The original algorithm for cystic fibrosis (CF) newborn screening (NBS) used 2 serial

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1 DIAGNOSTIC DILEMMAS RESULTING FROM THE IMMUNOREACTIVE TRYPSINOGEN/DNA CYSTIC FIBROSIS NEWBORN SCREENING ALGORITHM RICHARD B. PARAD,MD,MPH, AND ANNE MARIE COMEAU,PHD Objective To quantitate the proportion of infants ientifie through cystic fibrosis (CF) newborn screening (NBS) by an immunoreactive trypsinogen (IRT)/DNA screening algorithm who have an unclear iagnosis as efine by the finings of an elevate IRT level an either 1) 2 CF gene (CFTR) mutations etecte an sweat chlorie level <60 meq/l; or 2) 0 or 1 CFTR mutations an a borerline sweat chlorie level $30 an <60 meq/l. Stuy esign Using the 4-year cohort of CF-affecte infants recently escribe by the Massachusetts CF NBS program, we ientifie an escribe the number of infants with the iagnostic characteristics (iagnostic ilemmas) aforementione. Results Of infants with positive results on CF NBS who ha 1 CFTR mutation etecte an a borerline sweat chlorie concentration, nearly 20% isplaye a secon CFTR mutation on further evaluation. Of all infants with positive CF NBS results consiere affecte with CF, 11% ha a iagnosis that fell into 1 of the iagnostic ilemma categories aforementione. Conclusions Four problematic iagnostic categories generate by CF NBS are efine. In the absence of ata on the natural history of such infants, careful follow-up is recommene for infants in whom a efinitive iagnosis is elusive. (J Peiatr 2005;147:S78-S82) The original algorithm for cystic fibrosis (CF) newborn screening (NBS) use 2 serial rie bloo spot (DBS) immunoreactive trypsinogen (IRT) values (specimen collection separate by weeks) to confirm persistent neonatal IRT elevation before referral for efinitive CF iagnosis with pilocarpine iontophoresis, or sweat testing. 1,2 With the ientification of the gene associate with CF, the Cystic Fibrosis Transmembrane Conuctance Regulator (CFTR), Gregg propose using DNA testing in a new IRT/DNA 2-tier CF NBS algorithm that uses only a single DBS collection. 3 This allowe for faster turnaroun of screening results an a iminishe risk of losing track of an infant who might not have the secon DBS submitte for testing. Although the IRT/DNA algorithm improves some aspects of CF NBS (eg, positive preictive value 4 ), it raws attention to new issues. With over 1300 pathogenic mutations reporte in the CFTR gene from a variety of racial an ethnic groups, any screening algorithm that is applie to a iverse population such as that in the Unite States may nee to tailor the choice of the particular mutations inclue in the IRT/DNA screening assay. Use of only the most common CFTR mutation, DF508, in a subpopulation with a low DF508 allele frequency coul miss a significant number of affecte infants. The Massachusetts (MA) CF NBS algorithm, 5,6 which inclues a multiple CFTR mutation panel, uses a single, early DBS, an emonstrates a low false-negative results rate for ientifying CF. The use of multiple mutations allows for DNA-base iagnosis from the screening test, leaing to earlier initiation of therapy. In aition, use of a multiple rather than single CFTR mutation panel lowers the risk that the etection of only 1 CFTR mutation in the CF NBS will be associate with positive sweat test results, which allows for more reassuring pretest counseling. CF CFTR [Cl 2 ] Cystic fibrosis Cystic fibrosis transmembrane conuctance regulator Chlorie concentration DBS IRT NBS QNS Drie bloo spot Immunoreactive trypsinogen Newborn screening Quantity not sufficient From the New Englan Newborn Screening Program, University of Massachusetts Meical School, Boston, Massachusetts; Brigham an Women s Hospital, Department of Newborn Meicine, Boston, Massachusetts; an Chilren s Hospital, Boston, Massachusetts. Dr Para has receive grant support from the Cystic Fibrosis Founation. Drs Para an Comeau are avisors to the Cystic Fibrosis Founation on cystic fibrosis newborn screening. Reprint requests: Richar Para, MD, MPH, Co-Director, Massachusetts Cystic Fibrosis Newborn Screening Program, New Englan Newborn Screening Program of University of Massachusetts Meical School, 305 South St, Jamaica Plain, MA richar.para@umassme.eu /$ - see front matter Copyright ª 2005 Elsevier Inc. All rights reserve /j.jpes S78

2 Table I. A cohort of newborns with elevate serum IRT concentrations ivie into iagnostic categories by results of sweat Cl 2 concentration an CFTR mutation analysis (total number of infants with positive CF NBS = 1338, but sweat ata only available on 1214). Total number consiere CF true positives = 110 (ata not inclue on 2 false negative an 1 not screene affecte infants who woul bring the total known affectes to 113). Four shae areas contain infants consiere iagnostic ilemmas Sweat Chlorie (meq/l) Elevate serum IRT (>95%) Number of mutations etecte on CFNBS IRT > 99.8% $60 (Abnormal) CF (n = 75 * ) CF (n = 21) CF (n = 2) (Borerline) CF Spectrum y Possible CF Spectrum Possible CF Spectrum Group I (n = 4) Group III (n = 4) Group IV (n = 1),30 (Normal) CF Spectrum y Carrier (n = 904) z Normal (n = 324) z Group II (n = 3) *10 infants in this group with 2 Pancreatic Insufficient CF mutations (e.g., DF508 homozygote) have not yet complete sweat testing but are inclue in this group. ycf Spectrum refers to a CFTR ysfunction relate phenotype which might range from severe multi-organ to mil single organ involvement. Classic CF an Atypical or Variant CF are inclue. z114 infants inclue in these 2 categories have missing sweat ata, but are presume not to have CF for this analysis because they have not come to clinical attention at a CF center. The 2 main consierations in applying DNA testing in the CF NBS are: 1) choosing an IRT cutoff value that prompts DNA testing an 2) choosing a mutation panel appropriate to a given population that will minimize the false-negative results rate an ieally only ientify infants in whom classic CF will evelop. When single mutation testing is chosen, a higher false-negative results rate occurs with the DNA component of the algorithm. There is an option to use a failsafe for ientifying affecte infants with rare mutations by referring infants with an extremely high IRT concentration for sweat testing 5 regarless of DNA results. Although the gol stanar for CF iagnosis is still the sweat test, this test is also not perfect. 7,8 For a small percentage of infants with positive CF NBS results, testing too early might result in inaccurate reaings because sweat chlorie concentration ([Cl 2 ]) falls within ays of birth 9 or because too little sweat is obtaine to generate a reliable measurement. Repeating the test at a later time can solve these problems. In some frustrating instances, the sweat [Cl 2 ] value is in a borerline range. For infants, this has been efine in the MA CF NBS program as [Cl 2 ] values from 30 to 59 meq/l, because 30 meq/l is approximately 5 SDs higher than the mean of infants who are known CF carriers. 10 The natural history of these infants with borerline results is not well efine, which suggests the nee for a follow-up protocol to better unerstan the risks in this group. All infants with positive IRT/DNA CF NBS results who have unergone sweat testing will fall into 1 of 9 outcome categories (Table I). Most of them are preicte to have clear-cut outcomes. In this paper, we report the frequency of outcomes that fall into 1 of 4 categories that constitute iagnostic ilemmas (shown by the shae cells in Table I). Diagnostic Dilemmas Resulting From The Immunoreactive Trypsinogen/DNA Cystic Fibrosis Newborn Screening Algorithm Figure. Diagnostic ilemma follow-up protocol. METHODS Massachusetts began a CF NBS as a supplementary, optional program with approval for research from Human Subjects Committees on February 01, 1999, which require informe parental consent for CF NBS testing. The IRT/ DNA algorithm using a multiple mutation panel is escribe elsewhere in etail. 5 IRT an DNA ata were maintaine in an Access (Microsoft) atabase at the New Englan Newborn Screening Program (NENSP), where testing was performe. More than 99% of sweat tests require on infants with positive CF NBS results were performe at 1 of the 5 MA CF Founation centers in a National Committee for Clinical Laboratory Stanars-certifie sweat laboratory. A sweat [Cl 2 ]of >60 meq/l on $75 mg or $15 ml of collecte sweat resulte in recommenation for treatment at a CF care center. The presence of 2 CF-causing mutations or a sweat [Cl 2 ]of30 S79

3 Table II. Diagnostic Dilemma Groups I-IV: Initial characteristics of iniviual infants from the 110 infant affecte cohort. Shae cells inicate mutations later etecte on extene genotyping. Two infants with DF508/5T an borerline sweat chlorie values were not inclue in the count of the true positive cohort, however follow-up continues Group IRT (mg/ml) IRT % CFTR Allele 1 CFTR Allele 2 [Cl 2 ] meq/l Sex I DF508 R117H-7T 34 F DF508 R117H-7T 33 F DF508 R117H-7T 49 M W1282X kb 54 M II DF508 R117H-7T 24 F G85E R117H 21 F G551D R117H-7T 27 M III DF508 unknown 58 M * G85E R117C 33 F G551D R117C 46 F DF508 L206W 35 M IV G85E y R117C 41 M *Ientifie twin sibling has [Cl 2 ] > 60 meq/l. ythis mutation was not initially etecte because G85E was not inclue in the original MA CF NBS program multimutation panel. to 59 meq/l was consiere abnormal (borerline or ineterminate) an prompte entry into a follow-up protocol for which agreement from all 5 CF center irectors was obtaine (Figure). All follow-up ata (clinical iagnoses, sweat test ata, genetics evaluation) were reporte by the CF centers for entry into the central atabase at NENSP, where outcomes of infants with positive screening results were prospectively tracke. The MA CF NBS program follow-up on infants with positive CF NBS results assigns the infants to one of the 9 categories in Table I, 4 of which are consiere to be iagnostic ilemma categories. These 4 categories are: group I = IRT >95%, 2 CFTR mutations, an a borerline sweat test result; group II = IRT >95%, 2 CFTR mutations, an a negative sweat test result; group III = IRT >95%, 1 CFTR mutation, an a borerline sweat test result; an group IV = IRT >99.8%, 0 CFTR mutations, an a borerline sweat test result. A protocol for follow-up of both quantity not sufficient (QNS) an borerline sweat values (Figure) was agree on by the MA CF NBS Workgroup (a group incluing newborn screeners an CF center irectors) before the initiation of the MA CF NBS program. the parents ha refuse CF NBS. Results of 1214 sweat tests were available. Missing ata were cause either by eath before sweat testing, parental refusal to perform sweat test, sweat testing performe out-of-state, loss to follow-up (testing not requeste), or multiple QNS sweat tests without resolution. CF-Affecte Infants The CF NBS resolve 1338 infants who were screenpositive in one of 9 categories efine in Table I (1 normal an 8 others). The available sweat test results showe most infants with a positive CF NBS result (1117/1214 or 92%) ha normal sweat tests ([Cl2] <30 meq/l). For the purposes of subsequent consierations, in this Table, the 114 infants etecte with 0 or 1 mutations who i not have sweat test ata available were assigne as having a negative sweat test result. A iagnosis of CF was mae in 8% of the infants with a positive CF NBS result. Within this group, presentations inclue: 2 mutations etecte (82/929), 1 mutation etecte an a [Cl2] $60 meq/l (n = 21), or no mutations etecte, but an IRT >99.8% (2/327). RESULTS Between February 1, 1999, an January 31, 2003, 323,506 newborns were screene for CF in Massachusetts over 98% of infants born in that perio. Of the infants screene in 4 years, 1338 infants ha a positive CF newborn screen result, 5 an in 110 of these infants CF was iagnose. Two infants with CF (1 newborn with meconium ileus, an a 4- month-ol infant with respiratory an gastrointestinal symptoms) were ientifie as having false-negative results through the follow-up system. CF was iagnose in 1 infant after Borerline Sweat Test Results Forty-two infants (3.4%) ha borerline sweat [Cl2]on their initial visit to a CF center. Of these, 23 unerwent the repeat sweat testing suggeste by our guielines. This low level of compliance with guielines was often cause by the peiatricians choice not to pursue an ambiguous result in a healthyappearing infant, but in some cases appeare also to be cause by suboptimal communication of the follow-up protocol between the sweat laboratories an the peiatricians. Of the 23 infants who unerwent repeat sweat testing, 8 (approximately 1/3) roppe into the normal sweat [Cl 2 ] range, 1 ha a S80 Para an Comeau The Journal of Peiatrics September 2005

4 positive sweat test result ($60 meq/l), an 14 remaine in the borerline range. Diagnostic Dilemma Group I an Group II These groups inclue infants in whom 2 CFTR mutations are efine, but the sweat [Cl 2 ] is not efinitively abnormal. Some of these infants may have atypical forms of CF that will be associate with miler presentation an present at a later than average age. Diagnostic Dilemma Group III an Group IV In aition to the recommenation for repeat sweat testing, the follow-up protocol (Figure) also recommens using an expane mutation panel for infants with persistently elevate sweat [Cl 2 ]. Twenty-three of the 42 infants with borerline sweat test results (not all in the group that ha a repeat sweat test performe) ha expane genetic testing performe. Five of 24 (20%) unientifie chromosomes in these 23 infants (4 infants) ha a secon mutation etecte on the Genzyme 86 or 87 CFTR mutation panel (Genzyme Genetics, Framingham, Mass). One of these infants later ha [Cl2] >60 meq/l. The other 3 infants coul be recategorize from group III to group I. An aitional 2 infants ha the intron 8 5T allele etecte, presumably trans to the DF508 mutation. These 2 infants were not efinitively consiere as having a secon CFTR mutation in this stuy, but were eeme appropriate to maintain uner the follow-up protocol. Such infants might possibly be categorize in group I or III if 5T was shown to be acting as a true mutation. With the etection of a secon mutation in the setting of an elevate IRT, it appears that at least 17% (4/23) of infants in groups III an IV (Table II) with borerline or normal sweat [Cl2] may be iagnose as having CF or belonging to the CF spectrum of isease (a CFTR ysfunction-relate phenotype that might range from severe multiorgan to mil single-organ involvement; classic CF, an atypical, variant, or non-classical CF are inclue). Seven of the 110 infants (6.4%) reporte as having positive screen results by the MA CF NBS program in this 4-year pilot interval an who were given a CF iagnosis ha 2 CFTR mutations on the CF NBS, but sweat [Cl 2 ] that i not allow a classic CF iagnosis (groups I an II). An aitional 5 (groups III an IV) of the 110 infants reporte with positive screen results an given a CF iagnosis i not have 2 mutations ientifie by the initial MA CF NBS an ha sweat [Cl 2 ] in the 30 to 59 meq/l range, an 4 of those ha expane mutation analysis that etecte a secon mutation. DISCUSSION NBS, by efinition, is esigne to etect newborns who are affecte but have no symptoms. Although the CF Founation Consensus Guielines 14 mae provisions for iagnosis in infants with positive NBS results, not all group I to IV infants are covere by that scheme. The guielines state that when the NBS results are abnormal an 2 CFTR mutations are etecte, a iagnosis of CF can be mae. The consensus panel state,.the iagnosis of CF shoul be base on the presence of one or more characteristic phenotypic features, a history of CF in a sibling, or a positive newborn screening test result plus laboratory evience of a CFTR abnormality as ocumente by elevate sweat chlorie concentration, or ientification of mutations in each CFTR gene known to cause CF or in vivo emonstration of characteristic abnormalities in ion transport across the nasal epithelium. 13 Twelve (sum of infants in groups I-IV) of 110 of the infants (11%) etecte by the CF NBS with elevate IRT concentrations ha DNA or sweat [Cl 2 ] results that suggest the presence of atypical CF. The infants were esignate as having true-positive results through a combination of the 3 tiers of CF NBS testing an the evaluation of a CF center. These infants might go on to have severe, life-threatening, morbiitycausing ramifications of their CFTR abnormalities. However, they o not fit neatly into the classic gol stanar iagnostic guielines of having a sweat [Cl 2 ] $60 meq/l. It is also possible that some of these infants will go on to have such minimal mil phenotypes that they woul never cross the threshol to come to clinical attention as part of the CF spectrum. Because the borerline group we have monitore ([Cl 2 ]= meq/l) oes appear to contain a significant number of infants ultimately with 2 CFTR mutations, we have aopte this lower sweat chlorie threshol for infants (30 meq/l) for follow-up within the MA CF NBS algorithm. It is also possible that, within the cohort of babies with positive CF NBS results from the 4-year stuy perio, an infant resolve to the carrier or normal status (sweat [Cl 2 ] <30 meq/l) may actually harbor 1 or 2 mutations that were misse by the NBS mutation panel. That infant might have symptoms later in life that suggest CF spectrum an an atypical form of CF associate with a low or borerline sweat [Cl 2 ]. Because our evaluation of the infants in group III with borerline sweat [Cl 2 ] was not complete, an aitional 4 infants who coul be move to group I (assuming that 20% of unientifie chromosomes in the 19 who i not receive expane genotyping woul have a secon mutation) might be etecte if further genotyping were performe. There may also be iniviuals present in the population with IRT less than the 95% cutoff value who have 2 CFTR mutations an in whom symptoms will evelop. This aitional group will inclue both iniviuals with false-negative results who have classic CF an iniviuals with mil atypical forms of CF. Late ientification of any of these iniviuals coul change the size of the affecte cohort. There may be an increase in the number of patients with CF ientifie in the population because of those infants etecte who will be at the mil or atypical en of the CF spectrum. Critics of the ientification of such iniviuals through CF NBS programs claim that this is a risk or harm, because it strays from ientifying only the esire patients with classic CF, for whom the CF NBS was intene, in whom early severe isease will evelop. To put the atypical CF problem into perspective, nearly 90% of the infants ientifie with the MA CF NBS have genotypes an sweat [Cl 2 ] that are consistent with becoming patients with classic CF, an those infants Diagnostic Dilemmas Resulting From The Immunoreactive Trypsinogen/DNA Cystic Fibrosis Newborn Screening Algorithm S81

5 who might be at the atypical en of the spectrum are a relatively small number. Although the ultimate outcomes of these atypical patients is currently unclear, there is some evience to support concerns that significant problems may evelop in them, 15 an thus early intervention coul affect outcome. It will be important to evaluate the impact of this information on both the psychosocial an the meical outcome of infants with non-classical CFwho are etecte early, because only very limite ata are currently available. The concern that harm is being one, because there is not a clear answer to present to parents about whether the infant actually has CF, nees to be put into the perspective that the relative number in this group is small. Although one coul moify cutoff values (IRT or sweat [Cl 2 ]) in a way that woul iminish the etection of such infants, those changes coul lea to a higher false-negative result rate in infants who will turn out to have classic CF. Moification of the mutation panel to exclue pancreatic sufficient mutations not associate with classic CF (eg, R117H) so that elevate IRT level an at least 1 severe mutation became the screening gate that woul allow infants to procee to sweat testing is another potential solution. The latter moification assumes either the risk/benefit ratio oes not warrant ientifying this group or that limite resources are better focuse on infants with classic CF. Our workgroup of newborn screeners an CF center irectors evelope recommenations for the follow-up of infants with uncertain iagnosis (groups I-IV) until more ata are available on their outcome (Figure): Maintain a consistent follow-up approach between CF centers via a stanarize protocol; Follow sweat [Cl 2 ] with time, an consier resolution criteria (eg, sweat [Cl 2 ] falling into the reference range leas to ischarge of the infant as a carrier); Perform expane genotyping on infants with persistently elevate sweat [Cl 2 ] to search for a secon mutation using total gene screen approaches; Withhol a efinitive iagnosis of classic CF, but explain to parents that a CF iagnosis may surface with time; Perform regular (every 6-12 months) clinical follow-up with a CF specialist who will work with the primary care provier to monitor the chil for early CF symptoms an guie appropriate treatment. Accumulation of long-term outcome ata on infants followe with this protocol coul allow the ientification of factors that will preict who will ultimately have classic CF or atypical CF. Such information may also allow for fine-tuning of treatment protocols use to treat infants etecte with CF through CF NBS programs. REFERENCES 1. Crossley JR, Elliott RB, Smith PA. Drie-bloo spot screening for cystic fibrosis in the newborn. Lancet 1979;1: Hammon KB, Abman SH, Sokol RJ, Accurso FJ. Efficacy of statewie neonatal screening for cystic fibrosis by assay of trypsinogen concentration. N Engl J Me 1991;325: Gregg RG, Wilfon BS, Farrell PM, Laxova A, Hassemer D, Mischler EH. Application of DNA analysis in a population-screening program for neonatal iagnosis of cystic fibrosis (CF): comparison of screening protocols. Am J Hum Genet 1993;52: Gregg RG, Simantel A, Farrell PM, Koscik R, Kosorok MR, Laxova A, et al. 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