Incidence of Neonatal Hyperphenylalaninemia Based on High‑Performance Liquid Chromatography Confirmatory Technique in Mazandaran Province, Northern Iran (2007–2015)
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[Downloaded free from http://www.ijpvmjournal.net on Monday, November 27, 2017, IP: 176.102.233.15] Original Article Incidence of Neonatal Hyperphenylalaninemia Based on High‑performance Liquid Chromatography Confirmatory Technique in Mazandaran Province, Northern Iran (2007–2015) Abstract Ali Abbaskhanian1,2, Background: Classic phenylketonuria (PKU) is a metabolic disorder. The purpose of this study Daniel Zamanfar1,3, was to assess epidemiological factors of PKU phenotypes in a neonatal screening program for Parvaneh Mazandaran, Iran. Methods: In this descriptive‑retrospective study from 2007 to 2015, neonates 4 PKU level was conducted by phenylalanine level based on a biochemical technique by ELISA and Afshar , Einollah 5 then by confirmatory methods high performance liquid chromatography. Results: Of the 407,244 Asadpoor , Hamed screened newborns (48.7% girls and 51.3% boys), 14 girls and 13 boys were diagnosed definitely Rouhanizadeh1,6, from 465 suspicious cases of PKU. The incidence of PKU was 0.66 in 10,000, which was noted in Ali Jafarnia7, different severity (severe PKU ‑ 1:67,874, mild PKU ‑ 1:45,249, and HPA ‑ 1:33,937). In addition, Mohammad we did not detect any cases of nonclassic PKU. Conclusions: Although the consanguineous marriage 8 pattern is a major cause of hyperphenylalaninemia (HPA) particularly in Iranian, there was no Shokzadeh 1 significant difference between groups in this study. Now, screening should be executed for all of the Department of Pediatrics, School of Medicine, Mazandaran family that they have the familial history of PKU in Iran. According to varies actual of prevalence University of Medical Sciences, and incidence rate of PKU reported a real patient and taking PKU with mild PKU and HPA, it is Sari, Iran, 2Clinical Research recommended, the will provide the PKU reports based on the severity of the disease. Development Unit of Bou Ali‑Sina Hospital, Mazandaran Keywords: Chromatography high‑pressure liquid, Iran, neonatal screening, phenylketonurias University of Medical Sciences, Sari, Iran, 3Diabetes Research Center, Mazandaran University and deficiency of its cofactor THB causes of Medical Sciences, Sari, Iran, Introduction 4Research and Development Unit malignant PKU.[10] If the serum tyrosine Phenylalanine (Phe) is an essential of Referral Laboratory, Deputy of and urine THB levels were normal and Health Management, Mazandaran amino acid. The phenylalanine sera phenylalanine levels were ≥20 mg/dl, University of Medical Sciences, hydroxylase (PheOH or PHA) enzyme Sari, Iran, 5Deputy of Health, between 10 and 20 mg/dl, and between and its cofactor tetrahydrobiopterin (THB) Mazandaran University of Medical 2 and 10 mg/dl, the newborns were Sciences, Sari, Iran, 6Deputy of catalyze the conversion of phenylalanine diagnosed as having severe PKU, mild Health Management, Mazandaran to tyrosine, and their deficiency results in University of Medical Sciences, PKU, and hyperphenylalaninemia (HPA), 7 accumulation of phenylalanine in body Sari, Iran, Deputy of Health, [4] Babol University of Medical [1,2] respectively. fluids and central nervous system. In Sciences, Babol, Iran, 8Department affected patients, excessive phenylalanine There are several methods to PKU of Pharmacology, Faculty of is metabolized to phenyl ketones that are identify in blood‑dried specimens Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran excreted in the urine.[3,4] The PAH gene is (DBS) screening laboratory; such located on the long arm of chromosome as fluorometric,[11] enzymatic, 12 in the region q22‑q24. In 98% of PKU colorimetric,[8,12] high‑performance liquid patients, defects of the PAH enzyme are chromatographic (HPLC),[8,13] and more Address for correspondence: due to mutations in the PAH gene on recently tandem mass spectrometric Parvaneh Afshar, chromosome 12q23.2.[5,6] In 98% of cases, methods.[14,15] Research and Development Unit of Referral Laboratory, the damages of central nervous system are The first pilot study for the assessment Deputy of Health Management, due to mutations in the gene encoding the of neonatal HPA in Iran was started in Mazandaran University of [4,7,8] Medical Sciences, Sari, Iran. enzyme L‑PHA (EC 1.14.16.1). [16] Tehran from 1982, and the first National E‑mail : [email protected] Elevated phenylalanine, if not treated in Neonate Screening Program (NNSP) in Iran Access this article online the 1st days of the life, causes irreversible was begun from 2002 in Fars province[17] Website: brain and mental damages (microcephaly and afterward in Mazandaran Province www.ijpvmjournal.net/www.ijpm.ir [9] and seizures). Deficiency of PHA in 2007, based on the law; all infants DOI: causes severe phenylketonuria (PKU) should be screened for three diseases 10.4103/ijpvm.IJPVM_24_17 Quick Response Code: How to cite this article: Abbaskhanian A, Zamanfar D, This is an open access article distributed under the terms of the Afshar P, Asadpoor E, Rouhanizadeh H, Jafarnia A, Creative Commons Attribution‑NonCommercial‑ShareAlike 3.0 et al. Incidence of neonatal hyperphenylalaninemia License, which allows others to remix, tweak, and build upon the based on high‑performance liquid chromatography work non‑commercially, as long as the author is credited and the confirmatory technique in Mazandaran Province, new creations are licensed under the identical terms. Northern Iran (2007–2015). Int J Prev Med For reprints contact: [email protected] 2017;8:93. © 2017 International Journal of Preventive Medicine | Published by Wolters Kluwer - Medknow 1 [Downloaded free from http://www.ijpvmjournal.net on Monday, November 27, 2017, IP: 176.102.233.15] Abbaskhanian, et al.: Neonatal hyperphenylalaninemia in Mazandaran, Iran including hypothyroidism, PKU, and glucose‑6‑phosphate was <0.08 and for standard 32 concentration was >0.240. dehydrogenase deficiency. As well as, we used blood spot for controls (low and high level) in duplicate form. Acceptable ranges were The purpose of this survey was to evaluate the development for low control spot 1.5–3.4 mg/dl and high control and organization of phenylalanine level in newborn spot 6.5–12.3 mg/dl. Normally (reference limit), screening programs and their limitations and expectations DBS phenylalanine concentrations in neonates ranged in Mazandaran Province in the northeast of Iran (Sari) based from ~1.8 mg/dl to ~3.9 mg/dl. In referral laboratory, on biochemical ELISA and HPLC technique. Its geographical a phenylalanine cutoff point of <4 mg/dl (0.24 mmol/l) coordinates are 36° 34’ 4” North and 53° 3’ 31” East and was used. This cutoff level increases the sensitivity of its original name (with diacritics) is Sari. Mazandaran the screening test and reduces the risk of missing the is located in the Northern Iran and the southern coast of diagnosis of disorders of the phenylalanine metabolism in the Caspian Sea [Figure 1]. It is bordered clockwise by subjects with less pronounced phenylalanine‑circulating Golestan, Semnan, and Tehran Provinces. This province levels at the first investigation. also borders Qazvin and Gilan to the west.[18] Mazandaran province with an area equivalent to 23,842 km2, about All the newborn’s cases with phenylalanine levels >3.9 mg/ 1.46% of the Iran country’s area and eighteenth considered dl initially an additional sample were retested in parallel with in these respect 31 provinces in the country[19] and with the first DBS suspect sample, then reported immediately around 3.1 million inhabitants seventh considered in was re‑tested at the time off between the 9th and 16th day these respect 31 provinces in the country.[20] The probable of life with another DBS specimen collection. Subjects correlation between the PKU values in newborn in with normal phenylalanine concentrations (<3.9 mg/dl) in positive samples and with related potential risk factors and this second DBS specimen were classified as false positives environmental factors was evaluated. and those with a phenylalanine concentration >3.9 mg/ dL as positives. Afterward, they were referred to the Methods pediatric endocrinologist, the scientific adviser of this plan Sample collection in Mazandaran University of Medical Sciences for further evaluation. In this descriptive‑retrospective study, total newborns population were screened through an NNSP, in all 21 cities Phenylketonuria analysis in neonatal samples by of Mazandaran in 2007–2015. During the days 3–5 after high‑performance liquid chromatography technique birth, heel blood samples were taken based on Schleicher About 3 mL of the venous blood was obtained in the and Schuell 903 (Bioscience, Germany) papers with heparinized tube from these newborns. Their sera were sent [21] Clinical Laboratory Standards Institute and Ministry for the evaluation of phenylalanine and tyrosine by HPLC [22,23] of Health of Iran newborn PKU Screening Program method. If the serum phenylalanine level was ≥10 mg/dl and protocols by an experienced technician, and the samples tyrosine level was normal, the newborns were diagnosed were sent to the special screening laboratory in the Referral as having HPA. In those with serum phenylalanine levels Laboratory Mazandaran University Medical Science. between 7 and 9.9 mg/dl, another blood sample was Analysis phenylketonuria in neonatal samples by ELISA checked 1 week later, and if the serum phenylalanine levels technique were equal or >7 mg/dl, they were diagnosed definitely as having HPA. The newborns with serum phenylalanine The valid DBS specimens were punched at least 5 mm levels between