Original Article

Newborn screening for cystic fibrosis in Serbia: A pilot study

Danijela Radivojevic,1 Aleksandar Sovtic,2 Predrag Minic,2 Sanja Grkovic,3 Marija Guc-Scekic,1 Tanja Lalic1 andMarijana Miskovic1

1Laboratory of Medical Genetics, 2Department of Pulmonology and 3Laboratory for Testing of Inherited Disorders ofMetabolism, Mother and Child Health Institute of Serbia, Belgrade, Serbia

Abstract Background: We performed a pilot study of neonatal screening for cystic fibrosis (CF) in order to introduce it to thenational screening program in Serbia.Methods: Immunoreactive trypsinogen (IRT) concentrations were analyzed in dried blood spot samples. Patients wererecalled for repeated measurements in case of high IRT levels. Persisting high IRT levels resulted in DNA testing for the29 most common mutations in the CF transmembrane regulator (CFTR) gene (IRT/IRT/DNA method). Sweat chloridemeasurements and clinical assessment were further performed for newly diagnosed patients.Results: Of 1000 samples, three were initially positive and were further analyzed for the presence of the most commonCFTR mutations in the Serbian population. DNA analysis revealed two patients being homozygous for F508delmutation. One sample was false positive, as the genetic test proved to be negative and associated with normal sweatchloride concentration and unremarkable clinical presentation.Conclusions: The results of our pilot study justified the expanding of the routine neonatal screening program in Serbiawith CF. Data could be used in future in order to obtain accurate incidence of CF and carrier prevalence in our country.

Key words cystic fibrosis, DNA testing, genetic counseling, immunoreactive trypsinogen, newborn screening.

Cystic fibrosis (CF, MIM#219700) is the most frequent auto-somal recessive hereditary disease in Caucasians, with an inci-dence of 1:2000 to 1:3500 liveborn infants. It is a systemicdisease with most prominent respiratory symptoms and failure tothrive, usually presenting in infancy. Since the discovery of theCFTR gene (MIM#602421) in 1989, more than 1800 mutationshave been described with the most common being F508del (oraccording to HGSV c.1521_1523delCTT, p.Phe508del). Earlierstudies showed that results of molecular genetic analysis of CF inSerbia were heterogeneous, as it was observed in other easternand southern European populations.1 The incidence of CF in theSerbian population is not precisely known, because the nationalneonatal screening program has not started yet.

The primary goal of newborn screening (NBS) is to decreasemorbidity, mortality and associated disabilities in affected infantsearly in life. Early diagnosis could reduce morbidity and prolonglife, especially if the patients are treated in specialized CFcenters.2

In the late 1970s, Crossley et al. introduced the testing ofimmunoreactive trypsinogen (IRT) for CF screening. They devel-oped a method of IRT measurement in dried blood. This test isstill widely used in CF screening protocols. IRT concentration is

high in the blood of infants with CF, presumably from leakage ofthe protein into the circulation after exocrine pancreatic injury.3,4

The National Newborn Screening Program was initiated inour country in 1982, and performed by the Mother and ChildHealth Institute of Serbia (MCHI). At first, the screening wasonly performed for phenylketonuria. Screening for congenitalhypothyroidism was added a year later.

The aim of our pilot study was to introduce the screening forCF into the national screening program. It also provided a chanceto evaluate the reliability and expenses of procedures defined bythe protocol, in order to further expand the current NBS program.

Methods

Sample preparation and analysis

Blood samples were taken from newborns by heel prick andspotted on Scheisher and Schuell 903 filter paper sampling cards(Guthrie cards, S&S 903, Whatman, Maidstone, UK ) at 72 h oflife. Thereafter, blood samples were dried and sent by mail to thelaboratory in the national pediatric center as a routine step of theneonatal screening protocol, which is currently in use.

The initial procedure in the protocol was a “two-step” (IRT/IRT) measurement. Concentration of initial IRT, with an arbi-trarily fixed cut-off value of �50 ng/mL, was examined in driedblood spots (Guthrie cards) with commercial reagents (Quan-tase Neonatal IRT Screening Kit, Bio-Rad Lab, Perth, UK),a monoclonal antibody-based enzyme immunoassay using a96-well microtiter plate enzyme-linked immunosorbent assay

Correspondence: Danijela Radivojevic, PhD, Laboratory of MedicalGenetics, Mother and Child Health Institute of Serbia, 6 RadojaDakica St, 11070 Belgrade, Serbia. Email: badane@beotel.rs

Received 7 August 2012; revised 3 October 2012; accepted 30October 2012.

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Pediatrics International (2013) 55, 181–184 doi: 10.1111/ped.12009

© 2012 The AuthorsPediatrics International © 2012 Japan Pediatric Society

technique. If analysis showed elevated IRT level, the secondsampling of a blood spot (“recall”) was requested at the age of4 weeks.5,6

Direct gene analysis

In case of two positive IRT values, direct gene analysis wasperformed. Genomic DNA, eluted from dried blood in Guthriecards, was amplified and screened by heteroduplex analysison PAGE for the presence of F508del (c.1521_1523delCTT,p.Phe508del) and by standard multiplex PCR-amplification-refractory mutation system (ARMS) technique for simultaneousin vitro qualitative detection of 29 frequent CFTR mutations inEurope (Elucigenetm CF 29v.2, Tepnel (Tepnel Diagnostics,Abingdon, UK) [D1152H, 1717-1G>A, G542X, W1282X,N1303K, F508del, 3849 + 10kbC>T, 394delTT, 621 + 1G>T,S1251N, G551D, R117H, R1162X, R334W, A455E, 2183AA>G, 3659delC, 1078delT, I507del, R347P, R553X, E60X, 3120 +1G>A, 2789 + 5G>A, 1898 + 1G>A, 711 + 1G>T, G85E,2184delA and R560T]).7,8

Results of mutation analysis were available within 2 weeksafter receipt of the sample.

Sweat chloride measurements

Sweat chloride measurements were done in all patients whoeither had two positive IRT values, or two CF-causing alleles intrans. Normal value for sweat chloride, obtained by pilocarpineiontophoresis, was <60 mmol/L, as it was defined by Gibson andCook. Values higher than 40 mmol/L required retesting becauseof the possible false negative results.9

Clinical assessment and genetic counseling

Newly diagnosed CF patients were referred for clinical assess-ment to the Department of Pulmonology in the Mother and ChildHealth Institute of Serbia. Furthermore, all families at risk under-went genetic counseling and were offered “cascade” CFTR muta-tion screening to their direct relatives.

Results

The pilot study was performed in 2009 and 1000 dried bloodsamples were screened. This number represents 1.39% of all livebirths in Serbia in 2009.10

Increased values of IRT were registered in three patients. Intwo patients, who proved to be positive for double IRT testing,DNA analysis revealed identical genotype (F508del/F508delor according to HGSV c.[1521_1523delCTT] + [c.1521_1523delCTT]). The parents of these two neonates were alsotested and they were identified as carriers of this cystic fibrosismutation. They were referred to the Genetic Counseling Service,MCHI with advice for genetic testing in further pregnancies andcascade testing for their relatives. The calculated cost per patientin this pilot screening protocol (IRT/IRT/DNA) was $4.15.

The third double IRT-positive patient was negative for thepresence of most frequent CFTR mutations mentioned above.Unremarkable clinical presentation associated with normal sweatchloride and negative genetic tests were inconclusive with diag-nosis of CF, thus it was the only false-positive case (Table 1).

Discussion

The development and expansion of NBS programs for inheriteddiseases is considered to be a public health mission in manycountries.11 Although substantial benefits of screening on growth,nutritional status and median survival have been documented, theadvantages for pulmonary status have been more difficult toevaluate. Some skepticism remains regarding the current rapidprogress toward mass screening in several countries.12 Neverthe-less, in its latest report, the Centers for Disease Control andPrevention published data with affirmative and undoubted benefitof newborn screening for CF.3

It was shown that protocols that had been used in differentEuropean countries varied from the sweat test after the first IRTto those involving IRT/IRT/DNA.13 The median number of muta-tions analyzed was 31.14

Serbia is located in the Balkan Peninsula. Its estimated popu-lation is 7 320 807, with a birth rate of 70 299/year.10 Becausethere has not been a national screening program so far, the CFincidence for the Serbian population is not accurately known.Because of extensive ethnic assimilation throughout history,Serbia’s population could not be identified as homogeneous.

The MCHI is a tertiary care pediatric hospital and national CFcenter, currently providing care for more than 160 CF patients.Median survival age of these patients is 31 years.

Table 1 Clinical characteristics of patients with cystic fibrosis detected by neonatal screening in Serbia

Patient A Patient BEthnic origin Caucasian CaucasianSex Female MaleAge at diagnosis 35 days 45 daysOnset of symptoms 3 months Since birthSymptoms Cough, persistent wheezing, increased

stool frequencyMeconial ileus, greasy stools, decreased

weight gainBirthweight/length 3.300 g/51 cm 3.270 g/52 cmWeight at diagnosis 3.650 g 3.395 gIRT1 (ng/mL)/child’s age 413/4 days old 287.6/3 days oldIRT2 (ng/mL)/child’s age 392/30 days old 275/30 days oldSweat chloride (mmol/L)/child’s age 114/55 days old 78/50 days oldGenotype F508del/F508del F508del/F508del

IRT, immunoreactive trypsinogen.

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Previous studies investigating molecular basis of CF in Serbiashowed that 18 different CFTR mutations were accountingfor 82.57% of CF alleles. In this group, F508del (c.1521_1523delCTT, p.Phe508del) had a frequency of 72.28%.1 Using themethods mentioned above, 96% of patients with CF in thescreened Serbian population would be expected to carry at leastone of these mutations. It was most efficient to organize NBS forCF simultaneously with two other tests currently performed inSerbia. The existing network, which has been in place for almost30 years, provided efficient and reliable material transfer andoffered the least expense.

We decided to use the IRT/IRT/DNA testing method. Theadvantage of this method over a single IRT/DNA methodology isthat it reduces the number of children who need to undergo sweattesting, and the number of parents who had to be informed thattheir child potentially has an inherited disease and needed geneticcounseling. Furthermore, single IRT/DNA testing fails to detectrare mutations within ethnic minorities and increase the numberof patients referred to sweat testing. None of the patients werefirst IRT positive/second IRT negative. This is probably due to therelatively small number of analyzed samples in regard to thebirth rate in our country in the year when the pilot study wasperformed.

The total cost of the screening program for CF per patientusing the IRT/IRT/DNA method mainly depends on DNA analy-sis. It was shown that the IRT/IRT screening algorithm reducedthe cost, but IRT/DNA testing had an advantage in fewer delayeddiagnoses.15 The protocol we used led to the diagnosis withgreater certainty and with fewer false positive results.

In a cohort of 1000 analyzed paper filter cards, one samplewas false positive (0.1%), which led to a specificity of 99.9%.Diagnosis of CF was confirmed in two neonates who had highIRT values, and were homozygous for F508del mutation, so thesensitivity was 100%. These patients eventually had elevatedchloride sweat test consistent with typical CF phenotype. Thisis in accordance with data recently published by Vernooij-vanLangen et al.16

A third patient with false positive IRT values, normal sweatchloride and negative DNA testing thrived normally, withoutsigns and symptoms of CF on follow up. High IRT levels may beassociated with intrapartum asphyxia, neonatal infection or res-piratory distress.17,18 High initial values are usually normalizedwithin 4 weeks, but some continue to be elevated, probably dueto the genetic variability of newborns.19

Parents of affected children were informed about the diseaseand all its consequences, including necessity of careful andregular follow up, despite the possible current absence of severesymptoms. They were counseled at the Genetic CounselingService, MCHI about their testing for carrier status, prenataldiagnosis of CF in potential pregnancies and possible cascadetesting for their relatives, as it was suggested previously.13

Despite the fact that two newly diagnosed CF patients werefound during this pilot study, the number of analyzed sampleswas not big enough to assume the true incidence of CF in Serbia.

The future of neonatal CF screening is challenging. The defi-nition of the best local practices should serve to optimize screen-

ing programs for the individual population, and it is different toone established elsewhere. Besides screening and early diagno-sis, recognition of centralized care had been one of the factors,which improved survival rate and decreased incidence of possiblecomplications of the disease.12,20

More than 25 screening programs have been developed inEurope so far, with quite marked variations in protocol designs.Because of geographic and ethnic variations and different eco-nomic statuses, ideal harmonization of protocols is not fullyachievable.13 The protocol used in our pilot program was efficientand reliable, associated with a good specificity and sensitivityratio.

In our opinion, the introduction of the neonatal screeningprogram for CF is of high importance for the patient’s treatment,as the early diagnosis results in better survival and lower costs forthe health-care system. Data obtained by screening could also beused in population-based studies to evaluate the incidence of CFin our country in the future. We hope that these results willencourage our health authorities to provide support to include CFscreening in the national newborn screening program.

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