Following the discovery of the CFTR gene,⁹ most screening programs have modified their screening strategy.¹⁰ In regions where more than 90% of patients with CF have at least one ΔF508 mutation, analysis for ΔF508 is performed on week 1 neonatal blood spots with an elevated IRT level.¹¹ This strategy has been further modified in regions (particularly southern Europe) where ΔF508 is only present in 40%–60% of patients, by screening for an extended panel of non‐ΔF508 mutations (usually severe class I, II or III) common to that region.^12,13 These IRT/DNA screening strategies are, however, not without problems. For instance, in regions where ΔF508 is responsible for CF in over 90% of cases, up to 10% of patients have non‐ΔF508 mutations with a preponderance of PS patients¹⁴ and therefore screening for ΔF508 alone would not detect such patients.

Currently, only limited data are available on the pancreatic phenotype of infants with CF diagnosed by IRT/DNA screening from a study¹⁵ restricted to 27 infants with only class I or II mutations. The present study was thus undertaken, firstly to determine the pancreatic phenotype of infants in either a ΔF508 predominant or non‐dominant region, and secondly, to determine what proportion of infants initially with PS later transitioned to being PI and whether this was determined by the presence of two severe mutations.

The basic strategy of the two screening programs was similar: 1) an IRT measurement was taken on dried blood spots obtained from infants in the first week of life; 2) if the IRT was elevated (at Verona >100 μg/l until May 1995 and >95 μg/l thereafter, and at Westmead >99th percentile), a mutation analysis was performed; and 3) the diagnosis was confirmed by sweat chloride estimation. The major difference between the two programs pertained to the mutational analysis: at Verona, a region where at least one copy of ΔF508 was present in only 40%–60% of patients, the blood spots were analysed for ΔF508, R1162X and N1303K prior to March 1995 and ten additional mutations (2183AA→G, 3849+10KbC→T, G542X, 1717‐1G→A, R553X, Q552X, G85E, 711+5G→A, 3132delTG, 2789+5G→A)¹⁶ thereafter; at Westmead, only ΔF508 was screened for. The other minor difference in the screening strategy was that patients at Verona had a meconium lactase test performed and if either a CF mutation was detected and/or the lactase level was >0.5 U/l, a sweat chloride test was carried out. It is noteworthy that nine of the 13 mutations screened at Verona were severe mutations.^7,17,18 At both centres, elevated IRT, the presence of at least one CF mutation (or elevated meconium lactase in infants from Verona) and an elevated sweat chloride 60 mmol/l were diagnostic of CF. Infants at both centres with borderline sweat chloride values of 30–59 mmol/l underwent repeat sweat testing and a more extensive panel of mutations was analysed. At Verona this included denaturing gradient gel electrophoresis of coding regions and at Westmead 16 non‐ΔF508 mutations: Δ1507, R117H, G551D, A455E, G542X, N1303K, W128X, 1717‐1G→A, R560T, R347P, R334W, R553X, R1162X, S549N, 3849+10KbC→T and 621+1G→T.¹⁹ Excluding ΔF508, six of the other mutations were common to both centres.

At Verona, IRT was assayed by the Pharmacia (Turku, Finland) standardised Delfia Neonatal IRT kit,²⁰ and a variation was used at Westmead.²¹ Sweat chloride testing was performed by the Gibson and Cooke method²² at both centres and meconium lactase testing as described previously.²³ The procedure used to prepare DNA for PCR from the dried blood spots on Guthrie cards was similar at both centres.^24,25 Between January 1993 and February 1995, mutation analysis was performed by restriction enzymes²⁶ or heteroduplex analysis²⁷ and thereafter by a reverse dot blot assay.²⁸ At Westmead, DNA for mutation analysis was extracted from blood spots by isopropanol fractionation.²⁹

The 3–5‐day fat balance results are shown for each centre in fig 1​1,, and according to whether the infant was bottle or breast fed (panels A and B, respectively). Faecal fat varied widely among the PI patients, ranging from 11% to 70% of fat intake in the bottle‐fed group and from 2.2 to 19 g/day in the breastfed group. All infants at Verona had fat balance studies. At Westmead 124 (75%) patients had their pancreatic status at neonatal diagnosis determined by fat balance studies (113 infants) or PST (11 infants). Of the 11 who had a PST, 10 had colipase secretion values >10% of average normal values and were PS and one had a value <1% of average normal colipase secretion and therefore was PI.⁴ There was a delay in evaluating pancreatic function in an additional 13 patients at Westmead due to parent preference. In 10 of these 13 patients at an average age of 2 years, faecal fats ranged from 11% to 58%, and in the remaining three patients faecal fats were 7.9%, 5.1% and 5.6% at 6 months, 1 and 2 years, respectively. A group of 29 (18%) patients at Westmead did not undergo pancreatic testing. They included 12 with MI, 16 with symptoms of malabsorption and one without symptoms of malabsorption who was a sibling of a PS patient. As those with MI or malabsorption symptoms had severe mutations, we have considered them as PI patients.

The genotype/pancreatic phenotype relationships were evaluated for the three general genotypic groups: ΔF508 homozygotes, ΔF508 compound heterozygotes and non‐ΔF508 compound heterozygotes as shown in table 2​2.. The compound heterozygote groups were further subdivided into subgroups according to whether mutations were type I–III severe (designated as F/x or x/x), type IV and V mild (F/m or x/m) or unknown (F/uk or x/uk). For both clinics and the combined data, two horizontal rows of data are given. The first row provides the absolute numbers of patients with that specific genotype, noting the bracketed numbers associated with the subtotals represent the subtotal number as a percentage of the total number (n) of patients at that clinic. For example, under the column for ΔF508 homozygotes for Verona 37/149 (25%) and for Westmead 97/166 (58%) were ΔF508 homozygotes. The second row provides the absolute number of PS patients for that genotype and in brackets the percentage of the subtotal for that clinic; for example, for ΔF508 homozygotes 4/37 (11%) at Verona and 16/97 (16%) at Westmead were PS. As expected, Westmead had a significantly greater number of ΔF508 homozygotes than Verona (97 vs 37, p<0.001 by χ² test). There were similar numbers of ΔF508 compound heterozygotes (67 vs 64), whereas Verona had a significantly greater number of non‐ΔF508 patients (45 vs 5, Verona vs Westmead, respectively, p<0.001). Among the three general genotypic groups, ΔF508/ΔF508, ΔF508 compound heterozygotes and non‐ΔF508 patients, in the combined clinics the proportions with PS were 15%, 34% and 32%, respectively. Notably, from the severe mutation groups ΔF508/ΔF508, ΔF508/x and x/x, 34 of 231 (15%) infants at this early age were PS. Not unexpectedly, of those with the mild mutations F/m and x/m, 26 of 28 (93%) were PS. The two exceptions had the ΔF508/3849+10 KbC→T genotype.

The observed reduction in the proportion of PS patients in the current study was not unexpected. At Westmead only ΔF508 is initially screened for and thus infants without at least one ΔF508 mutation would not be detected except for those with either MI or a family history of CF, as shown by the Westmead data in table 2​2.. Previously, older ΔF508 predominant populations have demonstrated up to 10% of patients without ΔF508 mutations and 66% of this subgroup were PS.¹⁴ Missing these patients in addition to another subgroup of ΔF508 compound heterozygotes with low or borderline sweat chlorides as described previously¹⁹ would account for the decrease in absolute PS numbers in the current study. Although as yet we are uncertain as to whether a similar phenomenon exists in Verona, the data should alert community physicians/paediatricians that regardless of the mutation screening strategy, children presenting de novo at an older age with recurrent or persistent chest symptoms and infections, bronchiectasis and/or malabsorption, should be investigated for CF despite negative neonatal screening.

It is of interest in the current study that at the time of neonatal diagnosis, 34 of 231 infants with two severe PI‐associated mutations (ΔF508 homozygotes, ΔF508/x and x/x) were PS. This is an unusual finding in older patients with CF,^7,14 but previously we have demonstrated that this subgroup loses their pancreatic function with age.⁶ Walkowiak et al have demonstrated a similar phenomenon in a small group of 27 infants with class I or II mutations, noting that all eight with PS at diagnosis had become PI within the first year of life.¹⁵ In the current study, 20 of the 34 PS patients with class I, II or III mutations at neonatal diagnosis developed PI and as shown in table 3​3,, the age of onset of PI varied considerably, ranging from 2 months to 11 years of age. In contrast, to date none of the 26 patients with mild class IV or V mutations (designated as ΔF508/m and x/m in table 2​2)) have developed PI in their follow‐up investigations, a finding consistent with the data from an older population where only one of 49 patients with class IV or V mutations had developed PI.⁷ These findings are important for clinical management and clearly indicate that one should not assign the PI or PS phenotype to infants with CF based on the results of phenotype/genotype studies in older patients. Ideally, patients should be assessed with fat balance studies, as in the current study, or with other appropriate indirect tests to determine the necessity for enzyme therapy.

In summary, the present study demonstrates that infants with CF diagnosed by two different IRT/mutation screening strategies have a comparably higher proportion of PS patients at the time of newborn diagnosis than older populations with CF. The proportion is, however, approximately 33% lower than in populations screened by the older repeat IRT non‐mutation strategy. At least in programs where only ΔF508 is screened for, the lower proportion is in part related to the non‐detection of PS patients without a ΔF508 mutation and physicians in regions with such a population should be aware that these patients remain undiagnosed in their community. The current findings also demonstrate that 34 (42%) of the PS patients at the time of neonatal diagnosis had severe class I, II or III mutations, and nearly half of this subgroup suffered deterioration of their pancreatic function and became PI before 5 years of age. Clearly, this subgroup with severe mutations requires careful follow‐up with regard to their pancreatic function.