ARDPHE) de Bretagne, Rennes, France V. Scotet : M. P. Audrezet : C. Férec Laboratoire de Génétique Moléculaire, Université de Bretagne Occidentale, and INSERM U613, Brest, France M. Blayau Service de Biologie Moléculaire, Rennes, France V. David Centre de Ressources et de compétences de la mucoviscidose (CRCM), Nantes, France J. L. Giniès Centre de Ressources et de compétences de la mucoviscidose (CRCM), Angers, France M. Laurans Centre de Ressources et de compétences de la mucoviscidose (CRCM), Caen, France V. Moisan-Petit : P. Vigneron Centre de Ressources et de compétences de la mucoviscidose (CRCM), Bretagne-sud, France G. Rault Centre de Ressources et de compétences de la mucoviscidose (CRCM), Roscoff, France M. Roussey (*) CRCM Pédiatrique, CHU Anne de Bretagne, 16 Bd de Bulgarie, Rennes cedex, France michel.roussey@chu-rennes.fr

2 2 JIMD Short Report #066 (2007) Online Abbreviations CBAVD congenital bilateral absence of the vas deferens CF cystic fibrosis FEV 1 forced expiratory volume in 1 second FVC forced vital capacity IRT immunoreactive trypsin NBS newborn screening ST sweat test Introduction After various experiences in French areas and in other countries, in 2002 France decided to extend newborn screening (NBS) of cystic fibrosis (CF) to all newborns (Munck and the French Association for Neonatal Screening 2003). The programme is based on a strategy combining immunoreactive trypsin (IRT) assay, and, in case of elevated IRT, analysis of DNA mutations (Elucigene CF30 Orchid ARMS) in dried blood samples obtained at 3 days of age. However, diagnosis is not always straightforward and difficulties arise when exhaustive study of the gene, following a borderline sweat test (ST), detects a mild mutation or a previously unreported mutation of unknown effect. The problem is to know whether it is a case of CF that will follow the usual clinical course or a form linked with a CFTR abnormality that will become pathogenic in the years to come. The first scenario involves early preventive medicine in order to avoid or at least delay the classical complications of CF through early care. The second scenario involves probabilistic medicine and it is unclear whether diagnosis in the newborn is warranted: what therapeutic management should then be offered to the parents? Advances in genetics have led to an unexpected variability of the clinical spectrum of CF and has forced a review of the diagnostic criteria (Rosenstein and Cutting 1998). Diagnosis is usually based on a suggestive clinical picture and at least two abnormal ST results, or a sibling history of CF, or a screenpositive result in NBS with identification of two mutations in the CFTR gene, or demonstration of abnormal nasal epithelial ion transport even if the ST is normal. A mild mutation is theoretically dominant (Kulczycki et al 2003; Wallis 2003). Is it still CF when there are two CFTR mutations, one of them being mild? Several terms are used to define this situation: atypical CF (congenital bilateral absence of the vas deferens (CBAVD), mild pulmonary CF form, chronic idiopathic pancreatitis), pre-cf or subclinical CF, borderline CF, CFTRopathies, CF-like disease or CFTR-related diseases (chronic sinusitis, allergic bronchopulmonary aspergillosis, asthma) (Boyle 2003; LeGrys and Wood 1988). The European Diagnostic Working Group (De Boeck et al 2006) proposes the following terminology. On the one hand are patients diagnosed with classic or typical CF if they have one or more phenotypic characteristics and a sweat chloride concentration >60 mmol/l; patients can have exocrine pancreatic insufficiency or pancreatic sufficiency; the disease can have a severe course with rapid progression of symptoms or a milder course with very little deterioration over time. On the other hand, patients with non-classic or atypical CF have a CF phenotype in at least one organ system and a normal (<30 mmol/l) or borderline (30 60 mmol/l) sweat chloride level. In these patients confirmation of the diagnosis of CF requires detection of one disease-causing mutation on each CFTR gene or direct quantification of CFTR dysfunction by nasal potential difference measurement (Sermet-Gaudelus et al 2006). Non-classic CF includes patients with multiorgan or single-organ involvement. Most of these patients have exocrine pancreatic sufficiency and milder lung disease. Diagnostic dilemmas following newborn screening (NBS) are not rare. From the beginning of the nationwide CF French NBS programme, 14% (87/621) of the NBS CF patients had such case presentation (Munck et al (2006). The aim of our study is to describe the clinical outcome of the children screened at birth with an atypical CFTR genotype. To allow a significant follow-up, we conducted this study from regions where pilot experiences of NBS had been set up earlier. Population and methods The NBS started in 1980 in Normandy, in 1989 in Brittany, a western area of France where the incidence of CF is particularly high, and later in Pays de Loire. A two-tier strategy was used in earlier years (repeat IRT assay three weeks after first high IRT value), and onestage strategy thereafter (IRT assay combined with molecular analysis for CF mutations), from 1991 to 1995 according to the centre. This particular strategy (i.e. extended analysis) led to the identification of new CFTR mutations and some children with an atypical genotype were therefore identified. The incidence of atypical CF diagnosed by NBS is calculated only with data from the Brittany CF register, including children born between 1 January 1989 and 31 December 2004 whose genotype was suggestive of a

3 JIMD Short Report #066 (2007) Online 3 mild form of CF (mild genotype). The involved mutations generally belong to class IV or V, the most frequent of which is R117H (Rowntree and Harris (2003). Also included were children whose genotype at the time of diagnosis was of uncertain outcome and which we have called Fproblematic genotype_. To study the outcome of these children identified by NBS, we thought it of interest also to consider the children born in Pays-de-Loire and Normandy (some of them born before 1989 in Normandy), because of their longer follow-up. These children are monitored with a similar protocol in one of the following six CF centres: three in Brittany (Rennes, Roscoff, Bretagne-sud) and three in Normandy and Pays de Loire (Angers, Caen, Nantes). We studied several parameters in the medical records to assess outcome in these children: Nutritional, pancreatic, hepatic, respiratory, bacteriological: screening for Staphylococcus (S.) aureus, Haemophilus (H.) influenzae, Pseudomonas (P.) aeruginosa by throat swabs or sputum. Shwachman score included general activity, physical examination, nutrition and radiographic findings (maximum score 100) (Shwachman and Kulczycki 1958). Brasfield score used for longitudinal assessment of patient chest radiographs taken at annual review. The score is obtained by subtracting total points from 25, i.e. 25 would be the best possible score (Brasfield et al 1979). Number of annual consultations, frequency of respiratory physiotherapy, need or otherwise for pancreatic extracts, inhaled treatment, nutritional support, oral or intravenous antibiotic therapy. Results Study of incidence (data from Brittany) From 1989 to 2004, neonates were screened for CF in Brittany and, over this period, 196 cases of CF were diagnosed, corresponding to an incidence of 1/ 2885 births. Among these children: 169 (86.3%) presented with classic CF: 26 (15.5%) were detected because of meconium ileus, 137 (81%) by NBS, and six (3.5%) were false-negatives. 23 children (11.7%) had a genotype indicative of a mild form: 19 (9.7%) of these were detected by NBS, and 4 (2%) were false-negatives. None of these 23 cases of atypical CF was detected because of meconium ileus. 4 children (2%) had a problematic genotype: three (1.5%) were detected by NBS, one was a false-negative. Over this period, 11 false-negatives of NBS were therefore identified, i.e. an incidence of 5.6%. The causes are as follows: two human errors (misreading of IRT result; blood sampling error), five false-negatives of two-stage screening, three false-negatives of onestage screening, one false-negative of the ST. According to the overall incidence, which is close to the theoretical, it is reasonable to think that most falsenegatives were identified (13% and 3%, respectively, for two- and one-stage screening). As we did not know whether the disease progression in children with a problematic genotype at the time of NBS would be classical or mild, we deliberately studied their outcome separately from that of children with a mild genotype. Study of outcome in children identified by screening (data from the six CF centres) Overall Thirty-two children (17 girls, 15 boys) found to have a mild genotype (26/32) or a problematic genotype (6/ 32) were monitored in the western part of France. All were born at term following unexceptional pregnancies, without fetal distress and with normal neonatal measurements (mean weight 3400 g, mean length 49.7 cm, mean head circumference 35 cm). Over the studied period, six other children with a mild genotype (5/6) or a problematic genotype (1/6) who had gone undetected by the two-stage neonatal CF screening were born: One F508del/D1152H patient was not a falsenegative NBS since he was born in a region where such screening was not practised. Diagnosis was made at 6 months on the basis of CBAVD detected during surgery for inguinal hernia. At this time the ST was positive (100 mmol/l chloride) and it was discovered that the patient_s elder brother, who was born 9 years before in Brittany, had been screen-negative but had the same two mutations as his brother, with a borderline ST (56 mmol/l) and a clinical condition that was and remained normal at 15 years of age. Four other patients were diagnosed because of recurrent and/or persistent infections of the airways: One F508del/R347L (initial ST 35 mmol/l at 1 month, then 82 mmol/l at 2 years)

4 4 JIMD Short Report #066 (2007) Online One N1303K/F311L (initial ST 80 mmol/l at 4 months, then borderline on several occasions, and then constantly positive from 12 months, the age at which the F311L mutation was discovered) One F508del/S945L (first ST 70 mmol/l at 5 years) One F508del/3272j26A>G (first ST 102 mmol/l at 5 years) Outcome in 26 children (14 girls and 12 boys) with a mild genotype (Table 1) The mean age at the last consultation was 4 years 7 months (range 3 months to 19 years 10 months). One female patient was treated only by her attending physician, the other 25 patients were followed up in a CF centre: 22 (88%) had regular consultations (average of three a year), and the three others (12%) were seen episodically, about once a year. Sweat tests: Ninety-one ST were done, i.e. an average of 3.5 per child. After the first ST, performed on average at an age of 2 months (range 12 days to 6.5 months; median 1.5 months): Only 6 children (23%) had a positive ST (Q60 mmol/l) Seven children (27%) had a borderline ST (40 59 mmol/l); two became positive later Thirteen others (50%) had a ST <40 mmol/l; three became positive later and three became borderline CFTR mutations: One patient (nb 21) has two mild class V mutations ( kbC>T/3272j26A>G); the ST was 37 mmol/l but became positive at 7 months. At 18 months of age, this child is still asymptomatic. Twenty five patients are compound heterozygotes with one mild mutation: one class V (nb 14) ( kbC>T, with a ST of 58 mmol/l) and the other 24 class IV: Thirteen R117H associated with the IVS8, 7T variant (7 thymidines in intron 8); ST was below 30 mmol/l in five (nb 2, 7, 22, 24, 25) and between 40 and 59 mmol/l in the others (nb 3, 9, 13, 15, 16, 19, 20, 23) and became positive in two at ages 8 (nb 19) and 16 months (nb 13). Five R347H: three with ST positive immediately (nb 10, 11, 17), two with negative ST (nb 4, 26). One R117C (nb 1) with a initial ST of 33 mmol/ L which rose to 73 mmol/l at 9 months. One R1070W (nb 6) with a ST of 26 mmol/l, and then 40 mmol/l at 5 months. One S977F (nb 18) with a ST of 51 mmol/l, and then 60 mmol/l at 3 months. One S945L (nb 5), one R1066H (nb 8) and one L206W (nb 12), all three with a positive ST straight away. At the end of the observation period: Nutritional status of the children was normal: mean z-score of weight (range j1.2 to +3.3); mean z-score of height (range j1.7 to +2.5); mean BMI 16.3 kg/m 2 (range 12.9 to 22.1 kg/m 2 ); weight/ mean ideal weight 98.6% (range 77.1% to 122.1%).No child presented with pancreatic insufficiency or an episode of pancreatitis, diabetes or abnormal liver function tests. Respiratory status: 8 children (30%) presented with chronic functional signs of asthma, cough, or recurrent bronchopulmonary infections. Twenty children (77%) had at least one chest radiograph with a mean follow-up of 5 years. The mean Brasfield score is Eight (31%) underwent pulmonary function tests (PFT) with a mean follow-up of 10 years, the results of which are expressed as a percentage of the theoretical value for age and height. Only one patient, F508del/ S945L (nb 5), had abnormal PFT values: forced expiratory volume in one second (FEV 1 ) 48%, forced vital capacity (FVC) 64%. Bacteriological status: 13 children (50%) had at least one positive throat swab and/or sputum. The infection rate is probably underestimated, because 6 never underwent bacteriological tests and 5 had fewer than one examination every 2 years. Of the infected children, five (39%) had a single organism (S. aureus or H. influenzae), six (46%) had two organisms (S. aureus + H. influenzae) and two (15%) had in addition P. aeruginosa infection, one at 2 years 9 months (nb 13), the other at 6 years 5 months (nb 5), treated by intravenous antibiotics and with no chronic colonization. No child presented with colonization by Burkholderia cepacia, Stenotrophomonas maltophilia or Alcaligenes xylosoxidans. Lastly, 11 serological tests for Aspergillus and 8 for P. aeruginosa were negative.

5 JIMD Short Report #066 (2007) Online 5 Table 1 Mild genotypes Nb Sex CFTR mutations AST1 ST1 Age Z score for weight Z score for height Brasfield score Shwachman score FVC FEV 1 FEF 25j75 Bacteria 1 F W846X/R117C NR NR NR Sa 2 M DF508/R117H j0.4 j NR NR NR Hi 3 F DF508/R117H j1.1 j NR NR NR 4 F DF508/R347H j0.8 0 NR NR NR NR NR 5 M DF508/S945L j % 48,6% 56% Sa, Hi, Pa 6 F DF508/R1070W j % 102,% 109% Sa, Hi 7 F DF508/R117H NR NR NR 8 F DF508/R1066H j NR NR NR 9 F G551D/R117H NR NR NR NR NR 10 F DF508/R347H % 110% 121% Sa, Hi 11 M DF508/R347H % 121% 118% 12 M 3659delC/L206W j0.3 j % 112% 85% Sa, Hi 13 M DF508/R117H % 115% 86% Sa, Hi, Pa 14 F DF508/ kbC>T j1 j NR NR NR Sa, Hi 15 M G551D/R117H NR NR NR 16 M DF508/R117H NR NR NR Hi 17 M DF508/R347H NR NR NR Sa, Hi 18 M DF508/S977F j1.2 j % 84% 75% Sa 19 F DF508/R117H j NR NR NR Sa, Hi 20 M DI507/R117H j1.1 j % 118% 97% Hi 21 F A-G / kbC>T j0.2 j NR NR NR 22 F DF508/R117H NR NR NR Hi 23 F DF508/R117H NR NR NR NR NR 24 M DF508/R117H NR NR NR NR NR 25 F DF508/R117H j0.8 j0.5 NR NR NR NR NR 26 M DF508/R347H j NR NR NR NR NR Nb=patient number; F=female; M=male; ST1=first sweat test (mmol/l); AST1=age when first sweat test is realized (years); Age=age at the end of period observation (years); FVC=forced vital capacity; FEV 1 =forced expiratory volume in 1 second; FEF=forced expiratory flow; Bacteria=lower respiratory tract pathogens: NR=not realized. Sa=Staphylococcus aureus; Hi=Haemophilus influenzae; Pa=Pseudomonas aeruginosa.

6 6 JIMD Short Report #066 (2007) Online Therapeutic management of these children varied. Fifteen children had respiratory physiotherapy regularly, on average twice a week (range: 0.5 to 5). Concomitant long-term treatment of asthma has been necessary in seven children (nb 7, 12, 13, 16, 18, 20, 26). No patient has had aerosolized dornase alfa (rhdnase) or antibiotics. Finally, no child is taking pancreatic extracts, but two have nutritional supplements. Vitamin and trace element supplementation varies from one prescriber to another. Outcome in six children with a Fproblematic genotype_ (Table 2) These three girls and three boys of mean age 3 years 3 months (median 1.5 years; range 1 year 3 months to 10 years 6 months) were seen regularly in a CF centre from two to six times a year (mean of three annual consultations). Sweat tests: Twenty-five STs were done, i.e. a mean of four tests per child. The first ST was done on average at the age of 2 months (range 26 days to 4 months): it was negative in 2 children, borderline in 2 and positive in 2. One of the two ST-negative subjects (nb 3) and one of the two ST-borderline subjects (nb 5) became positive later, at 3 and 6 months, respectively. Overall, four of these six patients had a positive ST on average at 3 months (mean 71 mmol/ L, range mmol/l), one remained borderline, and one remained negative. CFTR mutations: The six children are compound heterozygotes with at least one classical class I (G542X, 1078delT), class II (F508del) or class III (G551D) mutation. For the second mutation: In three the mutations were unclassified at the time of CF NBS, and were subsequently defined as class III (P574H, I601F or R553G). In those patients the first ST was, respectively, 54 mmol/l, 49 mmol/l (then 64 mmol/l at 6 months), and 24 mmol/l (then 66 mmol/l in the following weeks). In two the mutations belonged to class IV: F311L and P205S with positive ST. In one there was a class V mutation: 5T variant with a negative ST. At the end of the observation period: Nutritional status: No child was suffering from malnutrition. Pancreatic and liver function tests were normal. One patient presented with chronic cough. Table 2 Unknown genotypes FVC FEV 1 FEF 25j75 Bacteria Shwachman score AST1 ST1 Age Z score for weight Z score for height Brasfield score Nb Sex CFTR mutations 1 M DF508/P574H j1.2 j NR NR NR Hi 2 M 1078delT/F311L j1.4 j NR NR NR Sa,Hi,Pa 3 M G551D/R553G j % 99% 108% Hi 4 F G542X/5T NR NR NR 5 F DF508/I601F j NR NR NR 6 F DF508/P205S j NR NR NR Sa Nb=patient number; F=female; M=male; ST1=first sweat test (mmol/l); AST1=age when first sweat test is realized (years); Age=age at the end of period observation (years); FVC=forced vital capacity; FEV 1 =forced expiratory volume in 1 second; FEF=forced expiratory flow; Bacteria=lower respiratory tract pathogens: NR=not realized. Sa=Staphylococcus aureus; Hi=Haemophilus influenzae; Pa=Pseudomonas aeruginosa.

7 JIMD Short Report #066 (2007) Online 7 Respiratory status: The mean Brasfield and Shwachman scores were 23 and A single patient underwent PFT: at the age of 10.5 years, FVC was 87.5% of the theoretical value, FEV 1 99% and FEF 25j75 108%. Bacteriological status: All children had a throat swab and/or sputum sample, on average 3.5 times a year. Two children were not infected; two had S. aureus infection, one of which was chronic; one child had P. aeruginosa infection at the age of 2 years 2 months (nb 2). The three serological tests for Aspergillus and P. aeruginosa were negative. Therapeutic management was varied. Three children had respiratory physiotherapy on average three times a week. Only one patient received a course of inhaled antibiotics and of oral antibiotics for a primary P. aeruginosa infection, and required nutritional support. Outcome in six false-negatives of CF NBS Although these six children are not part of our study, we include them in order to enrich the discussion. Five of the six had a mild genotype, but their clinical presentation varied greatly: one F508del/D1152H patient and another F508del/R347L were asymptomatic at, respectively, 15 and 13 years of age. Another F508del/D1152H child presented with FEV 1 103%, P. aeruginosa colonization, and pancreatic insufficiency at 7 years, while one F508del/S945L child had FEV 1 80% with pancreatic insufficiency at 11 years. Finally, a F508del/ A>G female patient died at 12 years of age while awaiting lung transplantation. The sixth child has a problematic genotype (N1303K/F311L) that combines episodes of pancreatitis, P. aeruginosa infection and normal values of PFT. Discussion The frequency of detection of mild or problematic genotypes by NBS for CF is quite high: respectively 9.7% and 1.5% over 16 years in Brittany. The results of the French NBS programme (Munck et al 2006) show that 87 children (14%) had a dilemma case presentation: 32 with a normal ST, 55 with a border line ST. The most frequent mild mutation is R117H, 47 from 87 (54%). Taking all methods of diagnosis together, the literature reports frequencies of 11.3% for mild genotypes and 0.93% for problematic genotypes (Storni et al 2001). The sweat test remains the gold standard in diagnosis, but, when the result is borderline, it complicates the diagnostic procedure and generates anxiety, incomprehension and stress in families, all of which must be taken into account. The ST should, of course, be done according to the reference technique of Gibson and Cooke (Gibson and Cooke 1959), but it has long been known that there are authentic cases of CF with a normal ST and others in which the sweat chloride concentration rises with age (LeGrys and Wood 1988; Massie et al 2000a). With the discovery of the CFTR gene, it was soon noted that IRT levels were significantly higher in heterozygous patients, i.e. those with a normal ST (Castellani et al 1999; Curnow et al 2003; Massie et al 2000b; Parad and Comeau 2005). Such patients may be found to have a second mutation if the gene is studied exhaustively, but these are mild mutations (R117H, A455E, kbC>T, D1152H or ISV8, 5T among the most frequent) or there may be simple polymorphism (Castellani et al 1997; Massie et al 2000b; Padoan et al 2002a,b). The mutations have been divided into five or even six classes depending on whether their molecular mechanisms cause partial or total loss of CFTR function. In theory, the mild mutations are class IV, V or VI, dominant, and a small fraction of CFTR function is preserved, leading to a clinical picture with no or few symptoms (Kulczycki et al 2003; Rowntree and Harris 2003). The Cystic Fibrosis Foundation cohort study of genotyped patients with CF shows that the mortality rate is significantly lower in F508del/ kbC>T or R117H or G>A compound heterozygotes than in F508del homozygotes or compound heterozygotes with classical mutations (McKone et al 2003). In the United Kingdom, a retrospective cohort study was carried out comparing the frequency of bacterial isolates and clinical outcomes in 11 compound heterozygotes for F508del and a second mild mutation, mainly R117H, with a matched group of F508del homozygotes. Shwachman scores, radiological scores and FEV 1 were significantly higher in patients with mild mutations. But most patients with mild variant CF will have bacterial isolates from airway cultures requiring antibiotic therapy three to four times a year. S. aureus was isolated in 8 of the 11 patients with mild variant disease and P. aeruginosa was found in 7 (64%), although the frequency of positive cultures was significantly less (2.8/year) than in the F508del homozygotes (6.1/year) (Lording et al 2006). However, as for classical mutations, genotype phenotype correlation is complicated by the existence of polyvariant mutant CFTR genes and modifier genes (Acton and Wilmott 2001; Drumm et al 2005; Rowntree and Harris 2003; Salvatore et al 2002), and the

8 8 JIMD Short Report #066 (2007) Online Table 3 Mild mutations with severe phenotype Mutation Author [ref] Genotype Sweat test Phenotype R117H Massie et al (2000b) Ellis et al (2002) 15 with 5T allele 15 positive or border line 25 with 7T allele 3 positive and 8 border line 13 F508del/R117H 7T 4 positive and 4 border line 2 deaths at 19 and 43 years 3 asymptomatic 4 with respiratory symptoms 2 with bronchectiasis and FEV 1 54% and 65% at ages 54 and 49 years 4 CBAVD kbC>T D1152H O_Sullivan et al (2006) Boyne et al (2000) Dreyfus et al (1996) Stewart et al (1995) Castellani et al (2000) Padoan et al (2002b) Padoan et al (2002b) Castellani et al (2000) Lebecque et al (2001) Mussaffi et al (2006) 3 F508del/R117H 7T NBS Positive Pa in oropharyngeal cultures 9 F508del/R117H 7T Negative. NBS 3 respiratory symptoms (unknown age) kbC>T / kbC>T Negative Death at age 29 years 1 F508del/ Negative Death at age 27 years kbC>T 1 F508del/ Negative. NBS Positive Pa. Bronchiectasia kbC>T FEV 1 42% at 12 years 1 D1152H/ Negative Respiratory symptoms in the first kbC>T months of life 1 D1152H/R1066C Border line Acute pancreatitis, recurrent abdominal pain at 8 years 1 R553X/D1152H Negative. NBS Positive Pa. Bronchiectasia FEV 1 57% at 14 years 1 F508del/D1152H Negative. Positive Bronchorrhea from age of 4 years nasal TPD Pulmonary lobectomy at 12 years Pa colonization at 13 years 2 D1152H/D1152H 1 prenatal dilated bowel 7 compound 1 infant with pulmonary symptoms heterozygotes 3 adults with chronic Pa and FEV 1 of with D1152H 20 55% 1 F508del/ A>G Death at 21 years A>G Bienvenu et al (1995) Our study 1 F508del/ A>G Positive Death at 12 years S945L Feldman et al 1 F508del/S945L Pulmonary symptoms at 1.5 month (2003) Our study 1 F508del/S945L Positive positive Pa. FEV % at 12.9 years Our study 1 F508del/S945L Positive. Negative NBS Diagnosis at 5 years. Repeated respiratory infections. FEV 1 80% with pancreatic insufficiency at 11 years Positive sweat test: >60 mmol/l. Borderline sweat test: mmol/l. FEV 1 : forced expiratory volume in 1 second; CBAVD: congenital bilateral absence of the vas deferens; Pa: Pseudomonas aeruginosa; TPD: transepithelial potential difference. literature describes classical outcomes in subjects with mild mutations (R117H, kb C>T, D1152H) and a persistently normal ST (Table 3). For example, the phenotypic expression of the missense mutation R117H may vary (Castellani et al 1997; Massie et al 2001) because it is associated with the polythymidine variant of intron 8 (IVS8jnT) on the same allele (cis position). In the presence of the 5T allele, it is typically associated with CF and normal pancreatic function. With the 7T allele, it is either asymptomatic (notably in females) or associated with sterility due to CBAVD in males. The 5T allele acts as a genuine deleterious mutation, but the 7T allele is not always free of respiratory symptoms (Table 3). More recently, it has been shown that the effect of the 5T variant is worsened by 12 or 13 TG repeats upstream (Groman

9 JIMD Short Report #066 (2007) Online 9 et al (2004). Whereas the 5T allele has been found to be more frequent among carrier newborns with elevated IRT values than in controls (Scotet et al (2001), an associated high number of TG repeats in hypertrypsinaemic infants with normal ST has also been reported: among 20 CFTR-identified alterations from 24 hypertrypsinaemic newborns with borderline ST, the 5T 12TG haplotype was the second most frequent mutation (14.6%) over F508del (Padoan et al 2006). The 3272j26A>G mutation is usually associated with delayed diagnosis, better growth, and moderate respiratory insufficiency, notably with less frequent P. aeruginosa colonization (Amaral et al 2001; Feldmann et al 2003). But sometimes the phenotype is severe; the same situation may occur with the S945L mutation (Table 3). We also found three mutations (R347H, R117C, L206W) that seem to be associated with progression to a mild form of CF, as also reported in the literature (De Braekeleer et al 1997; Desgeorges et al 1995; Feldmann et al 2003; Kosztolanyi et al 1996; Lebecque et al 2002; Lyon and Miller 2003; Rozen et al 1995). However, two mild CFTR mutations in cis can act in concert to dramatically alter CFTR function, contributing to the wide phenotypic variability of CF disease. For example, the R347H mutation is associated with mild defective chloride channel activity and the D979A defect leads to misprocessing; the mutant R347H/ D979A combines both defects to produce a dramatic decrease in chloride current (Clain et al 2001). These studies show that caution should be exercised when discounting CF diagnosis in such children. Yet to avoid creating anxiety it is important to be able to reassure the parents of a child whose ST result is normal. While recognizing that there are rare exceptions, the professional should reassure parents when the ST is below 30 mmol/l, but should exercise caution when it is between 30 and 60 mmol/l and repeat the ST later and conduct an exhaustive study of the gene. Farrell and Koscik, who initially set the lower safety limit at 40 mmol/l, subsequently reduced it to 30 mmol/l (Farrell and Koscik 1996). Therapeutic management of mild CF detected by NBS Management of so-called mild forms, whose outcome is theoretically favourable but which can also be unpredictable, must be individualized and adapted to the patient_s clinical picture (Rosenstein 2003; Wallis 2003). Care is founded on the same principles as management of classical forms, i.e. prevention of malnutrition and bacterial respiratory infections, but must also be flexible and yet systematic should functional signs arise, so as to slow disease progression. Long-term follow-up of children seems necessary, when possible in a specialized centre, as the clinical picture is complex and the family is best managed by specialists, even if some members of the medical team (physiotherapist, dietitian,...) will not systematically play a part. Other authors, however, consider that such specialized follow-up is too restrictive and harrowing and instead advocate monitoring by the family physician (Curnow et al 2003)]. Follow-up by the family physician alone may be justified in the setting of a care network, and in the absence of clinical signs and symptoms of CF the child can be referred to the specialized centre annually for clinical and laboratory work-up (ST, chest radiography, sputum or throat swab, pancreatic function tests, routine laboratory tests). We feel it is important to gain parental confidence in order that the child will be brought in for examination as soon as any suggestive signs appear and if the ST becomes unequivocally positive, so that more conventional therapeutic management of CF can be proposed (more frequent follow-up visits, respiratory physiotherapy, suitable antibiotic therapy,...). In the absence of signs, we feel that such therapeutic management is exaggerated and generates unjustified restrictions and unnecessary stress. In the absence of an annual work-up at the specialized centre, the risk is that the child will only be seen if there are lingering clinical signs, with the attendant risk of non-resolution. The parents of one of our patients, having DF508/S977F with a first ST of 51 mmol/l and a later value of 60 mmol/l, did not wish to return to the specialized centre once the diagnosis had been made, and the child was next seen at the age of 5 years, but some time after several lung infections. Admittedly the child_s FEV 1 was 95%, but marked bronchiectasis remained in one lung lobe and the Brasfield score was 20. Genetic counselling is another fundamental issue in therapeutic management, and raises a difficult ethical question to which there is no unanimous response. We have seen how the genotype alone does not make the disease, particularly if it contains one or two mild mutations whose penetrance is variable and whose phenotypic consequences are unpredictable and vary even between two patients carrying identical mutations. What should be done in the case of prenatal diagnosis? Like an Australian team (Curnow et al 2003), we recommend a multidisciplinary, case-by-case approach adapted according to the clinical status of older siblings and the parents_ decision, taken after provision of accurate information which makes it clear that a degree of uncertainty remains. Given the

10 10 JIMD Short Report #066 (2007) Online uncertain outcome of these mild forms of CF, some authors think that mild mutations should not be screened for (Boyne et al 2000; Massie et al 2000a; Scotet et al 2006), while others take the opposite view (Lording et al 2006; O_Sullivan et al 2006). A European working group is currently writing guidelines for the investigation and management of infants with an equivocal diagnosis following NBS for CF. Conclusion We do not really know the outcome of mild forms of CF as this problem has only arisen in recent years and follow-up is short. Diagnosis of CF in such cases is no doubt questionable in theory, but it is currently difficult to predict clinical progression and to offer the family satisfactory genetic counselling. For the R117H mutation, the most frequent mild mutation, we need to know straight away whether it is associated with the 5T, 7T or 9T allele in intron 8 and TG repeats, since its deleteriousness depends in part on the allele. In compound heterozygosity, the presence of mild mutations has occasionally and sometimes more consistently been linked with a ST below 60 mmol/l, or even under 30 mmol/l in very rare cases. To minimize parental anxiety, reassurance should be given when the value is below 30 mmol/l, but care should be exercised if it is between 30 and 60 mmol/l and the genetic study should be continued and the ST repeated, while recognizing that we cannot foresee the exact outcome in children for whom the diagnosis of CF is confirmed later by positive ST but who remain asymptomatic in the first years of life. Opinions differ regarding the value of exhaustive study of the CFTR gene, because of the psychological impact on the family. The ST is still the diagnostic gold standard, but its reliability is sometimes thrown into doubt by in-depth study of the CFTR gene. 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