International Journal of Human and Health Sciences Vol. 03 No. 02 April’19 58 Review article: Diagnostic Advancement in Evaluating Inborn Errors of Metabolism: Past, Present and Future: A systematic review Nazmin Fatima1, Shalini Tripathi2, Roshan Alam3, Mohammed Haris Siddiqui4, Abbas Ali Mahdi5, Gyanendra Kumar Sonkar6 Abstract: Metabolism is a delicately coordinated entity of chemical reactions. Inborn Errors of Metabolism (IEM) are rare congenital disorders that are mainly due to gene defect of enzymes or cofactors participating in a metabolic pathway or the transport of metabolites within a cell or between cells. The development of knowledge in basic sciences together with technology development in medical field has helped to better understand the molecular and biochemical basis of IEM. Environmental factors, ethnicity, race, consanguinity and genetic factors contribute to the increased prevalence of genetic disorders. The analytical methods have evolved over the years from thin layer chromatography (TLC), high performance liquid chromatography (HPLC) to tandem mass spectrometry (TMS) including gas chromatography mass spectrometry (GC/MS). Their applications for È¡7�Ø‹5�g of IEM has opened the door for screening of conditions that previously required molecular testing or another methodology that was not practical for population-based screening. Future technologies such as Matrix-assisted laser desorption/ ionization time- of-flight mass spectrometry (MALDI-TOF MS), has the potential for rapid and reliable identification of small metabolites and disease biomarkers in daily clinical laboratories, whereas DNA based screening by DNA microarrays or gene chips will allow much more improved diagnosis.These can be the boon to screening programs which will require excellent detection and follow-up services Keywords: IEM, TLC, HPLC, TMS, MALDI-TOF MS, GC/MS Correspondence to: Dr. Gyanendra Kumar Sonkar, Associate Professor, Department of Bio- chemistry, King George’s Medical University, U.P., Lucknow, India. Contact No.: +91- 9453817641. Email: gyanendrakrsonkar@kgmcindia.edu, gettwinklestar@gmail.com 1. Research Scholar, Department of Biochemistry, Integral Institute of Medical Sciences & Research, Integral University, Lucknow, Uttar Pradesh, India. 2. Associate Professor, Department of Pediatrics, King George’s Medical University, U.P., Lucknow, India. 3. Prof. & Head, Department of Biochemistry, Integral Institute of Medical Sciences & Research, Integral University, Lucknow, Uttar Pradesh, India. 4. Associate Professor, Department of Bioengineering, Integral University, Lucknow, Uttar Pradesh, India. 5. Former-Head, Department of Biochemistry, King George’s Medical University, U.P., Lucknow, India. Presently, Vice Chancellor, Era University, Lucknow 6. Associate Professor, Department of Biochemistry, King George’s Medical University, U.P., Lucknow, India. Introduction:Metabolism is a delicately coordinated entity of chemical reactions. For maintenance of tissue, growth and reproduction, the organism through various metabolic processes tries to balance between energy and nutrient intake, consumption and storage. It can be disturbed in either inherited or acquired situations. Inborn errors of metabolism (IEM) was first described by Sir Archibald Garrod in 1908, during the famous Croonian lectures on the inborn errors of metabolism, i.e., alkap tonuira, cystinuria, albinism and pentosuria.1 Following this, the world Short title: Diagnostic advancement in evaluating IEM International Journal of Human and Health Sciences Vol. 03 No. 02 April’19 Page : 58-63 DOI: http://dx.doi.org/10.31344/ijhhs.v3i2.78 59 International Journal of Human and Health Sciences Vol. 03 No. 02 April’19 of IEMs has been ever expanding, both in terms of diagnosing new affected phenotypes and in their therapeutic measures. They have also been coined different names, e.g., inherited metabolic disorders (IMD), hereditary metabolic disorders (HMD), or congenital metabolic diseases (CMD). Most of them cannot be cured and leads to fatal outcome with episodes of acute metabolic decompensation. Treatment is limited and most often supportive and experimental. Search for new therapeutic approaches is possible by gathering new information about its pathogenesis.2 IEM are rare congenital disorders that are mainly due to a gene defect of enzymes or cofactors participating in a metabolic pathway or the transport of metabolites within a cell or between cells. This results in either accumulation of substrate, loss of end products, accumulation of normally minor metabolites or secondary metabolic consequences.3 It can be classified in various forms and one of the ways to classify it is based on its pathophysiology as disorders. According to this, it is classified as (i) Group-I disorders, which gives rise to intoxication, (ii) Group-II disorders, involving energy metabolism, and (iii) Group- III disorders, involving complex molecules.4 Accumulation of the toxic metabolite, proximal to the metabolic block including aminoacidopathies, organic acidemias, urea cycle disorders, porphyrias, mineral metabolism disorders, sugar intolerances, as well as synthesis defects of neurotransmitters etc. are categorized under first group of disorder. The second group comprises mitochondrial and cytoplasmic energy defects. The last group is the most diverse and includes lysosomal storage disorders, peroxisomal biogenesis disorders, congenital disorders of glycosylation, cholesterol synthesis defects etc.4 Among the three groups of disorders, the first group of disorders can be diagnosed by simple tests such as blood and urine amino acids, organic acid and acylcarnitine profiling while the second group of disorders are identified by enzyme analysis, tissue biopsies or molecular testing and the third group is diagnosed only by molecular testing methods. Additionally, among the three groups, the disorders of first group can be treated either by specific dietary patterns or medications. Earlier the second and third group of disorders were untreatable but now days therapy is available in some cases.5-7 Initially the pathogenesis of IEM was not known. However advancement of technology helped in solving this problem. In 1957, Dorfman and Lorincz first revealed the biochemical basis of mucopolysaccharides by reporting excretion of mucopolysaccharides in the affected patient’s urine. Later in 1961, Guthrie developed screening of phenylketonuria using microbiological inhibition assay. The gradual increase of basic knowledge in IEM made it possible to discover over 500 disorders. Advancement in laboratory techniques helped detection of some disorders, even before the presentation of symptoms. Introduction of tandem mass spectrometry (TMS) in 1998 added new approach to screening program. IEM is now a subject of interest for research worldwide and continues to be a scientific challenge to modern medicine.8-10 Literature search and review: The literature was reviewed by performing database search such as MEDLINE, EMBASE, Science Direct, Cochrane library and Web of Science, using the keywords – ‘Inborn errors of metabolism’, ‘Inborn metabolic disorder’, ‘history of IEM’, ‘diagnostic’, ‘incidence’, ‘advancement’, ‘technology’, ‘TMS and IEM’, Tandem mass spectrometry’, ‘Gas chromatography-mass spectrometry’, ‘High performance liquid chromatography’, ‘screening’, ‘past’, ‘present’, future’, and ‘DNA microarrays’. Searches were limited to English language. Incidence of IEM: Environmental factors, ethnicity, race, consanguinity and genetic factors contribute to the increased prevalence of genetic disorders. This is the reason for such a wide variation of prevalence of IEM from country to country and also within the different regions of a country.11,12 Individual disorders of IEM are rare but collectively numerous, leading to substantial patient burden13 with the current incidence of IEMs standing at 1:800 live births. It may present at any age, from infancy to adult and can affect either individual or multiple organs. Depending on the severity of the disorder, they usually affect several organs.14 Sanderson et al.15 has reported 1 in 784 live births as an overall incidence of IEM in a five year retrospective study in United Kingdom. Dionisi-Vici et al. from Italy has reported an incidence of 1 in 3,707 live births from their 12 years of study. However using advanced technique such as TMS, reduced the incidence as 1 in 6200.16 Another study from Canada has reported an overall incidence of 1 in 2500 with disorders of amino acid metabolism, organic acid metabolism, urea cycle disorder, glycogen storage disorder etc. as more prevalent.17 A study from middle east country like Saudi Arabia has reported a high International Journal of Human and Health Sciences Vol. 03 No. 02 April’19 60 incidence of 1 in 666 live births.18 Another study from the same area reported a prevalence of 1.25% of symptomatic newborn babies were found to be suffering from IEM.19 Asian and South East Asian countries20 also reported a varied incidence such as 1 in 4000, 1 in 5,800 in Mainland China21, 1 in 5882 in Taiwan22, 1 in 2000 in Korea23 and 1 in 9330 in Japan.24 Scarce report is available from India as we still do not have mandatory screening program for IEM in newborns and infants. Only few private hospitals and health centers are providing diagnostic facilities, hence only few Indian studies have addressed the incidence of IEM.25,26 One study has reported incidence of 1 in 360027 and another study by Nagaraja et al.28 has reported a prevalence rate of 2.3%. Screening of IEM – Past, Present and Future: The history of screening for IEM started in 1959, when Prof. Guthrie first demonstrated the detection of phenylalanine level in dried blood spot based on bacterial inhibition assay.29 This technique later came to be known as Guthrie test which was easy, cheap and reliable and followed by mass screening of phenylalanine in children. This mass screening was opposed by medical fraternity at that time. However in 1962, a pilot study was done for screening of phenylketonuria (PKU).30 In the following years, screening for more conditions such as maple syrup urine disease (MSUD), galactosemia and homocystinuria were started.31 Later in 1968, the Wilson and Jugner criteria for screening of IEM was framed keeping in view its clinical validility, clinical utility, analytical validity, social and ethical issues to cost effectiveness.32 Finally in 1975, the United States of America made screening of PKU compulsory for all newborns. The Massachusetts Medical School also started the screening program with addition of more congenital disorders including congenital hypothyroidism, congenital toxoplasmosis, hemoglobinopathies, congenital adrenal hyperplasia, biotinidase deficiency and cystic fibrosis.31 Soon other countries like Canada, Portugal and Australia started screening of IEM in newborns.32-34 In India, the state of Karnataka was the first to start screening of IEM in neonates in 1980 by Appaji Rao and his team. The burden of IEM in neonates was reported to be 0.04% whereas it was 3.2% in high risk population and a bit higher (5.75%) in mentally retarded children.35,36 Another study from Kerala has reported a high incidence of aminoaciduria, organic aciduria and other IEMs in their studied population which extended over a period of five years.37 Similar report of high incidence (1 in 1000) has been published from Andhra Pradesh. The common disorders that have been detected were congenital hypothyroidism (CH), congenital adrenal hyperplasia (CAH) and hyperhomocystinemia38, using techniques such as chromatography, electrophoresis, ELISA and TMS. Study from North India shows a slightly different incidence from South India. Homocystinuria, alkaptonuria, MSUD and non ketotichyperglycinemia were common in North, whereas homocystinuria, MSUD, PKU, mucopolysaccharidoses were common in South11,39,40 using advanced techniques such as GCMS and TMS. Diagnostic advancements: Screening for IEM involves metabolic profiling of blood and urine samples. The analytical methods have evolved over the years from thin layer chromatography (TLC), high performance liquid chromatography (HPLC) to tandem mass spectrometry (TMS) including gas chromatography mass spectrometry (GC/MS). TLC is one of the oldest techniques introduced which is still in use. It is widely used as it simple to perform, cost effective, rapid and reproducible and requires less space.41 Stationary phases commonly used for TLC include silica, alumina and cellulose, which are coated onto a backing of aluminium, plastic or glass to provide physical support. It can be used to separate amino acids, organic acids, sugars, phenolic acids, ketoacids, imidazole, steroids, lipids, purine, pyrimidine and related compounds. Hence it is used in detection of aminoacidopathies in IEM cases This technique enabled Kaur et al. to report homocystinuria, MSUD, alkaptonuria, hyperglycinemia, phenylketonuria, cystinuria and general aminoaciduria in high risk infants and children from North India.42,43 This application has helped in screening more than twenty five different metabolic and transport disorders before the onset of the clinical symptoms. Thus, TLC was and still a useful tool in screening of IEM.41,44 HPLC is used to separate compounds on the basis of their chemical characteristics, such as polarity, molecular size and degree of charge in a pH gradient. It helps to quantify the individual components. There are many forms of HPLC—the most common is reverse phase HPLC. The amino acids are derivatized for carrying out HPLC. Earlier researchers used ion exchange chromatography in combination with post column ninhydrin detection, but now day’s methods have been simplified with 61 International Journal of Human and Health Sciences Vol. 03 No. 02 April’19 o-phthalaldehyde (OPA) method.45 However it can be used for primary amino acids but for a mixture of secondary amino acids, phenylisothiocyanate (PITC) method is used.46,47 It has helped in detection of homocystinuria, MSUD, tyrosinemia, phenylketonuria, histidinemia, citturullinemia, argininemia and hyperglycinemia.48 HPLC has also been used by researchers for detection of organic acids such as methyl malonic acid, lactic acid, isovaleric acid, glutaric acid, propionic acid etc. GC/MS is another chromatographic technique used to separate components in physiological samples as well as pharmaceutical, food and forensic samples using gas as the mobile phase (carrier) and a silicon based oil as the stationary phase. It accurately detects the volatile compounds in small samples. It has become one of the commonest methods used by researchers and laboratory personals for identifying organic acid metabolism disorders of IEM.49 The TMS technique was introduced by Millington et al. in 1990. Initially it was being used for detection of metabolic disorders such as phenylalanine and tyrosine.50,51 Now, this technique has gained popularity and it is being used to detect and analyze acyl carnitine profile, amino acids and organic acids simultaneously. Using this technology, one can detect more than 30 different types of IEM. Thus it has helped in screening, diagnosis and treatment52 and has been accepted in many developed countries leading to significant decrease in morbidity due to IEM.53,54 The technique has high sensitivity and specificity and comparable to other modern techniques like radio-immunoassay and GC/MS.55,56 The introduction of TMS has greatly influenced the screening of IEM, which can detect many treatable and untreatable disorder which might be useful to give therapy as well as guidance to affected families. A study by Wilcken et al.57, reported a twofold higher prevalence of IEM using TMS as compared to those diagnosed clinically. TMS based screening of IEM in newborns recorded an overall incidence of 1 in 9300 in Japan.58 Its application to newborn screening has opened the door for screening of conditions that previously required molecular testing or another methodology that was not practical for population- based screening.59,60 Future technologies: Advancement in technology will strengthen the diagnostic abilities. New technologies such as Matrix- assisted laser desorption/ ionization time-of- flight mass spectrometry (MALDI-TOF MS), has the potential for rapid and reliable identification of small metabolites and disease biomarkers in daily clinical laboratories, whereas DNA based screening by DNA microarrays or gene chips will allow much more improved diagnosis.61 Qualitative and quantitative changes in nucleic acid sequences such as mutations, single- nucleotide polymorphisms, insertion/deletion, alternative splicing, copy number variations, gene and allele expression, modifications brought about methylations of DNA, post transcriptional modifications of tRNAs and rRNAs can be done using MALDI-TOF MS.62 Recently it has been used for screening of IEM in newborns using dried blood spot samples. In a study by Hachani et al.63, this technique was used to screen sickle cell disease and thalassemia with the primary objective to determine the mass and relative abundance of primary hemoglobin (Hb) α and β subunits and of the HbS subunit, indicative of sickle cell disorder. They reported a decrease in mass of 30 Da in the HbS subunit. They concluded that this involved marked reduction in cost per unit analysis. DNA microarrays is another future technology which will help to screen IEM in newborns and infants for diseases arising due to mutations in genes.64 In conclusion, the upcoming techniques are becoming affordable, simple to handle, provides high throughput and very cost effective with high sensitivity and specificities. These can be the boon to screening programs which will require excellent detection and follow-up services. Funding: We are thankful to Council for Science & Technology, Uttar Pradesh, India, for providing us financial support vide sanction order no. CST/ YSS/D-2874, for carrying the research work related to Inborn Errors of Metabolism. Acknowledgement:We would also like to acknowledge Dr. Sangeeta Singh and Dr. Jamal. A. Ansari, both postdoctoral fellows of the department of Biochemistry, K.G.M.U. for their constant help and support. Conflict of Interest: None Authors Contributions: Conception and design: NF, ST, GKS, AAM Analysis and interpretation of data: NA Critical revision of the article for important Intellectual content: NF, GKS Final approval of article: NF, ST, RA, MHS, AAM, GKS Statistical expertise: NA Collection and assembly of data: NA International Journal of Human and Health Sciences Vol. 03 No. 02 April’19 62 References: 1. Scriver CR. Garrod’s Croonian Lectures (1908) and the charter ‘Inborn Errors of Metabolism’: albinism, alkaptonuria, cystinuria, and pentosuria at age 100 in 2008. J Inherit Metab Dis 2008; 31: 580–98. 2. Maher AD, Zirah SF, Holmes E and Nicholson JK. 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