Inborn errors of metabolism (IEMs) are a complex and heterogeneous group of monogenic disorders that exhibit clinical symptoms due to an error in a genetic code that results in a lowered or deficient activity of an enzyme in a single pathway of intermediary metabolism.- ¹ Due to severe clinical consequences of IEMs, they are an important cause of morbidity and mortality in clinical practice, especially in pediatrics. Delay in diagnosis and treatment of these disorders, leads to a variety of adverse outcomes, including moderate-to-severe neuropsychological dysfunction, mental retardation, and death. Each disorder is individually rare, but their cumulative incidence is relatively high, around 1 in 1500 to 1 in 5000 live births. ² , ³

Newborn screening is a vital process that identifies apparently healthy infants with serious inherited disorders, generally metabolic in origin, that are usually correctable by dietary or drug interventions before they suffer significant morbidity or mortality. ⁴ Carlson commented that, "newborn screening represents one of the major child health advances of this past century".- ⁵ In expanded newborn screening, a single test allows for early detection and treatment of a large number of disorders and it can potentially prevent serious consequences. ⁶ , ⁷

General aims of newborn screening and criteria for screening

The goal of newborn screening is to identify apparently healthy infants with severe congenital disorders that are relatively prevalent and treatable (or controllable). It originated with the work of Robert Guthrie, ′Guthrie test,′ in the 1960s, for detecting the metabolic disorder, phenylketonuria (PKU) and used for years to justify screening programs. ⁸ , ⁹ With the development of immunoassays for thyroxin and the thyroid stimulating hormone (TSH) in the 1970s, it became feasible to add congenital hypothyroidism (CH) to the Newborn Screening (NBS) panel. Many conditions are potential candidates for NBS, but this is not practical for all. ¹⁰ The World Health Organization (WHO) Wilson-Jungner criteria are the gold standard criteria that are used to determine the disorders that need to be included in the newborn screening program. These criteria cover the aspects of knowledge of the disease, its treatment, the scientific validity of the tests, and cost considerations associated with the screening program. ⁹ , ¹¹ , ¹² According to the WHO Wilson-Jungner criteria, a disease which has the following properties should be screened: (1) An important health problem; (2) the natural history of the condition should be adequately understood; (3) it should be recognizable in the early stages; (4) there should be a suitable test or examination; (5) the test should be acceptable to the population; (6) intervals for repeating the test should be determined; (7) there should be an accepted treatment for patients; (8) facilities for diagnosis and treatment should be available; (9) there should be an agreed policy concerning whom to treat as patients; and (10) the costs of case finding should be economically balanced against the benefits. ¹²

Brief history of expanded newborn screening

Historically, most NBS programs such as PKU were established without evidence-based evaluation. PKU screening was based on a prediction that dietary treatment would prevent mental retardation. ⁸ In the late 1960s, the WHO criteria established by Wilson and Jungner have served to justify NBS programs. Screening for PKU successfully fulfilled these criteria. Then programs were extended, with success, to other conditions, such as CH. Yet, an increasing number of conditions that can be detected by multiplex technologies do not fulfill all the Wilson and Jungner criteria. Since 2000, the progressive inclusion of tests for several diseases has been driven by the tandem mass spectrometry (MS/MS) technology, hence, national NBS programs differ widely now. In 2005, the American College of Medical Genetics (ACMG) proposed 29 core conditions that are considered appropriate for newborn screening, because they have a screening test, an efficacious treatment, and an adequate knowledge of natural history. ⁸ , ¹³ , ¹⁴

Newborn screening as a public health system

An NBS program is not just a panel of screening tests to identify whether a metabolite is found in abnormally high or low concentrations in a particular blood spot. Ideally, it also integrates five parts as follows: (1) Testing of newborn infants, (2) follow-up of an abnormal screening result, (3) diagnostic testing and confirmation by specialized laboratory testing, interpretation, and treatment, (4) lifelong disease management (5) the NBS system′s evaluation, which makes regular and timely communication between nurseries, screening laboratories, health authorities, pediatricians, and subspecialists. ¹⁵ , ¹⁶ Today, recommendations for the screening process also vary, for example, the time of blood sampling ranges from 24 hours (USA) and 48 to 72 hours (Germany) to 120 hours (UK). ¹⁴ In one cohort screened with MS/MS in Australia, involving 362,000 newborns, the prevalence of inborn errors, excluding phenylketonuria, was 15.7 per 100,000 births, as compared with the adjusted rates of 8.6-9.5 per 100,000 births before the MS/MS screening. ¹⁷ Follow-up reports provide a better outcome for patients at six years of age, by MS/MS screening, with fewer deaths and fewer clinically significant disabilities. ¹⁸ This strategy represents diagnosis and treatment before the onset of symptoms. ¹⁹ Economically, each Euro spent on the screening program saved more than 25 Euros in health and social costs. ²⁰ Results of many studies reflect a minimum burden and a better outcome for patients, with fewer deaths and fewer clinically significant disabilities achieved by expanded newborn screening. ¹⁸ , ²¹ , ²² , ²³ However, other types of screening such as carrier state, lethal conditions, disorders with delayed onset, and susceptibility testing are controversial. They need an extensive and ethical considerations approach, to allow us to find an optimal equilibrium between the potential benefits and the possible damages derived from neonatal screening. ²⁴

Tandem mass spectrometry in newborn screening

In recent times, advances in laboratory technology with MS/MS has enhanced the identification of newborns with an inherited metabolic disease using a single sample of a dried blood spot on filter paper. ¹¹ , ²⁵ , ²⁶ Tandem mass spectrometry is a powerful technology used for rapid identification and quantification of a large number of different analytes from a single sample, by separating the ions based on their molecular mass-to-charge ratio and measuring their intensities. ²⁷ A tandem mass spectrometer comprises of five basic components: An ion source, a mass analyzer (MS1), a collision chamber, a second mass analyzer (MS2) that separates the ions and fragments produced in the collision chamber, and a detector. ²⁷ , ²⁸ Samples are introduced into the instrument and then ionized to generate charged molecules. These molecular ions are extracted into the first analyzer (MS1) and separated by their mass-to-charge ratio. These molecular ions then enter the collision cell one at a time, where they collide with an inert gas and fragments. These fragments are separated by the second analyzer (MS2) and are detected as they emerge from the analyzer. The results are presented as a graphical mass spectrum, showing each ion by its mass-to-charge ratio and its relative intensity. MS/MS can be used for the rapid detection of a large number of IEMs, including some diseases that can not be readily detected by other methods, such as, Medium-chain Acyl-CoA Dehydrogenase (MCAD) deficiency. MS/MS has a number of advantages compared with the currently available analytical methods and in recent years its use for newborn screening has universally expanded. Some advantages of using MS/MS for newborn screening include the following: (a) The analysis can be performed on very small quantities of blood or other body fluids; (b) the analysis uses two mass spectrometers concurrently. The first is used to separate the components of the mixture, hence eliminating or minimizing prior chromatographic separation; (c) the time required for the analysis of each sample is therefore reduced to around two-to-three minutes; and (d) the process can be automated, permitting analysis of around 600 samples per 24 hours, with a very modest cost per sample. ¹¹ , ²⁹

Metabolic disorders detected by MS/MS

The application of MS/MS to newborn screening currently involves measuring mainly two groups of analytes, amino acids and acylcarnitine species, for the detection of aminoacidopathies, organic acidurias, and fatty acid oxidation defects, ³⁰ although other disease markers may also be added. ²⁶ Metabolic disorders detected by MS/MS can be categorized into four groups: Amino acid disorders, urea cycle disorders, organic acid disorders, and disorders of fatty acid oxidation Table 1. Around 40 disorders can be detected through the aforementioned analytes. The severity of metabolic disorders can range from mild to severe. ³¹

In view of the advances in the technology, recently, the American College of Medical Genetics (ACMG) has considered IEMs that are suitable for inclusion into newborn metabolic screening programs. Based on criteria such as: (1) Clinical characteristics (e.g., incidence, burden of disease if not treated, and phenotype in the newborn); (2) analytical characteristics of the screening test (e.g., availability and features of the platform); and (3) diagnosis, treatment, and management of the condition in acute and chronic forms, each disorder was assigned a score. Conditions with scores of >1200 met the key criteria and were preliminarily considered appropriate for inclusion in a core newborn screening panel. Conditions scoring <1000 were not considered appropriate for inclusion in the core newborn screening panel. Conditions with intermediate scores (1000-1199) were part of the differential diagnosis of a high-scoring core condition, but without an efficacious treatment or without a well-understood natural history. ⁸ , ¹³ , ¹⁴ Based on the ACMG recommendations, 29 core conditions that are detectable by MS/MS are considered appropriate for newborn screening Table 1. In the following section three representatives of these conditions, that is, PKU, MCAD deficiency, and methylmalonic acidemia (MMA) will be briefly described (characteristics of all these disorders may be found in general pediatric texts and further details on the rarer disorders are available). ³²

Phenylketonuria (PKU), one of the most commonly screened metabolic disorders, can readily and reliably be detected by MS/MS. It is an autosomal recessive disorder, most commonly caused by a mutation in the gene coding for phenylalanine hydroxylase. Classic symptoms of untreated PKU include; mental retardation, learning difficulties, spasticity, seizures, developmental delay, and congenital heart disease. Figure 1illustrates the blood amino acid profile from a patient with PKU analyzed by MS/MS, essentially as described elsewhere. ³³ , ³⁴ It is now clear that if Phenylketonuria is diagnosed immediately after birth, the irreversible mental retardation can be prevented. This requires comprehensive newborn screening in all populations around the world. Infants diagnosed with PKU are treated with a special low-phenylalanine formula and it is recommended that strict dietary therapy be continued during their entire lifetime. ³⁵ , ³⁶ , ³⁷ Figure 1

Blood amino acid profiles from a control subject and a patient with PKU analyzed by tandem mass spectrometry (neutral loss of m/z 102). Ion signals at representative m/z values correspond to butyl esters of phenylalanine (m/z 222), phenylalanine internal standard (m/z 227), tyrosine (m/z 238), tyrosine internal standard (m/z 242). The key diagnostic peaks are identified by arrows

Newborn blood spot screening started in the 1960s for detection of PKU. Over the years, screening programs have expanded to include other inherited diseases. Newborn screening is carried out to prevent significant morbidity, mortality, and mental handicap in infants. Newborns are tested to identify serious or life-threatening conditions before the symptoms begin. ⁶⁶ Natural history and the prevalence of each disorder shows remarkable variation among neonates from one country to another. Therefore, the data cannot be applicable directly to another locality. In developing countries like Iran, where a high rate of consanguineous marriages may lead to increased incidence of metabolic conditions, MS/MS screening for metabolic disorders will likely prove cost-effective. Furthermore, many families feel relieved that they are aware of their child′s condition in early infancy, although there might not be a specific treatment. ⁶⁷ On account of the importance of early diagnosis and treatment of inherited metabolic diseases and with respect to that little available information about the frequency and natural history of these disorders in Iran, establishment of the MS/MS neonatal screening program seems to be a major health priority in this country. In order to minimize morbidity, mortality, and disabilities associated with the inherited metabolic disorders, we suggest a national pilot study, to evaluate the establishment of the expanded newborn screening, in this country.