Introduction

Inborn errors of Metabolisms (IEMs) are inherited disorders occurring due to a metabolic block because of deficiency of an enzyme or cofactor leading to metabolic derangements in the body. IEMs are collectively not uncommon in our country and there is a need to consider these disorders in the differential diagnosis of a sick infant, as early detection has potential for treatment and thus prevention of death and irreparable brain damage. This article describes an approach for testing a sick neonate, as well as newborn screening. Diagnostic algorithms for a suspected case of IEM are given. For newborn screening, the evolving methods are discussed including tandem mass spectrometry.

Screening for Inborn Errors of Metabolism

Inborn errors of metabolism (IEMs) are genetic disorders occurring due to lack of an enzyme in the metabolic cascade in our body (1). This disrupts a biochemical pathway, leading to symptoms and signs due to accumulation of toxic metabolites prior to the block or deficiency of the end product beyond the metabolic block. Nearly all the inborn errors of metabolism are inherited recessively, mostly autosomal and some X-linked. They are individually rare but collectively numerous (2). They are increasingly being recognized because of the advancement in diagnostic facilities like blood gas analysis and automated biochemical tests, and the reduction in mortality related to infections and perinatal asphyxia (3). Other factors that lead to an increased frequency of IEMs in our country are a large population, high birth rate, and custom of consanguineous marriages among many communities (4). Limited studies have been published on the prevalence of IEMs in India (5, 6, 7). At Sir Ganga Ram Hospital, a study of 393 cases suspected to have an inborn errors of metabolism resulted in specific diagnosis in 56 (6.7%) cases (Table 1) (8). Table 1

IEMs Diagnosed at SGRH

Disorders detected	Number of children

Organic acidemias	38
Methyl malonic acidemia	12
Multiple Carboxylase deficiency	8
Maple Syrup Urine Disease (MSUD)	7
Glutaric aciduria type II	4
Propionic acidemia	3
3 methylglutaconic aciduria	2
Beta-ketothiolase deficiency	2
Urea Cycle Disorders	9
Citrullinemia	4
Argininosuccinic aciduria	2
Arginase deficiency	1
Ornithine Transcarbomylase deficiency	1
HHH syndrome	1
Other disorders including Tyrosinemia 2	9

Screening for IEMs in the Newborn

IEMs can be broadly categorized into three groups (9). The enzyme defects occurring in the metabolism of amino acids, carbohydrates, and fatty acids or in the mitochondrial energy metabolism are referred to as ‘small molecule disorders’. Clinical manifestations of these disorders are severe and non-specific, and occur early in life. In contrast, larger molecule or storage disorders the symptoms develop gradually due to accumulation of substrates, thus resulting in progressive organomegaly and coarse body features. A third group is that of other miscellaneous disorders of metals (Wilson and Menkes disease), lipoproteins, congenital adrenal hyperplasia and hyperinsulinism. The focus of this article is on the ‘small molecule disorders’ which comprise classical IEMs in the neonatal period. These disorders are dynamic, fluctuating with the patient’s metabolic state and require early diagnosis for therapeutic intervention to prevent the irreparable brain damage.

Approach to Inborn Errors of Metabolism

There are two approaches to screening for inborn errors of metabolism. The first is the testing of suspected cases in order to establish diagnosis in a sick infant. The second approach is to screen all newborns for selected group of disorders. The latter is an established practice in the developed countries, but has yet to take off in India. All countries need to establish diagnostic facilities for IEMs, because a majority of them are treatable. Once these facilities are set up and strong socioeconomic and health infrastructure has been built up, newborn screening should be initiated.

Screening of Sick Neonates

Enormous strides have been made in the past three decades in the recognition and understanding of metabolic illnesses. Progress on the reverse process - identifying specific metabolic disorders on the basis of signs and symptoms of disease - has been slower. The newborn appears to have a limited repertoire of ways to respond to insult, with the result that the clinical features of many inherited metabolic diseases are superficially indistinguishable from that of the commoner acquired conditions, such as infections and intoxications. Thus, a high index of suspicion is required in order to identify a sick neonate with an IEM. The diagnosis of IEM should be considered along with that of other common illnesses, and should not be a diagnosis of exclusion. Pointers to the presence of IEM are (10):

History of acute deterioration after a period of normalcy after birth. This is so because symptoms are caused by postnatal accumulation of toxic metabolites, which were getting cleared in utero by the placenta and maternal metabolism. Examples are urea cycle defects, organic aciduria and galactosemia. However, it is important to note that this period of normalcy may not always be present and obscured by other problems like perinatal asphyxia.

A family history of consanguinity or similar illness in a sibling.

Progressive illness showing non-specific signs of cerebral dysfunction such as poor sucking, limpness, vomiting, irritability, respiratory distress, lip smacking movements and hypothermia.

Unusual odors may offer an invaluable aid to the diagnosis. In Maple syrup urine disease (MSUD), there is a characteristic odor of maple syrup or burnt sugar. Performing a DNPH test in the urine of such a case can make the diagnosis fairly easily.

Some IEM are associated with concomitant sepsis and neutropenia like organic aciduria and galactosemia (E. coli sepsis).

Initial laboratory investigations (listed in Table 2) should be carried out as soon as possible. Some tests like blood ammonia and lactate are considered crucial for diagnosis of IEMs, and thus should be available to every neonatal care unit. Specialized investigations are done depending upon the clinical features and initial investigations. MRI brain is increasingly being recognised as an important modality for diagnosis of some IEMs such as molybdenum cofactor defects where there are extensive changes suggestive of the diagnosis. A simplified algorithm for diagnosis of Inborn errors of metabolism in an acutely sick neonate is given in Figure 1. Table 2

Investigations in a suspected case of IEM

A. Initial investigations	Blood
	Hemoglobin, white blood count, platelets
	Blood gases and plasma electrolytes (anion gap (Na+K)-(HCO3+Cl)= normal<20 nmol/L)
	Glucose
	Calcium, Magnesium
	Blood urea nitrogen, creatinine
	Liver function tests (Prothrombin
	time, liver enzymes, albumin etc.)
	Ammonia
	Lactate
	Urine
	Specific gravity, pH Proteins
	Sugar, Reducing substances (Benedicts and uristix)
	Ketones, Ketoacids (DNPH)
	Sulfites
	Microscopy
B. Specialized investigations	Blood
	Amino acid analysis - quantitative
	Galactosemia screening test
	Carnitine, total and free (Tandem Mass
	Spectrometry)
	Plasma for storage at −20°C: 2-5 ml
	Urine
	Organic acids (GC-MS)
	Succinyl acetone
	Urine for storage at −20°C: 10-20 ml
	Neuro imaging
	CT/MRI brain

Modified from Clarke JTR (10).

Figure 1Simple approch for Diagnosis of IEM

Metabolic acidosis is an important finding and narrows down the diagnosis to a few disorders including all the organic acidurias and primary lactic acidurias. A diagnostic algorithm is given in Figure 2. The common organic acidurias prevalent in our country are methyl malonic aciduria, propionic aciduria, multiple carboxylase deficiency and MSUD (8). Figure 2Flow chart for Metabolic acidosis

Hyperammonemia is a very useful clue, and often associated with encephalopathy and seizures. Urea cycle disorders occur without metabolic acidosis. These babies are sick and need immediate treatment with sodium benzoate or phenyl butyrate if available. Raised ammonia in presence of metabolic acidosis suggests organic aciduria, which can be diagnosed using approach given in Figure 3. Other causes of raised ammonia are lysinuric protein intolerance, liver failure and ‘congenital hyperinsulinism with hyperammonemia’. Figure 3Algorithm for diagnosis of hyperammonemia

Blood glucose is one of the first investigations performed on a sick neonate and thus hypoglycemia is commonly encountered. When common causes of hypoglycemia like maternal diabetes, small for gestational age baby and sepsis are ruled out, one can follow the algorithm given in figure 4 to make a diagnosis of IEM. Figure 4Flow chart for diagnosis of hypoglycemia

Neurologic dysfunction occurs most commonly in newborn in form of lethargy, poor reflexes and seizures. This encephalopathy may or may not be associated with acidosis. While the disorders related to acidosis are given above, the IEMs associated with encephalopathy without acidosis include:

Urea cycle disorders

Maple syrup urine disease

Homocystinuria

Pyridoxine dependent seizures

Xanthine-sulfite oxidase deficiency/molybdenum cofactor defects

Non-ketotic hyperglycinemia.

Electron transport chain disorder or Pyruvate dehydrogenase deficiency.

Fatty acid oxidation defects.

Peroxisomal disorders like Zellweger syndrome.

Hepatic involvement manifesting as jaundice, hepatocellular dysfunction and hypoglycemia, is a common presentation of many inborn errors of metabolism. The main disorders here are amino acidopathies – hepatorenal tyrosinemia, disorders of carbohydrate metabolism – hereditary fructose intolerance (HFI) and glycogen storage diseases (GSD), fatty acid oxidation defects – Medium chain acyl CoA dehydrogenase (MCAD), long chain acyl-CoA dehydrogenase (LCAD), carnitine palmitoyl transferase II deficiency (CPT II) and carnitine –acylcarnitine translocase deficiency (CAT) (10). These disorders are diagnosed by tandem mass spectrometry. Mitochondrial energy metabolism – cytochrome C oxidase deficiency and mitochondrial depletion syndromes also present with hepatic symptoms.

Non-immune hydrops may be due to IEMs and these should be suspected after exclusion of anemia and hemolysis and in the presence of significant organomegaly. Main IEMs to be looked for are GM1 Gangliosidosis, Gaucher Disease, Niemann-Pick disease, Sialidosis, Galactosialidosis, I-cell disease, Morquio (MPS IV) and Sly disease (MPS VII).

Cardiac involvement occurs as rhythm disturbances, shock like symptoms, cardiomegaly or cardiomyopathy leading to cardiac failure (2). Some IEMs such as glycogen storage disease type 2, fatty acid oxidation defects – MCAD, LCAD, CPT I and II, CAT deficiency, tyrosinemia type 1, and electron transport chain disorders may present with cardiac symptoms.

Collection, Storage and Transport of SamplesUrine

Collect 10-15 ml of urine, preferably first morning specimen. Tests include

Routine biochemical tests, protein, glucose, reducing substances, pH.

Chemical tests like DNPH, Ferric Chloride test, Sulfite dipstick test etc.

Amino acid chromatography and quantitative estimation.

Organic acids (collect on filter paper)

Blood

For ammonia estimation, a free flowing fresh sample should be drawn in vial and transported directly to lab on ice, as soon as possible. This is critical, as ammonia tends to rise falsely if sample is not processed immediately.

For lactate and pyruvate, a fluoride vial is used and same precautions as for ammonia are to be taken.

Collect in EDTA if plasma is required for testing or for storage.

For galactosemia and tyrosinemia testing, heparinised blood is required.

For enzyme assays on blood leukocytes, blood sample should be collected in EDTA vial.

For Tandem Mass Spectrometry, take fresh blood on filter paper, or 1 ml of blood in heparin or EDTA vial and put on filter paper.

Every suspect case of IEM who is very sick should have following samples stored for diagnostic purpose and to carry out prenatal diagnosis in the family’s future pregnancies.

DNA - collect blood in EDTA tube. All gene studies require DNA sample. DNA to be stored at 4 –8 degree Celsius.

Plasma to be stored at –20 degree Celsius.

Urine to be also stored at –20 degree Celsius.

Other body tissues

Liver, muscle, bone biopsy, kidney etc. may be utilized later for diagnostic purpose and therefore should be stored in culture medium for enzyme assays and DNA studies. Transport of samples on filter paper (blood spots and urine) and DNA can be done in room air while plasma samples and blood for enzyme assays need to be cooled during transportation.

Therapy, Genetic Counselling and Prenatal Diagnosis

Management of inborn errors of metabolism is out of scope of this article. Books on IEM may be consulted (1, 2, 9, 10, 11).

It is essential to make a diagnosis in a given suspected case of IEM for genetic counseling and prenatal diagnosis in the couple’s subsequent pregnancies. This holds importance in our country since the burden of these disorders is high in view of the costly treatment and lack of government assistance. Blood and urine sample should be stored in anticipation of a adverse outcome as the diagnosis can be made on these samples later. Prenatal diagnosis can be offered after an accurate diagnosis by DNA technology or enzyme assay on chorionic villi or cultured amniocytes, as there is a 25% risk of recurrence in all the autosomal and X-linked recessive disorders.

Newborn screening for Inborn Errors of Metabolism

A novel idea struck Robert Guthrie in 1961 and the world of the newborn changed like never before. He successfully performed a test on dried blood spots on filter paper for phenylketonuria (12). Medical science took a major leap forward and newborn screening emerged as a preventive health program to prevent mental retardation. Newborn screening is mandatory in many developed countries as part of their public health programs. The time has come to introduce newborn screening programs in India.

WHO task force committee defines newborn screening as a “public health program aimed at the early identification of conditions for which early and timely interventions can lead to the elimination or reduction of associated mortality, morbidity and disabilities. It involves the following components : screening, short-term follow-up, diagnosis, treatment/management, and evaluation” (14). Thus, it is not just diagnosis but also about treatment to prevent disabilities and reduce the burden of such illnesses. Neonatal screening service involves many professionals such as nurses, laboratory technicians, clinical biochemists, pediatricians, neuropediatricians, social workers, obstetricians, medical geneticists, but some one is required to coordinate all these activities.

Since the introduction of screening for phenylketonuria, many other disorders have been added for analysis of dried blood spots on filter paper. Newborn screening for congenital hypothyroidism was introduced in 1974 by Dussault et al (15). Later on with the advent of newer technologies, several changes in the newborn screening were incorporated for detection of many other in-born errors of metabolism, notably by TMS. Presently as many as 30 disorders amongst the 600 odd IEMs are detectable using the filter paper methodology (16).

Principles and Criteria for Newborn Screening

Screening is the application of a test to people who are as yet asymptomatic, with the purpose of classifying them with respect to their likelihood of having a particular disease. Since the tests are carried out on normal individuals, this raises certain ethical issues. The WHO (1968) had outlined certain criteria for a screening program based on principles formulated by Wilson and Junger (17). These are:

Clinically and biochemically well-defined disorder.

Known incidence in the population

Disorder associated with significant morbidity or mortality

Effective treatment available

Period before onset during which interventions improves outcome

Ethical, safe, simple and robust screening test

Cost-effectiveness of screening is established

Newborn Screening Methodology

The newborn screening methods have evolved over the last 4 decades from the basic bacterial inhibition assay used for phenylketonuria by Robert Guthri,e to the more sophisticated tandem mass spectrometry today. Methods such as ELISA and Time resolved fluorometry are being used in many countries for newborn screening because of its low cost and rapid analysis of multiple samples. Conditions included in Newborn screening programs are given in Table 3 (13). The various analytes tested for screening of above disorders and the evolving methodologies from time to time are enlisted in the Figure 5 (13). Table 3

Conditions for New Born screening

Common disorders

Phenylketonuria

Endocrine

Congenital Hypothyroidism

Congenital Adrenal Hyperplasia (21 hydroxylase deficiency)

G-6-phosphate dehydrogenase deficiency

Galactosemia

MSUD

Biotinidase deficiency

Homocystinuria

Tyrosinemia

Cystic fibrosis

MCAD

Alpha-1 antitrypsine deficiency

Other disorders

Infections

HIV

Toxoplasmosis

Tumors

Neuroblastoma

Ambiental toxicity

Lead exposure

Sickle cell anemia

DMD

Hypercholesterolemia

Figure 5Analytes that have been determined in dried blood samples collected on filter paper, and more frequently employed screening methods

Tandem Mass Spectrometry

Tandem Mass Spectometry is rapidly gaining popularity a method for newborn screening because of its simplicity, rapidity and ability to detect a number of disorders in the one sample. It has its own controversy because of the ability to screen some disorders also which do not have any treatment at present.

Mass spectrometers are an analytical tool used for measuring the molecular weight (MW) of a given sample, be it blood, urine, water etc (18). They are used in industry and academia for both routine and research purposes. Most commonly they are employed in biotechnology, pharmaceutical, environmental and geological fields and also in clinical field such as neonatal screening.

Mass spectrometers can be divided into three fundamental parts, namely the ‘ionisation’ source, the ‘analyzer’, and the ‘detector’ (19). The sample once introduced into the ionization source gets ionized which are separated according to their mass to charge ratios (m/z). The separated ions are detected and this signal sent to a data system. The Tandem (MS-MS or TMS) mass spectrometers are instruments that have more than one analyzer and so can be used for structural and sequencing studies. The TMS is extremely useful as it gives accurate results in terms of detecting and quantifying biomolecules.

Tandem mass spectrometry expands neonatal screening to several disorders in amino acid, fatty acid oxidation and organic acid metabolism, more than 60 in number. TMS can improve the detection of PKU also. A high sensitivity (95.4%) and specificity (99.7%) is reported with a combined prevalence of 1:2336 (in Germany) (20). Treacy et al argue that after diagnosis using TMS, response to treatment was complete in 12% of IEMs, there was no response in 34%, and in more than half (54%) there was a partial response, which justifies some intervention (21). Thus, TMS provides an early diagnosis and facilitates early intervention, so that ‘partial benefits’ can become ‘substantial benefits’. Further, once an IEM is diagnosed in a family, a prenatal diagnosis can be performed to prevent birth of a child if it’s a disorder which is not treatable. This perhaps has more relevance in our country with little resources and health care system not being completely state funded.

Indian Scenario

India has progressed immensely in the field of medical technology and health care. Therefore, this is the time to implement a newborn-screening program. Pilot studies are required to determine the prevalence of the disorders in different parts of the country before starting a national program.

Conclusion

It is the fundamental right of a human being to live a disease free life as far as possible. The newborn screening systems can help in achieving this goal. Let us benefit from the medical advancements and give our newborns a disease free life.