NEONATAL SCREENING FOR INBORN ERRORS OF METABOLISM OUR EXPERIENCE AT CABRI, GULF MEDICAL UNIVERSITY

GMJ GULF MEDICAL JOURNAL ORAL PROCEEDINGS NEONATAL SCREENING FOR INBORN ERRORS OF METABOLISM OUR EXPERIENCE AT CABRI, GULF MEDICAL UNIVERSITY I.A. Shaafie 1 *, A.D. Vijay Raju 2, P.K. Menon 3 1Division of Clinical Biochemistry, 2 Analytical Chemistry, 3 Clinical Microbiology, Molecular Biology, Centre for Advanced Biomedical Research and Innovation (CABRI), Gulf Medical University, Ajman, UAE *Presenting Author ABSTRACT Neonatal screening is a vital process that identifies apparently healthy neonates with serious inherited disorders, which are generally metabolic in origin and correctable by dietary or drug interventions. Inborn errors of metabolism are rare in isolation, but collectively are common. They usually present in the neonatal period or infancy, but can occur at any time, even in adulthood. The purpose of this study is to review the results of a comprehensive newborn screening panel of 57 biochemical parameters measured in 4,408 dried blood spot samples collected 24 72 hours after birth by the heelprick method. The samples were analyzed using tandem mass spectrometry (LC-MS/MS), cation exchange chromatography, and the DELFIA time-resolved fluorescence (TRF) intensity technology. All the assays were validated and well controlled. Proficiency testing samples from the Centers of Disease Control and Prevention (CDC) were run along with the neonatal samples. The cut-off values used for various parameters were validated and found to be acceptable. The results showed that glucose-6-phosphate was the most common disorder (93 cases), followed by hemoglobinopathies (27 cases), congenital hypothyroidism (24 cases), cystic fibrosis (22 cases), and congenital adrenal hyperplasia (7cases). The overall incidence rate of inborn errors of metabolism was 4.31%, marginally higher than the national figures of the UAE. The types of disorders detected are identical to those found in surveys conducted in the UAE and other countries. In view of the high prevalence of genetic and metabolic disorders detected by the study, neonatal screening should be made mandatory in the UAE. This would enable early detection and management of genetic and metabolic disorders, and reduce the number of physically and mentally challenged children in the community. The role of governments, by providing administrative and financial support, is crucial for successful implementation of the neonatal screening program. Keywords: Neonatal screening, dried blood spots, inborn errors of metabolism, tandem mass spectrometry, DELFIA TRF technique, Newborn Screening Quality Assurance Program, cation exchange Citation: Shaafie IA, Raju ADV, Menon PK. Neonatal screening for inborn errors of metabolism our experience at CABRI, Gulf Medical University. Gulf Medical Journal. 2016;5(S1):S35 S42. INTRODUCTION Neonatal screening (NNS) is the practice of testing every newborn for certain genetic, endocrine, and metabolic disorders that are Correspondence: Dr. Ishtiyaq Ahmad Shaafie (MD, PGDHPE), Specialist Clinical Biochemist, CABRI, Gulf Medical University, UAE. Email: piashaafie@gmu.ac.ae typically not apparent at birth. All these disorders, except congenital deafness and critical congenital heart diseases, are detected by dry blood spot (DBS) screening. Inborn errors of metabolism (IEM) are a major group of heterogeneous monogenic disorders commonly identified by NNS. When dietary restriction or drug therapy is used at an early age, morbidity and mortality due to IEM are reduced 1. Any delay in the diagnosis and treatment leads to 35

I.A. SHAAFIE, ET AL adverse outcomes, including neuropsychological dysfunction, mental retardation, and ultimately death 2. Dr. Robert Guthrie, an American bacteriologist and physician, was the first to develop a test to detect phenylketonuria in 1960 for his own child 3. Later, Guthrie and his colleagues used tests to detect congenital hypothyroidism, maple syrup urine disease (MSUD), and classic galactosemia. In 1968, the World Health Organization (WHO) provided guidelines for screening of newborns for specific disorders using the 10 criteria of Wilson and Jungner 4. However, the introduction of tandem mass spectrometry (MS/MS) in early 1990 and its ability to simultaneously measure numerous metabolites changed the old concept about NNS as one test for one disorder to one test for many disorders, i.e., a multiplex test 5. The WHO guidelines have been revised, and a number of new disorders that could be detected from a single sample have been added to the list. This expanded newborn screening has led to the detection of disorders for which there is currently no treatment. However, NNS helps families/relatives by providing early diagnosis for the neonate and planning for future pregnancies 6, 7. In 2006, the Advisory Committee on Heritable Disorders in Newborns and Children from the US Department of Health Resources and Services Administration (HRSA) recommended a uniform screening panel of 29 core (primary) and 25 secondary disorders among neonates for all US states on the basis of scientific and medical evidence from the American College of Medical Genetics and the American Academy of Pediatrics. Of these 54 disorders, 42 can be detected by MS/MS, while others, such as hemoglobinopathies, congenital hypothyroidism, congenital adrenal hyperplasia, biotinidase deficiency (BIOT), galactosemia, cystic fibrosis, and congenital hearing loss, are detected using other methods. The National Academy of Clinical Biochemistry has categorized the strengths of recommendations for the adoption of MS/MS for IEM 8. False positive results and relatively poor positive predictive values for some MS/MS tests have led to the development of second-tier tests that require separate testing protocols 9, 10. NNS has been adapted as a coordinated comprehensive public health program in the US, Canada, and several European countries. These programs involve education, screening, detection, and follow-up of abnormal and unsatisfactory results; confirmation of positive results; treatment and follow-up of neonates with genetic disorders; quality assurance and program evaluation; validation of testing systems; and assessment of long-term benefits to the patients, families, and society. Most developing countries do not have a government-funded preventive public health program for NNS, resulting in many cases of inherited disorders going undetected among the population. In 1995, a national NNS program was established in the UAE for early identification and treatment of IEM, so as to prevent morbidity and mortality due to these disorders. The program started with initial screening for phenylketonuria and was made comprehensive in 2013 11. Centre for Advanced Biomedical Research and Innovation (CABRI) at Gulf Medical University started NNS in January 2015, providing a comprehensive screening panel for 57 biochemical and hematological genetic disorders. The current study reviews the outcomes of this screening, and summarizes the validation process for establishing reference ranges of all 57 analytes using the LC-MS/MS and dissociationenhanced lanthanide fluorescence immunoassay (DELFIA ) techniques. MATERIALS & METHODS Blood Specimens DBS samples were collected at Thumbay Hospitals and other clinics from 4,408 apparently healthy neonates 24 72 hours after birth with parental consent. The samples were collected by the heel-prick method using standard Whatman 903 TM Specimen Collection cards from GE Healthcare, USA. All guidelines for collection, transport, and processing of samples were followed. 36

NEONATAL SCREENING FOR INBORN ERRORS OF METABOLISM OUR EXPERIENCE AT CABRI, GMU Methodology The DBS samples were analyzed for various biochemical and hematological parameters using LC-MS/MS with electrospray ionization (ESI), DELFIA [a time-resolved fluorescence (TRF) intensity technology], and cation exchange HPLC techniques (refer Tables 1 3). The test results were checked for precision and accuracy using the quality control materials provided by the kit manufacturers for HPLC and DELFIA assays. All the results were found to be precise and accurate. Table 1. Amino acid disorders detected by LC-MS/MS Biomarker Phenyl alanine Phenyl alanine/tyrosine ratio Arginine Citrulline Valine/leucine/isoleucine Disorder(s) related Phenylketonuria (PKU) Argininemia/arginase deficiency Citrullinemia (argininosuccinic acid synthetase deficiency) Maple syrup urine disease (MSUD) Table 2. Acylcarnitine/fatty acid disorders detected by LC-MS/MS Biomarker C3 C3-DC + C4-OH C4 C4-DC + C5-OH C4 & C5 C5 C5-DC + C6-OH C0 C6, C8, C8/C10 ratio, C10:1 C14, C14:1 C14:1 C14-OH C18 Disorder(s) Propionic acidemia Methyl malonic acidemia Malonic aciduria Short-chain acyl-coa Methylmalonic acidemia Methylmalonic acidemia with B12 defect and homocystinuria Ethylmalonic encephalopathy Isovaleric acidemia Glutaric acidemia type I Carnitine transporter deficiency Medium chain acyl-coa (MCAD deficiency) Very long-chain acyl-coa Multiple acyl-coa (MAD deficiency)/glutaric acidemia type II (GA-2) Long-chain hydroxyacyl-coa Very long-chain acyl-coa Table 3. Disorders detected by DELFIA and HPLC (cation exchange) techniques Biomarker Disorder(s) Glucose-6-phosphate Glucose-6-phosphate dehydrogenase dehydrogenase (G6PD) deficiency Immunoreactive trypsinogen (IRT) Cystic fibrosis 17-hydroxy progesterone (17-OH-P) Congenital adrenal hyperplasia Thyroid stimulating hormone (htsh) Congenital hypothyroidism Thyroxine (T4) Congenital hypothyroidism Galactose-1-phosphate uridyl transferase (GALT) Classic galactosemia Biotinidase BIOT Disorders detected by HPLC Hemoglobinopathies HbS, HbC, HbD Cut-off Values The cut-off values for our tests were adapted from the kit inserts; operation manuals of the analyzers; NeoGen Labs (India); California Department of Public Health s cutoff/reference ranges for acylcarnitines and amino acids; and reference ranges used at the Biochemical Genetics Laboratory, King Faisal Specialist Hospital & Research Centre, Saudi Arabia. Quality Control Internal quality control tests were run with each batch of tests. The results were evaluated for acceptable criteria. According to the requirements of the College of American Pathologists (CAP) for alternate proficiency testing, CABRI s laboratory participates in the Newborn Screening Quality Assurance Program (NSQAP) run by the Centers of Disease Control and Prevention (CDC), USA. The results showed 100% accuracy for 2015 and 2016 (Table 4). Ethical Issues All samples were collected after parental consent. The data presented is from deidentified samples. 37

I.A. SHAAFIE, ET AL Table 4. NSQAP - Year: 2015 (Cycles: 3 and 4) and 2016 (Cycle 1) Sr. No. Component No. of analytes Total no. of challenges per cycle Cycle 3 2015 Cycle 4 2015 Cycle 1 2016 No. of challenges passed Accuracy (%) Acceptable limit 1 Acylcarnitines 18 90 90 90 270 270 100% 80% 2 Amino acids 7 35 35 35 105 105 100% 80% 3 GALT 1 5 5 5 15 15 100% 80% 4 IRT 1 5 5 5 15 15 100% 80% 5 BIOT 1 5 5 5 15 15 100% 80% 6 Hormones 3 15 15 15 45 45 100% 80% Total RESULTS & DISCUSSION The biomarkers of IEM detected by various techniques are depicted in Tables 1 3. The results show that the majority of neonates (62%) observed were from South Asia, followed by East Asia, the Middle East, and Africa. (Figure 1). This indicates the ethnic background of the patients who usually visit Thumbay Hospitals. The cut-off values used for 57 analytes were internally verified by 80 randomly collected normal DBS samples. The mean and median values were calculated for each analyte. The calculated values were below the cut-off limits, found to be acceptable, and adopted (Tables 5 7). Tables 8 and 9 display the genetic and metabolic disorders identified, and ethnic and gender distribution of these disorders, respectively. G6PD deficiency was the most common disorder (2.1%), followed by hemoglobinopathies (0.61%), congenital hypothyroidism (0.54%), and cystic fibrosis (0.50%). The findings of a hospital-based study conducted by Saleh et al. on 15,995 cases at Abu Dhabi for the molecular characterization of G6PD deficiency showed that the deficiency was prevalent in both UAE (7.4%) and non- UAE nationals (3.8%), while the Mediterranean mutation, 563C T, was predominant among non-uae nationals 12. G6PD is not a part of the UAE s National Newborn Screening Program, although the Health Authority of Abu Dhabi (HAAD) included the condition in its comprehensive screening panel in 2010. Figure 1. Ethnic distribution of neonates subjected to DBS analysis 2,742 516 South East Asians Asians (China, (India, Philippines) Pakistan, Bangladesh, Sri Lanka) Number of samples 895 221 Table 5. Verification of amino acid cut-off values using LC-MS/MS Analyte Mean Median Cut-off value (µmol/l) Glycine 306.76 296 1,000 Alanine 192.22 184.5 583 Valine 144.44 141 255 Leucine-isoleu 135.96 133 310 Ornithine 113.64 108 454 Aspartic acid 46.63 41.85 420 Methionine 18.57 17.8 100 Phenyl alanine 68.87 67.5 180 Citrulline 13.99 13.4 70 Tyrosine 134.83 126.5 350 Proline 226.16 219.5 440 Glutamic acid 506.49 476.5 1,074 Arginine 12.21 7.86 45 34 Arabs Africans Europeans 38

NEONATAL SCREENING FOR INBORN ERRORS OF METABOLISM OUR EXPERIENCE AT CABRI, GMU Table 6. Verification of acylcarnitine/fatty acid cut-off values Analyte Mean Median Cut-off value (µmol/l) C0 35.21 33.95 50 C2 16.66 15.8 71.17 C3 1.59 1.35 6.0 C4 0.20 0.18 1.0 C5 0.13 0.12 0.4 C6 0.05 0.05 0.5 C5DC 0.18 0.17 0.4 C8 0.06 0.06 0.5 C10 0.08 0.08 0.5 C12 0.06 0.06 0.35 C14 0.17 0.16 0.6 C16 2.40 2.21 7.0 C18 0.70 0.61 4.0 C5:1 0.04 0.03 0.25 C5-OH 0.11 0.11 0.60 C8:1 0.05 0.05 0.77 C10:1 0.07 0.07 0.40 C14:1 0.06 0.05 0.43 C14-OH 0.01 0.01 0.15 C16:1 0.20 0.16 0.67 C16-1OH 0.05 0.03 0.50 C16-OH 0.02 0.02 0.25 C18:1 2.00 1.66 2.50 C18:1OH 0.06 0.04 0.20 C18-OH 0.02 0.01 0.20 Table 7. Verification of cut-off values of neonatal biochemical parameters measured using DELFIA TRF technique Analyte Unit Mean Median Cut-off value 17-OH progesterone nmol/l 5.27 3.72 >29.9 borderline high ; >89.9 very high G6PD U/g Hb 3.55 3.9 2.6 borderline deficient; 2.2 highly deficient Thyrotropin µu/ml 2.20 1.829 >9.9 borderline high; >24.9 very high IRT ng/ml 14.80 11.8 >40 borderline high; >80 very high Gal-1-phosphate uridyl transferase U/gHb 3.99 3.45 3.5 borderline low; 2.4 very low Biotinidase U 229.88 229.51 Thyroxine nmol/l 89.87 87.8 60 deficient 75 borderline deficient; 25 highly deficient 39

I.A. SHAAFIE, ET AL Table 8. Gender Distribution of genetic and metabolic disorders among neonates Disorder Gender Male (N = 2,165) Female (N = 2,243) G6PD deficiency 84 9 Hemoglobinopathies (HbC 4, HbS 12, HbD 11) 16 11 Congenital hypothyroidism 17 7 Cystic fibrosis 9 13 Congenital adrenal hyperplasia 4 3 BIOT 2 4 Phenylketonuria 1 1 Tyrosinemia - 2 Propionic acidemia/methylmalonic acidemia - C3 1 1 MSUD 1 - Glutaric acidemia type II - C4 1 - MCAD deficiency - C8 1 - Isovaleric acidemia - C5 1 - Classical galactosemia 1 - Positive cases (Total) 139 51 Positive cases (%) 6.42 2.27 Table 9. Prevalence of genetic disorders among neonates by ethnicity Disorder South Asians (N = 2,742) East Asians (N = 516) Ethnicity Arabs (N = 895) Africans (N = 221) Europeans (N = 34) G6PD deficiency 49 15 20 9 - Hemoglobinopathies 22-5 - - Congenital hypothyroidism 12 3 4 5 - Cystic fibrosis 14-2 6 - Congenital adrenal hyperplasia 5-2 - - BIOT 3 1 2 - - Phenylketonuria 1-1 - - Tyrosinemia - 1 1 - - Propionic acidemia/ methylmalonic acidemia 2 - - - - MSUD 1 - - - - Glutaric acidemia type II 1 - - - - MCAD deficiency 1 - - - - Isovaleric acidemia 1 - - - - Classical galactosemia 1 - - - - Total no. of positive cases 113 20 37 20 - % of positive cases 4.12 3.88 4.13 9.05-40

NEONATAL SCREENING FOR INBORN ERRORS OF METABOLISM OUR EXPERIENCE AT CABRI, GMU Currently, the UAE s National Newborn Screening Program includes the following 16 disorders: congenital hypothyroidism; sickle-cell disease; congenital adrenal hyperplasia; BIOT; and 12 amino acid, organic acid, and fatty acid disorders. Al Hosani et al. analyzed the data of NNS from January 1995 to December 2011; the results showed that the prevalence of screened disorders was 1:1,873 for congenital hypothyroidism; 1:14,544 for phenylketonuria; 1:3,526 for amino acid, organic acid, and fatty acid disorders; 1:9,030 for classical congenital adrenal hyperplasia; 1:8,300 for BIOT; 1:2,384 for sickle-cell disease; and 1:121 for sickle-cell traits 11. About 95% neonates were screened in the UAE in 2010. Although the sample size of our study is small, the results obtained are in line with the national figures. The findings of our study showed that 22 cases had high IRT activity, the most likely feature of cystic fibrosis. Some of these cases were confirmed positive for cystic fibrosis at Tawam Hospital, UAE. NNS is still not mandatory in the UAE, although HAAD has made it compulsory for all hospitals in Abu Dhabi to screen all newborns delivered in their facilities. The Ministry of Health and Dubai Health Authority hospitals perform free NNS for Emiratis. Private hospitals offer screening to their clients at a price, and the panel offered is not uniform. The barriers for uniform NNS in the UAE are high cost for NNS, improper treatment and followup of positive cases, births occurring at places other than hospitals, improper sample collection due to the lack of proper training, lack of knowledge among parents about the importance of screening, and difficulty in contacting parents in remote areas. CONCLUSION The results of our study showed that the prevalence of IEM among neonates was high, dominated by G6PD deficiency, hemoglobinopathies, and cystic fibrosis. Considering the rising incidences of genetic and metabolic disorders globally, and benefits of early diagnosis and treatment of affected neonates, it is imperative to screen every newborn for early detection of any congenital, genetic, or metabolic disorder to prevent any mental or physical derangement if the baby survives. Lack of knowledge about these disorders and inadequate facilities for LC- MS/MS at many hospitals in the UAE result in numerous cases of IEM in neonates going undetected, causing serious health issues. CABRI provides comprehensive testing and evaluation of 57 disorders using state-of-the-art technology for specimen analysis. Currently, the main limitation of our screening program is the inability to follow-up with neonates; this requires a concerted effort by the healthcare team comprising the obstetrician, neonatologist, pediatrician, educators, counselors, and parents. However, given the challenges and opportunities, screening itself is not enough. It is important to avoid complacency in assuming that every newborn who is screened positive would receive optimal service and care, irrespective of the lack of financial resources. Short-term follow-up and management of children with disorders, and long-term followup activities within the entire newborn screening system are crucial for an effective NNS program. In general, some factors that would make the newborn screening program successful and sustainable are government prioritization, full or partial government financing, public education and acceptance, cooperation of health practitioners, and institutionalization of the program. REFERENCES 1. Chen B, Mei J, Kalman L, Shahangian S, Williams I, Gagnon MB, et al. Good laboratory practices for biochemical genetic testing and newborn screening for inherited metabolic disorders. MMWR Recomm Rep. 2012;61(RR-2):1 37. 2. Zhang C, Xu K, Dave UP, Wang Y, Matsumoto I. Inborn errors of metabolism discovered in Asian department of pediatrics and mental retardation research center. J Chromatogr B Biomed Sci Appl. 2000;746(1):41 9. 3. Guthrie R, Susi A. A simple phenylalanine method for detecting phenylketonuria in 41

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