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Analysis of the entire coding region of the cystic fibrosis transmembrane regulator gene in neonatal hypertrypsinaemia with normal sweat test
  1. *Cystic Fibrosis Centre, Ospedale Civile Maggiore, Piazzale Stefani 1, 37126 Verona, Italy
  2. Department of Mother and Child, Biology and Genetics, Section of Biology and Genetics, University of Verona, Verona, Italy
  1. Dr Castellani, carlocastellani{at}

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Editor—Measurement of immunoreactive trypsinogen concentration (IRT) in dried blood spots is the most common technique for neonatal screening for cystic fibrosis (CF).1 Since a considerable number of newborns show raised IRT levels, several laboratories improve the screening specificity by testing infants with hypertrypsinaemia for the most common CF mutations. Diagnosis is established in neonates carrying two mutations, but a sweat test is required if only one mutation is found, in order to detect affected subjects with a second, unrecognised mutation. Infants with raised IRT, one CF mutation, and normal sweat electrolyte concentrations are usually considered to be just carriers.

Unexpectedly, a frequency of CF heterozygotes significantly higher than in the general population has been repeatedly reported among neonates with hypertrypsinaemia and normal sweat chloride levels.2 3 It is not clear whether having one CF mutation, perhaps together with some unknown pathogenetic factor, is sufficient to predispose to neonatal hypertrypsinaemia. Such a hypothesis is corroborated by the findings of Lecoqet al,4 who showed that the probability of a newborn being a carrier of the majorCFTR mutation, ΔF508, increases with neonatal IRT concentration, and suggested that heterozygotes may have early subclinical impairment of exocrine pancreatic function. Alternatively, it could be speculated that at least some hypertrypsinaemic newborns who, after testing for a limited number of CF mutations have been found to carry a CFTRgene mutation, have on the other chromosome an undetected mild mutation, and possibly suffer from an atypical form of CF, characterised by a negative sweat test. A DNA polymorphic sequence of five thymines in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is very commonly involved in the pathogenesis of a primarily genital form of CF called congenital bilateral absence of the vas deferens (CBAVD),5 has actually been found to be more frequent in carrier newborns with raised IRT values than in controls.3 6 Moreover, a complete scan by denaturing gradient gel electrophoresis of all 27 CFTRexons detected nine more mutations in a sample of 18 heterozygous newborns whose IRT was raised and sweat chloride normal.7

In order to contribute to the understanding of the correlation between CFTR mutations and neonatal hypertrypsinaemia, we analysed the CFTR gene in a larger group of IRT positive and sweat chloride negative heterozygous neonates and in a subset of neonates with similar characteristics but negative for a standardCFTR mutation panel.

The population under study consisted of 47 screened neonates with IRT at birth above the 99.5th centile and normal sweat test (<40 mEq/kg, Gibson and Cooke method8). The screening strategy was as previously described.3 As non-CF hypertrypsinaemia has been reported in perinatal asphyxia and prematurity,9 10no newborn with any of these conditions was included.

The 47 neonates were divided into three groups. Group A consisted of 13 newborns, with one identified CFTR mutation among the panel of 15 routinely sought in hypertrypsinaemic babies according to our neonatal screening protocol. In a previous study, all of them had been tested for the (T)5 allele in intron 8 and none of them carried it.3 Group B consisted of 19 newborns, again carrying one identified CFTR mutation out of the panel of 15, but not previously tested for the (T)5 allele. Group C consisted of 15 newborns carrying no CFTRmutation out of the panel of 15 and not previously tested for the (T)5 allele. Groups A and B included all the screened carrier newborns, born between July 1993 and January 1998, for whom a blood sample sufficient for genetic analysis was available. These two groups were meant to represent CFTR heterozygous hypertrypsinaemic neonates, as opposed to just hypertrypsinaemic neonates (group C); however, as A and B disagreed with regard to the previous 5T analysis, they were processed separately.

In all the neonates and in a control group of 15 healthy subjects whose IRT at birth was unknown but presumed to be normal (the chance of having raised IRT at birth using our cut off is 1 in 200),11 a complete gene search of all 27 exons and intronic flanking regions with denaturing gradient gel electrophoresis (DGGE) analysis was performed. PCR products that displayed altered behaviour in the gel were sequenced after cloning. DGGE primers and conditions in the three groups of newborns under study were as previously described.12 In addition, the alleles at the polymorphic loci IVS8(TG)m, IVS8(T)n (not in group A), and M470V were studied. M470V was analysed by PCR and HphI restriction enzyme analysis,13 and IVS8(TG)m and IVS8(T)n with fluorescent primers on an ABI PRISM 377.4

The Kruskal-Wallis test was used for statistical analysis of IRT and sweat chloride and sodium values and the Wilcoxon test to compare weight Z scores at birth and at the time of sweat testing. Comparison of mutation and polymorphism frequencies was performed by Fisher's exact test, including Yates's continuity correction.

Preliminary results of DGGE analysis, but not of the polymorphic loci study, of all newborns in group A and five newborns in group B have been previously published.7

The median IRT at birth of the three groups is shown in table 1. Sweat chloride and sodium and concomitant IRT values are also included. Trypsinogen levels showed a time related decrease; IRT values were below the cut off at 1 month of age (75 μg/l) in all cases in which data were available, with the only exception of subject 2B in table 2, who was later found to be a compound heterozygote for the mutations 1717-1G→A and D1152H. Table 1 also shows the median weight Z scores calculated at birth and at the time of sweat testing.

Table 1

IRT, sweat test, and weight Z score values in groups A, B, and C

Table 2

Characteristics of 24 neonates with CFTR mutations other than the 15 routinely sought in IRT positives or the 5T allele. Patients 1A, 2A, 3A, 4A, 5A, 6A, 1B, 8B, and 10B have been partially reported elsewhere7

Seven more CFTR gene mutations were found in group A, eight more in group B, and eight in group C. One mutation (L997F) was found in the control group. The 5T variant was carried by three neonates in group B and one in group C. Table 2 shows genetic and clinical data of the neonates carrying CFTRmutations other than the 15 routinely sought in IRT positives or the 5T allele.

Phase was determined in the three babies of group B carrying the 5T allele: in patient 8B, N1303K was in transwith (T)5-(TG)11-M470, as was ΔF508 in newborns 9B and 10B with (T)5-(TG)12-M470 and (T)5-(TG)11-M470, respectively.

The incidence of CFTR mutations and alleles at (T)n, (TG)m, and M470V polymorphic loci in groups A, B, and C and in controls are shown in table 3.

Table 3

Incidence of CFTR mutations and alleles at poly T, (TG)m, and M470V polymorphic loci among the chromosomes of patients in groups A, B, C, and in controls

A complete CFTR gene scan of the whole coding sequence has never before, to the best of our knowledge, been performed in unaffected hypertrypsinaemic neonates not carrying any of the commonest mutations, like the ones in group C. Among them, a high incidence of mostly rare CFTR mutations, significantly more than in the control group was found.

A high frequency of CFTR mutations has been detected in subjects showing some clinical manifestations which can also be found in patients with CF, namely CBAVD,5pancreatitis,15 16 disseminated bronchiectasis,11 and allergic bronchopulmonary aspergillosis.17 It is still not completely understood what the correlation between these conditions and carrier status could be. It is generally assumed that carriers ofCFTR mutations have about 50% of normal function, which is sufficient to remain free from disease. However, most of the studies tested only for a limited number of the more than 850 known CF mutations, and it is therefore possible that more comprehensive genetic analysis might detect additional mutations. Even without postulating a second CFTR mutation, the contribution of unidentified modifier genes could possibly be sufficient to cause isolated manifestations of CF in heterozygotes.

Similar speculations could also apply to some cases of neonatal hypertrypsinaemia with normal sweat test, and our results seem to confirm that the association of this condition and aCFTR mutation may have some phenotypic consequences. Both sweat chloride and sodium in the two groups of carriers, A and B, were higher (the latter with statistical significance) than in group C; similarly, weight Z score in groups A and B was worse at the time of sweat test than at birth.

An alternative, or perhaps complementary speculation to explain the high carrier frequency in hypertrypsinaemic newborns could be that at least some of them carry on the other chromosome an undetected mild mutation, associated with normal sweat chloride values, but able to raise trypsinogen levels. Such a hypothesis has been substantiated by a previous study showing that a few heterozygous newborns with raised IRT and normal sweat chloride do actually carry a secondCFTR mutation.7

The results of the present study confirm such a finding; in 14 out of 32 babies (groups A and B), together with the previously detected mutation, there were either one (in 13 cases) or two (in one case) moreCFTR gene mutations. The phase could be determined in 10 cases and in eight of them the newly found mutation was in trans with the one originally found. As deep intronic mutations remain uncharacterised after screening the coding sequence, we cannot exclude that more infants could be compound heterozygotes.

All missense mutations detected with DGGE analysis (R117H, Y301C, D1152H, M1137V, L997F, F1052V, R75Q) except one (E527G) are located in membrane spanning domains (MSD). It has been reported that some MSD mutations alter the pore properties of CFTRby decreasing the amount of current18; the residual channel activity may be sufficient to produce a mild phenotype, like the rise in newborn IRT levels.

A role in determining neonatal hypertrypsinaemia could also be played by the 5-thymidine allele in intron 8. The proportion of the correctCFTR gene transcript is inversely related to the length of the polythymidine tract in the sequence of the acceptor splice site of intron 8; three alleles ((T)5, (T)7, and (T)9) can be found at this locus, and the (T)5 variant results in a high proportion of abnormal, alternatively spliced CFTRmRNA.19 Subjects with one CF mutation on one chromosome and the (T)5 allele on the other have little normalCFTR, and their phenotypes are quite diverse, ranging from good health, to CBAVD, or even to mild CF.5 20 Former evidence suggests that possibly neonatal hypertrypsinaemia may also be included in this wide clinical spectrum, as the incidence of (T)5 in heterozygous newborns with raised IRT was found to be higher than in other carriers.3 6 The greater (T)5 frequencies in groups B and C compared to controls, even though not statistically significant, are compatible with such previous findings, and so is the fact that in the three infants in group B carrying the (T)5 allele this is always intrans with the originally found mutations.

As for the M470V polymorphism, in vitro studies showed that the V allele yields a lower functional amount of CFTR protein at both transcriptional and translational levels.21 Although one would have expected to find this allele more frequently among hypertrypsinaemic neonates, it was instead the M polymorphism which was significantly higher in groups A and B compared to group C and controls. This result may be explained by the strong association of the amino acid methionine with mutations located in one of the nucleotide binding folds, mutations which were well represented in both groups A and B.22

It is debatable whether the compound heterozygotes detected have or do not have CF. A diagnosis of CF can be made in the presence of a positive neonatal screening test plus the evidence ofCFTR dysfunction as documented by raised sweat chloride concentrations, or the in vivo demonstration of abnormal ion transport across the nasal epithelium, or identification of two CF causing mutations.23 None of the mutations found by DGGE were known to be CF causing (R117H would be if incis with (T)5, but in patient 1A, a compound heterozygote for R1162X and R117H, the polyT genotype was (T)7/(T)7).

However, one cannot exclude that neonatal hypertrypsinaemia, in the presence of some degree of genetic abnormality in both CF alleles, could be the first sign of some CFTR related disease. In approximately 2% of CF patients, there is an “atypical” phenotype, which often consists of mild chronic sinopulmonary disease, pancreatic sufficiency, and normal to borderline sweat chloride concentrations23; compound heterozygous infants could possibly develop a similar phenotype in time, or, if males, CBAVD.

In practice, it is not at present possible to predict the clinical outcome of our newborns, nor to provide satisfactory genetic counselling for the family. Close clinical follow up should help in clarifying the extent of the disease, if any, in these subjects.

Two mutations, D1152H and L997F, deserve further comment. Neonate 2B (table 2) carried, on different alleles, 1717-1G→A and D1152H. His IRT value at birth was unusually high, even for CF, and remained raised at the time of the sweat test as well; also, his sweat chloride level, even though under 40 mEq/kg, was the highest among the neonates under study. D1152H has been reported in association with isolated CBAVD, but also with mild, late onset lung disease and pancreatitis in conjunction with normal sweat values.24

Another peculiar result of the study was the finding on four occasions of L997F, a mutation usually rare in CF, but perhaps more common in idiopathic disseminated bronchiectasis,12 as well as in idiopathic pancreatitis. Unpublished data from our group show that L997H was found in four cases from a subset of 32 subjects suffering from idiopathic pancreatitis, but in none of 100 ΔF508 carriers.

In conclusion, standard mutation panels can detect a high prevalence ofCFTR mutations among subjects with neonatal hypertrypsinaemia and negative sweat chloride, but even more mutations can be found by a more thorough gene search. Close clinical follow up should help in clarifying the extent of the disease, if any, in compound heterozygous newborns.


This work was supported by grants from the Italian Ministry of University and Research and the Italian Ministry of Public Health, CF Project, law 548/93. M G Benetazzo received a fellowship from the Cystic Fibrosis Centre of Verona.