CLINICAL SPECTRUM OF INBORN ERRORS OF METABOLISM IN CHILDREN IN A TERTIARY CARE HOSPITAL

CLINICAL SPECTRUM OF INBORN ERRORS OF METABOLISM IN CHILDREN IN A TERTIARY CARE HOSPITAL

THE TAMIL NADU DR.M.G.R. MEDICAL UNIVERSIT

In partial fulfilment for the award of the Degree of

INSTITUTE OF CHILD HEALTH AND

MADRAS MEDICAL COLLEGE

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CLINICAL SPECTRUM OF INBORN ERRORS OF METABOLISM IN CHILDREN IN A TERTIARY CARE HOSPITAL

Dissertation submitted to the

THE TAMIL NADU DR.M.G.R. MEDICAL UNIVERSIT CHENNAI

In partial fulfilment for the award of the Degree of MD PAEDIATRICS

(BRANCH VII)

CHILD HEALTH AND HOSPITAL FOR CHILDREN MADRAS MEDICAL COLLEGE, CHENNAI

MAY-2020 REG. NO. 201717004

CLINICAL SPECTRUM OF INBORN ERRORS OF METABOLISM IN CHILDREN IN A TERTIARY CARE HOSPITAL

THE TAMIL NADU DR.M.G.R. MEDICAL UNIVERSITY,

In partial fulfilment for the award of the Degree of

HOSPITAL FOR CHILDREN

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CERTIFICATE

This is to certify that the dissertation titled “CLINICAL SPECTRUM OF INBORN ERRORS OF METABOLISM IN CHILDREN IN A TERTIARY CARE HOSPITAL” submitted by Dr.JAYASHREEK.R.to the Faculty of Pediatrics, THE TAMILNADU DR.M.G.R. MEDICAL UNIVERSITY, CHENNAI in partial fulfillment of the requirements for the award of M.D., DEGREE (PEDIATRICS) is a bonafide research work carried out by her under our direct supervision and guidance.

PROF.Dr.R.JAYANTHI, D.FRCP(Glasg), The DEAN,

Madras Medical College &

Rajiv Gandhi Govt. General Hospital, Chennai – 600 003.

PROF.Dr.S.ELILARASI, MD., DCH, Director & Superintendent,

Institute of Child Health &

Hospital for Children, Chennai – 600 008.

PROF.Dr.J.RUKMANI, MD, DCH Professor of paediatrics

Institute of Child Health & Hospital for children,

Chennai- 600 008

PROF.Dr.K.KUMARASAMY MD, DCH., DNB

Professor of paediatrics

Institute of Child Health & Hospital for children,

Chennai- 600 008

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DECLARATION

This dissertation entitled “CLINICAL SPECTRUM OF INBORN ERRORS OF METABOLISM IN CHILDREN IN A TERTIARY CARE HOSPITAL” is a bonafide work done by Dr.JAYASHREE K.R. at Institute of Child health Madras medical college Chennai during the academic year 2016-2019 under the guidance of Prof.Dr.J.RUKMANI & Prof.Dr.K.KUMARASAMY, Professor of Pediatrics, Institute of Child Health & hospital for children. This dissertation submitted to The Tamil Nadu Dr.M.G.R. Medical University, Chennai towards partial fulfilment of the rules and regulations for the award of M.D., Degree in Paediatrics, Branch(VII).

PROF.Dr.J.RUKMANI, MD, DCH Professor of paediatrics

Institute of Child Health & Hospital for children, Chennai- 600 008.

PROF.Dr.K.KUMARASAMY MD, DCH., DNB Professor of paediatrics

Institute of Child Health & Hospital for children, Chennai- 600 008.

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DECLARATION

I, Dr.JAYASHREEK.R, solemnly declare that the dissertation titled “CLINICAL SPECTRUM OF INBORN ERRORS OF METABOLISM IN CHILDREN IN A TERTIARY CARE HOSPITAL.”-An observational descriptive study has been prepared by me.

This is submitted to The Tamilnadu DR.M.G.R. Medical University, in partial fulfilment of the rules and regulations for the M.D Degree examination in Paediatrics.

Place : Chennai Dr JAYASHREEK.R.

Date :

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SPECIAL ACKNOWLEDGEMENT

My sincere thanks to Prof. Dr.R.JAYANTHI. M.D. FRCP (Glasg), Dean, Madras Medical College, for allowing me to do this dissertation, utilizing the institutional facilities

ACKNOWLEDGEMENT

It is with immense pleasure and privilege, I express my heartful gratitude, admiration and sincere thanks to Prof. Dr.S.ELILARASI, M.D., DCH., Professor and Head of the Department of Paediatrics, for his guidance and support during this study.

I am greatly indebted to my guide and teacher, Prof.Dr.J.RUKMANI., MD, DCH, Professor of Paediatrics, Prof.Dr.K.KUMARASAMY, M.D, DCH., Professor of Paediatrics for guidance and encouragement while undertaking this study.

I would like to thank Prof.Dr.PRAMILA, MD, Professor of Biochemistry, for guidance and encouragement while undertaking this study.

I would like to thank to my Assistant Professors Dr.V.PRABU, M.D, DCH, Dr.S.PERUMAL PILLAI, MD, DCH., Dr.HARSHITHA CHANDRAMOULI, MD, DCH., Dr.G.THANNOLI GOWTHAMI, MD, for their valuable suggestions and support.

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I also thank all the members of the Dissertation Committee for their valuable suggestions.

I gratefully acknowledge the help and guidance received from Dr.S.SRINIVASAN, DCH., Registrar at every stage of this study.

I also express my gratitude to all my fellow postgraduates for their kind cooperation in carrying out this study and for their critical analysis.

I thank the Dean and the members of Ethical Committee, Rajiv Gandhi Government General Hospital and Madras Medical College, Chennai for permitting me to perform this study. I thank all the parents and children who have ungrudgingly lent themselves to undergo this study without whom, this study would not have seen the light of the day.

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CERTIFICATE – II

This is to certify that the dissertation work titled “CLINICAL SPECTRUM OF INBORN ERRORS OF METABOLISM IN CHILDREN IN A TERTIARY CARE HOSPITAL” of the candidate Dr.JAYASHREE K.R with Registration Number: 201717004 for the award of M.D. PAEDIATRICS in the branch of VII. I personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contains from introduction to conclusion pages and the result shows 8% percentage of plagiarism in the dissertation.

Guide and Supervisor Sign with Seal

Guide and Supervisor Sign With Seal

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CONTENTS

SL.NO. TITLES PAGE.NO.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 49

3 STUDY JUSTIFICATION 53

4 AIM & OBJECTIVE OF THE STUDY 54

5 MATERIALS & METHODS 55

6 RESULTS 58

7 DISCUSSION 82

8 LIMITATIONS 85

9 CONCLUSION 86

10 BIBLIOGRAPHY 88

ANNEXURES

• Proforma

• Patient Information sheet

• Informed Consent form

• Patient Information Sheet (Tamil)

• Informed Consent Form (Tamil)

• Master Chart

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INTRODUCTION

Inborn errors of metabolism are the hereditary diseases which occurs due to disturbances in normal biochemical process. Although individual diseases are rare, they collectively cause significant amount of morbidity and mortality. The term INBORN ERRORS OF METABOLISM first being used by Sir ARCHIBALD GERROD in 1902. The disorders he initially described were Pentosuria, Alkaptonuria, Albinism and Cystinuria. After that in 1934, FOLLING described about Phenylketonuria. As IEM’s have been known for more than 100 years along with newer diagnostic tools and techniques now more than 500 IEM’s are uncovered.

Inborn errors of metabolism are group of disorders due to one or more defective or non functional enzymes or defect in transport of proteins. The defective enzyme disturbs the metabolic reaction that leads to deficiency of essential products for cell function, accumulation of substrates or toxic metabolites of alternative pathway. IEMs are congenital chemical alterations, also called as INBORN METABOLIC DISORDER (IMD).These are individually rare with average incidence of 1 in 100000.

But due to huge number of enzymatic derangements, they are collectively common with overall incidence of 1 in 800 to 1 in 2500. They are one of the major contributors to chronic diseases in childhood¹

Due to IEMs there will be

(a)Inadequate product as seen in Phenylketonuria-deficiency of tyrosine

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(b)There may be accumulation of substrates which may be harmful eg.Urea cycle disorder-ammonia which is toxic to brain

(c) Increased levels of alternative pathway products which might have toxic effects or interfere with other metabolic products eg. Formation of abnormal products in propionicaciduria because of accumulated propionylco A, partipating in reactions normally using acetyl coA.

Clinical manifestations depends on the concentrations of accumulated toxic metabolites. As a consequence of this, IEM may be exhibiting severe symtoms or may be asymptomatic. Frequently manifested symptoms which suggest that IEM may fall into several categories including hypoxia ,seizures, lethargy, vomiting, poor feeding and other changes that may often cause death if not promptly intervened.² Asymptomatic individuals become symptomatic following exposure to triggers such as fasting, diet, Fever, drugs, anaesthesia.

Usually early symptoms of IEM are non specific ,which makes it difficult to diagnose as they overlap with sepsis ,birth asphyxia, hepatitis ,viral infections, arthritis. Prompt diagnosis is required even in asymptomatic individuals to prevent sequlae. Proper diagnosis requires the use of biochemical markers, and the diagnosed cases may require lifelong therapy, so it imposes a substantial burden on the patients family which includes cost if diagnosis and treatment³.

One single test is not enough to identify the IEM. Different strategies are required to diagnose it. Accumulated biochemical substances can be estimated in blood or urine.

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Knowledge of analytical chemistry, enzymology and biochemistry helps to plan final work of IEM. To improve the establishment of diagnosis and management of long term effects ,Newborn screening(NBS) gas chromatography, mass spectrometry (GCMS)tandem mass spectrometry(TMS),molecular analysis and enzyme assay has been developed. With the above diagnostic techniques, the number of patients diagnosed with IEM are at a rise. Some of them are responsive to therapy if treatment stated early. Incase where the therapy is unavailable diagnostic methods will be useful for genetic and prenatal counselling. Asymptomatic newborn with IEM can later present with irreversible neurological damage ,hence NBS is widely accepted in developed countries. In contrast, less information is available from developing countries^4.

Though it is impossible for anyone to remember the possible symptoms or complexities of every disorder under the spectrum of IEM there lies a logic which forms a basic network or thread and by holding on to it, one can make a reasonable attempt to come to a diagnosis. The following basic characteristics helps in the diagnostic approach.

1.There are some disorders in which errors in the biochemical pathway will affect only one anatomical system or functional organ & symptoms are exclusive to that system.

2.In others, the biochemical pathway may affect many systems in the body, hence the presenting symptoms are variable, yet there could be an attempt at categorising the diverse signs or symptoms⁵.

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3.Biochemical abnormality may be a defect in intracellular trafficking

It can be explained by going through the basic biochemistry with the following illustration⁶.

A is a the precursor in the body which is to be converted to the product C through product B with one helping enzyme in the pathway the inadequacy or absence of that particular enzyme results in one of the following three things.

1. Absence of product C

2. Excess of substrate of A and B

A new pathway taken by product B to produce new products D and D1, which may be not in need for the body and may produce toxicity

CLASSIFICATION OF IEM

Due to the diversity of IEM is difficult to classify. Hence many classifications were proposed based on pathophysiology, accumulation of toxic products, affected organs and clinical symptoms.

A. Classification based on Pathophysiology

IEM occurs due to any deficiency or defective enzyme which involved in metabolic pathways, hence the classical IEM are the defects in enzymes of metabolism of aminoacids, carbohydrates, lipids, fatty acids or in mitochontrial energy metabolism. Based on this they are classified into

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Group1: which gives rise to intoxication

Group2: disorders involving energy metabolism Group 3: disorders involving complex molecules

Group1: This is due to block in the metabolism which causes accumulation of toxic metabolites. The disorders included in this group are aminoacidopathies such as PKU, homocystinuria etc., organic aciduria like glutaric aciduria, isovaleric aciduria etc., urea cycle disorders, metal intoxication such as Menkes Wilson’s, hemochromatosis etc. Usually these group of disorders will not affect embryonic and fetal development. The clinical presentation may be precipitated by infection, fever, food intake.

Group2 : These group includes symptoms due to deficiency in energy production or utilisation within liver, muscle, brain ,myocardium and other tissues. The disorders in this group includes congenital lactic academia, mitochondrial respiratory chain disorders ,fatty acid oxidation defects disorders of glycolysis, Glycogen metabolism, gluconeogenesis, hyperinsulinism.

Disorders of creatine and new inborn errors of Pentose phosphate pathway.

Group3:It involves disorders that interfere with metabolism of complex molecules, like Lysosomal storage disorder, peroxisomal disorder ,disorders of intracellular trafficking and processing such asalpha1-anti trypsin, congenital disorders of glycosylation(CDG)and Inborn errors of cholesterol synthesis.

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B. Clinical classification of IEM⁷. Category 1: Involving a functional system

Category 2: Involving metabolic pathways common to great number of cells or organs

Group1: defects in the synthesis or catabolism of complex molecules Group2: Defects in intermediary metabolism

Group3: Defects in energy production or utilisation

CATEGORY 1: This includes disease affect a functional or anatomical system or organs, such as the endocrine system, immune system, coagulation factors or lipoproteins. The symptoms are uniform and easy because the basis of biochemical defects incorporates the given sequence, e.g: tendency to bleed seen in cases of coagulation defects.

CATEGORY 2: Diseases in this includes the diseases in which the biochemical basis affects one metabolic pathway common to great numbers of cells or organs, such as storage disorders due to lysosomal disorders or is restricted to one organ with humeral and systemic consequences, such as hypoglycaemia in hepatic glycogenesis, hyperammonemia in urea cycle defects. These diseases can be divided into three groups from clinicopathological view, greatly assisting in diagnostic purposes.

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Clinical classification of IEM-GROUP 1:

LYSOSOMAL DISORDERS Mucopolysaccharidosis(MPS)

• I-H- Hurlers

• I-HS-Herler-Scheie

• I-S-Scheie

• II-Hunter

• III-SanFlippo

• IV-Morquio

• VI-Maroteaux-lamy

• VII-Sly

Sphingolipidosis

• GM1 gangliosidosis

• Tay -sachs

• Fabry disease

• Shindler disease Lactosylceramidosis

• Gauchers disease

• Farber disease

• Nieman Pick disease

• Krabbe disease

• Metachromatic leukodystrophy

• Multiple sulphatase deficiency Mucolipidoses

Glycoproteinoses

• Fucosidosis

• Mannosidosis

• Sialidosis

• Aspartylglucosaminuria Others

• Canavan disease

• Pompe disease

• Acid lipase deficiency

Disturbance of membrane transport

• Sialic acid storage disease

• Salla disease

• Cystinosis

Peroxisomal biogenesis disorders

• Zellweger syndrome

• Adrenoleucodystrophy

• Refsum disease

• Hyperoxaluria Type -1

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Clinical classification of IEM-GROUP 2:

Disorders of aminoacids

• Phenylketonuria

• Tyrosenemia

• Cystinuria

• Homocystinuria

• Non ketotichyperglycenemia

• Maple syrup urine disease

Urea cycle defects

• Carbamoyl phosphate synthetase deficiency

• Ornithine transcarbamylase deficiency

• Citrullinemia

• Arginosuccinicaciduria

• Argininemia

• Lysinuric protein intolerance Organic acidurias

• Glutaric academia Type1

• Isovaleric academia

• Propionic academia

• Methyl melanoic academia

• Multiple carboxylase deficiency

• 3-methyl crotonyl-coA carboxylase deficiency

• 3-methylglutaconic academia

• 3-hydroxy 3-methylglutaric academia

Carbohydrate intolerance

• Classical galactosemia

• Galactokinase deficiency

• Epimerase deficiency

• Hereditary fructose intolerance

• Hereditary fructose 1,6 biphosphatase deficiency

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Clinical Classification of IEM – Group 3:

Fatty acid oxidation defects

• Medium chain acyl coA dehydrogenase deficiency

• Long chain acyl coA dehydrogenase deficiency

• Short chain acyl coA dehydrogenase deficiency

• Long chain 3-OH acyl coA dehydrogenase deficiency

• Multiple acyl coA dehydrogenase deficiency(Glutaric academia type2)

• Carnitine plasma transport defect

• Carnitine palmityltransferase deficiency

Mitochondrial disorders

• Pyruvate dehydrogenase complex deficiency

• Oxidative-phosphorylation defects (MERRF) (MELAS)

• Phosphoenolpyruvate carboxylase deficiency

• Pyruvate carboxylase deficiency

Glycogen storage disorders

• Hepatic forms type 0,I,III,IV,VIII,IX,X

• Muscle forms type II,V,VII

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Classification Based on size of molecule:

Small molecule:

• Amioiacidurias, Urea cycle defects, Organic acidemias, Fatty acid oxidation defects

• Disorders of intermediary metabolism Large molecules:

• Storage disorders: Mucopolysaccaridoses, Glycogen storage disorders

• Accumulation of proteins, Carbohydrate, Lipids etc Classification based on metabolism of macromolecules:

Disorders of protein metabolism

• Aminoaciduria

• Organic aciduria

• Urea cycle disorder

Disorders of carbohydrate metabolism

• Carbohydrate intolerance disorders

• Glycogen storage disorders

• Disorders of Gluconeogenesis

• Glycogenolysis Lysosomal storage disorders:

• Gaucher’s disease

• Nienann-Pick disease

Disorders of lipid metabolism

• Fatty acid oxidation defects

• Spingolipidoses Mitochondrial disorders:

• Kearns-Sayre syndrome

Peroxisomal disorders:

• Zellweger syndrome

• Adreno leukodystrophy Trace metal disorders:

• Menke,s Kinky hair disease

• Williams disease

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CLINICAL EVALUATION AND DIAGNOSTIC APPROACH TO IEM

IEM is a condition which is rapidly progressive and can cause irreversible damage. Early diagnosis is important to prevent mental retardation, severe neurological damage, physical disability and infant mortality. Diognosis leads to initiation of proper treatment if available, antenatal diagnosis and genetic councelling in subsequent pregnancies. As the symptoms of IEM are nonspecific a strategy is required for its evaluation which includes careful study of history, clinical examination and relevant investigations.

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Approach to a case with a suspected metabolic disorder⁸

SUSPECTED METABOLIC DISORDER

(Poor feeding, persistent vomiting, seizures, floppiness, encephalopathy)

PLASMA AMMONIA

HIGH

NORMAL

BLOOD PH, HC03 AND ANION GAP

NO ACIDOSIS ACIDOSIS

NO ACIDOSIS OR KETOSIS

UREA CYCLE DEFECT

HIGH LACTATE HYPOGLYCEMIA

± KETOSIS

HIGH LACTATE NORMOGLYCEMIA

KETOSIS

NORMAL LACTATE NORMO- OR

HYPOGLYCEMIA KETOSIS

ORGANIC ACIDURIA FATTY ACID

OXIDATION DEFECT GSD TYPE-I

HEREDIARY FRUCTOSE INTOLERANCE

PYRUVATE CARBOXYLASE DEFICIENCY

MULTIPLE CARBOXYLASE DEFICIENCY

RESPIRATORY CHAIN OR MITOCHONDRIAL

DEFECTS

MSUD

SHORT CHAIN ACYL CoA

DEHYDROGENASE DEFICIENCY AMINOACIDOPATHIES

NONKETOTIC HYPERGLYCINEMIA GALACTOSEMIA

PEROXISOMAL DISORDERS

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The following steps are involved in the evaluation if IEM

• History

• Clinical symptoms

• General and systemic examination

• Base line investigations

• Advanced screening

• Confirmatory diagnosis

HISTORY:

The diagnosis of IEM relies on detailed reliable history and careful clinical examination. Parents will come with history and symptoms which have been noticed as unusual. It leads the clinician to advice subsequent investigations and special interventions. The important steps in history includes

• Family history

• Prenatal history

• Postnatal history

FAMILY HISTORY:

The detailed evaluation of IEM starts with family history. A positive family history of affected siblings or early death of siblings, neonatal death, consanguinity, miscarriages, siling with developmental delay, psychiatric diagnosis, congenital malformations increases the chances of presences of IEM.

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Treatment is considered before the appearance of clinical symptoms if investigations are suggestive of IEM⁹.

Consanguinity:

Early mortality is increased in consanguineous product than among children born to unrelated parents¹⁰. Most IEM are inherited as autosomal recessive manner and rate of IEM more in children born to consanguineous parents. In X – linked disorders even the affected relative’s history also gives important clues. Increased incidence of inherited disorders is seen in countries where consanguinity is high.

It is higher in South Europe, Latin America, and Japan. In India occurrence of consanguineous marriage is relatively frequent and rate of inbreeding is higher in South compared to North India where consanguinity is one of the risk factors in IEM¹¹.

PRENATAL HISTORY

It includes perinatal insults, history of gestational diabetes mellitus (GDM), pregnancy induced hypertension (PIH), abnormal scan, intrauterine growth restrictions (IUGR). In general, newbornwith IEM is normal during pregnancy and delivery, as the toxic metabolites accumulated are cleared through placenta and metabolised by mother. In some cases, history of complications during prenatal period also gives clues such as acute fatty liver of pregnancy (AFLP), HELLP Syndrome suggest that the condition may be due to disorders of fatty acid

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metabolism in the baby¹². Disorder that interfere with energy metabolism may impair fetal organ development.

POSTNATAL HISTORY

As toxic metabolites cleared by the maternal placenta most infants are born at full term with good weight. Cry at birth, birth weight, term delivery, suckling at breast are the important clues which guides to IEM¹³.

Age at presentation:

Clinical symptoms of different IEMs appear at different stages of growth of the child. They posses a symptom free period immediately after birth. But they become lethargy with poor feeding and weak cry when feeding introduced.

• Disorders of intoxication group: This includes aminoacidopathies, organic acidurias, urea cycle defects. Mostly they will develop progressive symptoms between 2^nd and 5^th days of life. The subsequent risk period includes 6 to 8 months. Hereditary fructose intolerance will manifest after introducing fruits and galactosemia after exposure to milk.

• Disorders of energy metabolism: These disorders usually symptomatic at birth. Depending on the severity of genetic defect and the organ involved, they may present at any age of life. Mitochondrial disorders and long chain fatty acid defects are most common in this group.

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• Disorders of metabolism of complex molecules: there are no precipitating factors for this group.symptoms usually present with variable and progressive organ dysfunction throughout all ages.

Symptoms and signs

Symptoms and signs are myriad. Hence when and how to suspect IEM' always lies in the hands of the primary physician and it encompasses all that is said initially starting with conception. So suspect IEM when there are¹⁴

• Coarse features, dysmorphism.

• Unexplained odor (abnormal urine odor can be detected on a dry filter paper or by suddenly opening the lid of the container of stored urine at room temperature).

•••• Isovaleric aciduria - Sweaty feet

•••• Maple syrup urine disease - Maple syrup

•••• Phenyl ketonuria - Musty

•••• Tyrosinemia - Cabbage

•••• Glutaric aciduria type II - Sweaty feet

•••• Multiple carboxylase deficiency - Cat urine

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• Persistent hiccoughs, change in tone, convulsions (hiccoughs when the child is well is not abnormal)

• Persistent tachypnoea, respiratory distress, apnea

• Poor feeding, vomiting, diarrhea and dehydration

• Unexplained shock

• Acute Reye like syndrome

• IEM mimic sepsis in the new born. ( certain IEMs are associated with risk of sepsis, eg. galactosemia, CAH, organic acidemias)

• Cholestatic jaundice, (not biliary)

• Unexplained cardiomegaly with decompensation

• Abnormal visceromegaly

• Temperature instability

• Persistent lethargy

• Abnormal neurological signs

• Regression of attained milestones

• Feeding problems

• Unexplained clinical deterioration following a period of normalcy

• Family history of fetal wastages, hydrops, severe IUGR, neonatal deaths and

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SIDS - not only for that family but for uncles, aunts both paternal and maternal and cousins. Negative family history does not rule out IEM.

• Abnormal acute laboratory abnormalities (biochemical and hematological) e.g.:

persistanthypoglycemia, persistant acidosis, abnormal hepatic and renal parameters, hyperammonemia, pancytopenia, thrombocytopenia, abnormal coagulation profile.

PRECIPITATING FACTORS:

When child presenting with signs and symptoms, what exactly did provoke the symptoms in the child who was well before that event.

Commonly Symptoms were trigger by:

Weaning

Fructose intolerance, Fructose diphosphatase deficiency, Urea cycle defects, Lysinuric protein intolerance, Triple-H syndrome, Maple syrup urine disease, Organic acidurias.

Fructose

Fructose intolerance, Fructose diphosphatase deficiency.

Galactose

Galactosemia

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Glycerol

Glycerol intolerance Protein

Urea cycle defects, Lysinuric protein intolerance, Triple H syndrome, Maple syrup urine disease, Organic acidurias, Hyperinsulinism (with hyperammonemia) Carbohydrate

Pyruvate dehydrogenase deficiency, Respiratory chain disorders, Hyperinsulinism

Catabolic circumstances Aminoacidopathies Infection

Organic acidurias Fever, fasting

Fatty acid oxidation disorders, Urea cycle defects, Gluconeogensis defects, Glycogenosis defects.

Anesthesia

Thromboembolic accident in homocystinuria

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Drugs

Porphyria, Glucose-6-phosphate dehydrogenase deficiency CLINICL EXAMINATION:

Detailed clinical examination to be needed for the proper diagnostic approach and plan for stepwise investigation work.

HEAD TO FOOT EXAMINATION

• Kinky steely hair in Menkes disease

• Seborrheic dermatitis in Biotidinase deficiency

• Fair skin, blond hair in Phenyl ketonuria

• Peau d’ orange appearance in Carbohydrate deficient glycoprotein syndrome

• Subcutaneous nodules over joints in Farber’s syndrome

• Angiokeratoma in Fabry’s disease

• Dysmorphic facies: Doll like facies in Glycogen storage disorder Coarse facies in MPS and storage disorders

• Microcephaly will be seen Krabbe’s disease, Phenyl ketonuria, Leukodystrophy, neuronal ceroid lipofuschinosis

• Macrocephaly will be seen in Tay sach’ s disease, Canavan disease, Alexander disease, Metachromatic leukodystrophy, Glutaric aciduria.

• Short stature will be associated with Hurler’s disease ,mucolipidosis, and storage disorders.

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• Ocular examination:

1) Cataract - Galactosemia, Zellweger syndrome 2) Corneal clouding - MPS, GM1 gangliosidosis 3) Lens dislocation - Sulfite oxidase deficiency

4) Cherry red spot – Niemann-Pick disease, GM1 Gangliosidosis, Sandhoff ‘s disease, sialidosis

5) Retinal pigmentary changes: Mitochondropathy, Congenital glycosylation disorders.

6) Ocular movement disorders- vertical gaze palsy in Niemann pick type C, Gaucher’s disease.

7) Vision loss – MELAS, X- Linked adrenoleukodystrophy 8) Optic atrophy- Krabbes, leukodystrophies.

• Bone and joint involvement: Dysostosis multiplex in mucopolysacchradosis, Tendon Xanthoma in bile acid synthesis defect.

• Abnormal posturing: Frog like position in Pompe’s disease, Dystonia in Phenyl ketonuria and Niemann – Pick, glutaric aciduria.

SYSTEMIC EXAMINATION:

GI: Isolated hepatomegaly, splenomegaly, hepatosplenomegaly in glycogen and lysosomal storage disorders, mitochondrial disorders, Fatty acid oxidation defects.

CVS: Hypertrophic obstructive cardiomyopathy in Pompe’s disease. Dilated cardiomyopathy also be there in IEM.

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CNS: 1) Higher function examination for metabolic encephalopathy.

2) Motor system examination for Hypotonia, hypertonia, ataxia, spasticity, to differentiate pyramidal and extra pyramidal involvement.

3)Peripheral nervous system examination 4) Cerebellar function examination

RS: To look for chest wall deformity, pattern of breathing.

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FLOW CHART FOR EVALUATION OF METABOLIC ACIDOSIS

METABOLIC ACIDOSIS WITH INCREASED ANION GAP

NORMAL LACTATE ELEVATED LACTATE

ABNORMAL ORGANIC ACID

ORGANIC ACIDEMIA ABNORMAL ORGANIC ACID NORMAL ORGANIC ACID

DICARBOXYLIC ACIDURIA

NORMAL OR LOW PYRUVATE

ELEVATED L-P RATIO ELEVATED

PYRUVATE

NORMAL L-P RATIO FATTY ACID OXIDATION

DEFECTS

METHYLMELONIC ACIDEMIA

PROPIONIC ACIDEMIA MULTIPLE

CARBOXYLASE DEFICENCY, OTHERS

RESPIRATORY CHAIN DEFECTS

PYRUVATE CARBOXYLASE DEFICENCY

NO HYPOGLYCEMIA HYPOGLYCEMIA

GSD TYPE-I

FRACTOSE 1,6DP DEFICENCY PEP CARBOXYLASE

DEFICENCY

PYRUVATE DEHYDROGENASE DEFICENCY

PYRUVATE CARBOXYLASE DEFICENCY

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DISORDERS OF CARBOHYDRATE METABOLISM:

This includes defects in glycogen metabolism, defects in gluconeogenesis which includes galactose and fructose metabolism, defects in pyruvate metabolism and defects in pentose metabolism.

• Defects in Glycogen metabolism causes accumulation of glycogen in the tissues called as GLYCOGEN STORAGE DISORDERS (GSD) which further divided to LIVER GLYCOGENOSIS and MUSCLE GLYCOGENOSIS according to the site of accumulation.

• Defects in the Gluconeogenesis or glycolytic pathway includes disorders of galactose, fructose, gluconeogenesis. In this there is no accumulation of glycogen in tissues.

• Defects in Pyruvate metabolism in the pathway of conversion of pyruvate to carbon monoxide and water via mitochondrial oxidative phosphorylation are mostly associated with lactic acidosis and some tissue glycogen deposition.

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GLYCOGEN STORAGE DISORDERS:

LIVER GLYCOGENOSES¹⁵: TYPE 1a / VON

GIERKE

GLUCOSE -6- PHOSPHATASE DEFICENCY

Common form. Severe hypoglycaemia,

hepatomegaly, short stature, gout.

TYPE 1b Glucose -6- phosphate translocase deficiency

Same as type 1a along with neutropenia and impaired neutrophil function. 10% of 1a.

Type III a / CORI or FORBE

Liver & Muscle Debranching enzyme

Hypoglycaemia, hepatomegaly, muscle weakness, liver failure.

TYPE III b Liver debranching enzyme defect with normal muscle function

Same as type III a with no muscle weakness.

15% of type III.

TYPE IV/ ANDERSON Branching enzyme deficiency

Failure to thrive, hypotonia, hepatomegaly,

progressive liver failure.

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TYPE VI / HERS Liver phosphorylase deficiency

Hepatomegaly, typically mild hypoglycaemia, ketosis.

Fanconi - Bickel GLUT 2 Failure to thrive, rickets, proximal renal tubule dysfunction,

hepatorenomegaly.

MUSCLE GLYCOGENOSES:

TYPE II / Pompe Acid α glucosidase Cardiomegaly, HOCM, hypotonia,

hepatomegaly, death by 1 to 2 years.

Type V / McArdle Muscle phosphorylase Exercise intolerance, muscle cramps, increased fatigability Type VII/ Tarui Phosphor fructokinase Exercise intolerance,

muscle cramps, hemolytic anaemia, myoglobinuria.

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PRKAG2 deficiency AMP activated protein kinase γ

Hypertrophic obstructive cardiomyopathy

(HOCM).

Phosphoglycerate kinase deficiency

Rare, x linked. As with type V

Phosphoglycerate mutase deficiency

Rare, in Africa and America

As with type V

Lactate dehydrogenase deficiency

Rare As with type V

DISORDERS IN PENTOSE METABOLISM:

Pentosuria L-xylulose reductase deficiency

Liver failure

Transaldolase deficiency Liver cirrhosis and failure,cardiomypathy Ribose-5 phosphate

isomerase deficiency

Progressive

leukoencephalopathy, peripheralneuropathy

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DISORDERS OF GALACTOSE METABOLISM:

Type 1 Galactose -1- phosphate

uridyltransferase deficiency

Failure to thrive, cataracts, vomiting, hepatomegaly, amino aciduria.

Type 2 Galactokinase deficiency Benign form, only cataract.

Type 3 Uridine diphosphate

galactose -4- epimerase

Similar to type 1 with hypotonia and nerve deafness.

DISORDERS OF FRUCTOSE METABOLISM:

Essential fructosuria Fructokinase deficiency Vomiting, sweating, lethargy, failure to thrive.

Hereditary fructose intolerence

Fructose -1- phosphate aldolase deficiency

Failure to failure, hepatic failure, good prognosis with fructose restriction.

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DISORDERS OF GLUCONEOGENESIS:

Fructose -1, 6 – diphosphatase deficiency

Episodic

hypoglycaemia, apnoea, acidosis.

Good prognosis, avoid fasting

PEP Carboxy kinase deficiency

Hypoglycaemia, hepatomegaly, hypotonia, failure to thrive

Rare disorder

DISORDERS OF PYRUVATE METABOLISM:

Pyruvate dehydrogenase complex defect

Severe lactic acidosis, psychomotor retardation, failure to thrive

X linked defect

Pyruvate carboxylase deficiency

Same as above Rare, autosomal dominant

Respiratory chain defect

Complex I – V defects,

multisystem involvement

Mitochondrial inheritance.

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MUCOPOLYSACCHARIDOSIS:

These are hereditary progressive diseases caused by mutation of genes, coding for lysosomal enzymes needed to degrade glycosoaminoglycans.

MPS type Enzyme defect Clinical features Inheritance I-H

Hurler

α -L-iduronidase Mental retardation, corneal clouding, death≤14 yrs

AR

I-S Scheie

α -L-iduronidase Joint stiffness, corneal clouding, normal IQ, survive to adulthood

AR

II Hunter

Iduronatesulfate sulfatase

Clear cornea,

milder form survive to aduldhood

XLR

III San Filippo

Heparan-s- sulfamidase

Behavioural problems, sleep disorders, progressive dementia, Clear cornea

AR

IV Morquio

N-acetyl

galactosamine 6- sulfatase

Short trunk dwarfism,

Corneal opacity, Bone dysplasia

AR

VI Maroteaux- Lamy

N-acetyl

galactosamine 4- sulfatase

similar to Hurler with normal intelligence

AR

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VII Sly

β- glucuronidase Varying from

fetalhydrops to mild dysmorphism

AR

IX Hyaluronidase deficiency

Periarticular mass AR

DISORDERS OF LIPID METABOLISM:

These group of disorders includes,

• Disorders of mitochondrial fatty acid oxidation defect

• Disorders of very long chain fatty acids

• Disorders of lipoprotein metabolism and transport

• Lipidoses (LSD)

• Mucolipidoses

Manifestation I-H I-S II III IV VI VII

Mental deficiency + - ± + - - ±

Coarse facies + (+) + - + ±

Corneal clouding + + - (+)

visceromegaly + (+) + (+) - + +

Short stature + (+) + - + + +

Joint contractures + + + - - + +

Dysostosis multiplex + (+) + (+) + + +

Leucocyte inclusions + (+) + + - + +

Mucopolysacchariduria + + + + +

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LIPIDOSES OR LYSOSOMAL STORAGE DISORDERS:

In disorders of lipid metabolism lysosomal storage disorders are more commonly diagnosed. Lysosomal storage disorders are diverse disorders each caused by specific hydrolase deficiency leading to intra lysosomal accumulation. Progressive lysosomal accumulation of glycosphingolipids in visceral organs leads to organomegaly, in central nervous system leads to neuro degeneration, in other organs leads to skeletal abnormalities ,pulmonary infiltration and other manifestations.It is classified as follows,

• GM 1 gangliosidosis

• GM 2 gangliosidosis(Tay sachs ,sandhoff disease)

• Gauchers disease

• Niemann-Pick disease

• Farber’s disease

• Fabry disease

• Krabbe disease

• Metachromatic leukodystrophy

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CLINICAL FEATURES OF COMMON LYSOSOMAL STORAGE DISORDERS:

CLINICAL FEATURES

GANGLIOSIDOSIS GM1

GAUCHER NIEMANN- PICK

TAY SACH’S

SANDOFF KRABBE MLD NCL

CHERRY RED SPOT

+ - + + + - ± +

VICEROMEGALY + + + - + - - -

SKELETAL CHANGES

+ + - - + - - -

DEVELOPMENTAL DELAY

+ + + + + + + +

BULBAR SIGNS - + - +/- +/- - - -

PROTEIN METABOLISM COMMOM AMINOACIDOPATIES:

DISORDER DEFECIENCY

CLINICAL FEATURE

DIAGNOSIS &

TREATMENT

Phenylketonuria Phenyl alanine hydroxylase

Profound intellectual disability,

microcephaly, musty odour, blond hair.

Plasma phenyl alanine >

1000µmol/L, enzyme assay, genetic assay.

Restrict

phenylalanine in diet with

tetrahydrobiopterin

34 Maple syrup urine

disease (MSUD)

Branched chain ketoacid

dehydrogenase complex

Poor feeding, encephalopathy, hypertonia, maple syrup urine odour, seizure,

developmental delay.

DNPH ketonuria, elevated leucine, isoleucine, valine, genetic testing. Diet restriction, thiamine.

Tyrosinemia type1 Fumaryl acetoacetate hydroxylase

Vomiting, hypoglycaemia, hepatomegaly, liver failure, change in mental status, peripheral neuropathy.

Elevated AFP, high tyrosine, methionine, phenyl alanine, high succinyl acetone in blood, urine

Homocystinuria Cystathionine β synthase

Developmental delay, seizure,

psychiatric problems, extra pyramidal signs, ectopia lentis, osteoporosis

Urine nitroprusside test, high plasma methionine, homocysteine, genetic testing.

Methionine

restriction, oral B6, B12, Vit – C

35 Alkaptonuria Homo gentisate

dioxygenase

Urine turns to black on standing, staining of diaper, grey sclera, ear, nose

GCMS can identify, vitamin C Prevents Ochronosis.

ORGANIC ACIDEMIAS:

• Autosomal recessive disorders

• Excretion of non-amino organic acids in urine.

• Deficiency of specific enzyme in pathways of amino acid degradation such as branched chain amino acids, tyrosine, homocysteine, methionine, threonine, lysine, tryptophan.

• Insidious in onset, metabolic encephalopathy precipitated by precipitated by fever, fasting or infection.

• Multiple carboxylase deficiency, and biotidinase deficiency have additional hair and skin abnormalities

• Diagnosis by abnormal basic metabolic screening, abnormal liver function test and neutropenia, abnormal plasma acyl carnitine profile.

• Confirmatory by genetic testing

• Treatment by adjuvant therapy with vitamins.

Eg: Methyl melonic acidemia by vit B-12, L- Carnitine Isovaleric acidemia by L-Carnitine & Glycine

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PHENYLKETONURIA

Phenylketonuria (PKU) is an inborn error of protein metabolism caused predominately by mutations that result from an impaired ability to metabolise the essential amino acid Penylalanine.PKU is an autosomal recessive inherited metabolic disease in which mtations in phenylalanine hydroxylase( PAH) gene or the gene coding for its cofactor,tetrahydrobioptrin (BH4) results in decreased catabolic pathway of phenylalanine.The lack of PAH or its cofactor BH4 which results in the accumulation of excess phenylalanine,that impairs intellectual abilities if untreated.The classical PAH deficiency is considered when the serum concentration of unchanged phenylalanine crosses the level of 1200µmol/L.The phenylalanine concentration in the range of 600-1200µmol/L is diagnosed as mild PKU,and values below 600µmol/L are classified as hyperphenylalaninemia (HPA).When the treatment program for the patient with PKU did not start at the early weeks of neonatal period developmental delay,mental retardation ,and microcephaly are caused by the accumulation of toxic byproducts of phenylalanine within its metabolic pathway.

Dr.Folling had disovered that phenyl pyruvic acid which is responsible for the disturbance in the metabolism of the amnoacid during 1934 called phenylalanine which was subsequently named as “phenylketonuria” later developed by Penrose a Quastel.Jervis in 1953 identified the metabolic error and Bickel,et al.in 1954 reported the success of low phenylalanine diet therapy.The incidence of PKU or HPA is highest among Cuacasians ,occurring in approximately 1 in 10,000 births.The

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prevalence of PKU varies by country ranging from between 1 in 10,000 and 1 in 20,000 births in USA and Europe .The error in the genetic information affects the metabolism of the protein module phenyl alanine ,an essential amino acid ,which is normally ion the liver turned into aminoacidtyrosine. In the process of biosynthesis the “wrong” aminoacid is assembled ,as a result of mutation .This leads to a missing or lacking activity of enzyme which turns phnylalanine into tyrosine. Thesulplus of phenylalanine accumulates in the blood and tissues and causes a brain damage .

The natural history of PKU in a progressive irreversible neurological impairment during infancy and childhood .The most common outcome is severe metal retardation often associated with a “mousy” odor, eczema and reduced hair skin and iris pigmentation; also reduced growth, microcepaly and neurological signs as tremor, epilepsy are present.

All untreated patients have behavioural problems as hyperactivity, sterotypy, and anxiety. The severity of the clinical phenotype directly correlates with blood phenylalanine levels that reflect the degree of enzymatic deficiency. The urine samples are tested for substances ,including ketones. When ketones are present urine usually develops a red-brown colour upon the addition of ferric chloride .

The PKU suggests that the metabolic abnormalities could cause the neurological effects and also shows the importance to treat the abnormalities and that would lead to positive clinical outcomes. The development of Guthrie’s screening test, and dietary treatment led to the prevention of intellectual impairment in affected

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children throughout the world. Furthermore, the PKU model has since been used as a template to shed light on over 200 other inborn errors of metabolism.

Patients with PKU should still be treated with diet therapy ,but in long term the introduction of wide array of new treatment approaches such as more palatable foods based on the use of GMP products or the administration of LNAA or BH4 could decrease the need for phenylalanine restriction in the diet. Patients surveys shows that GMP foods have improved taste and are preferred a standard formula. The foundation of PKU treatment is a low phenylalanine diet which, by reducing or normalizing phenylalanine concentrations, prevents the development of the neurological and psychological changes. Since neurological changes have been demonstrated within one month of birth, it is recommended that dietary restriction should be started early and to be continued through childhood when neural development is maximal.

After proper history, clinical symptoms, precipitating events, detailed clinical examination will guide for further evaluation with proper investigations. A step wise investigation approach to be needed for correct way to make a diagnosis. Physician should be judicious to choose in proper manner.

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BASIC METABOLIC TESTS:

Some tests performed at the bedside or quickly in a routine lab. These tests are not diagnostic by themselves but helpful in screening for many IEMs.

• Urine analysis:

As the excess pathological metabolites are excreted in urine, the urine is the excellent source of crucial metabolites. Urine should be examined for following parameters before subjecting into advanced testing.

A) Colour: colour of the urine will give clues to diagnosis of some disease in IEM. Interpretation of colour of urine as follows:

1) Dark brown or black - alkaptonuria, hemoglobinuria, myoglobinuria

2) Red - Hematuria, porphyria, red dyes, 3) Blue - Hartnup disease

4) Brown or blue - Alkaptonuria B) Odur of urine:

Odur of urine will be specific for some diseases in IEM, which are already discussed in clinical symptoms and sings.

C) Urine for metabolic screening (UMS):

1) Ferric chloride test for phenyl ketonuria

2) Reducing substances for galactosemia, fructose intolerance

3) DNPH Test ( Dinitrophenyl hydrazine ) for -oxo acids lactic acidosis, methionine malabsorption, MSUD.

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4) Nitroprusside test for sulphur containing amino acids like Cystinuria, Homocystinuria, hyperargininemia

5) Cetrimide test for Mucopolysacchradosis D) Urinary Ph

E) Urine specific gravity

• BLOOD TESTS:

1) CBC: Anaemia, thrombocytopenia, leucopenia, or leucocytosis is noted in organic acidurias. Mostly sepsis is the common presentation in IEM.

It will usually be abnormal.

2) Serum blood glucose: It is the useful indicator for glycogen storage disorders and carbohydrate metabolism. Hypoglycemia will be presenting symptom with most if IEM.

3) Serum ammonia: Metabolism of ammonia involves gut, muscle, liver, kidney and ammonia. Ammonia produced in our body by any mechanism is toxic to brain. IEM which causes severe hyperammonemia are either defects in enzyme involved in urea cycle, defects in its substrate carrier or secondary disruption of urea cycle or abnormal liver function. Secondary disruptions of urea cycle, defects in its substrate carrier or secondary disruption of urea cycle or abnormality of liver function. Secondary disruptions which causes hyperammonemia are organic acidurias, fatty acid oxidation defects mitochondrial disorders, pyruvate carboxylase deficiency and transient hyperammonemia.

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4) ABG: Normal blood pH is in narrow range i.e 7.35to 7.45. If it is less than 7.35 it is called as acidosis. Among IEMs Methyl melonic aciduria, Propionic acid urea, Isovaleric academia are organic acidemias characteristically associated with metabolic acidosis infancy. It also associated with defects in pyruvate metabolism, respiratory chain disorders, disorders of gluconeogenesis, glycogen synthesis.

5) LACTATE: Lactate is the metabolic end of products of anaerobic glycolysis. It is the main energy source for heart, kidney, and muscle. It is the non- carbohydrate precursor for gluconeogenesis during fasting.

Normal level of blood lactate is < 2 mmol/L. Elevate level known as lactic acidosis can be seen in inherited metabolic disorders such as mitochondrial disorders, pyruvate carboxylase deficiency, PDH deficiency, ischemic conditions or in thiamine deficiency. Increased level of lactate may give a clue to increased rate of anaerobic metabolism or impaired aerobic metabolism.

6) RENAL FUNCTION TESTS: Usually renal function will be altered in IEM, which is due to secondary manifestations of the diseases. As progressive vomiting, poor feeding, hyperammonemia, metabolic acidosis are inter related with renal function it will lead to abnormal renal function tests.

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FLOW CHART FOR EVELUATION OF HYPERAMMONEMIA¹⁶

NEONATALHYPERAMMONEMIA

Symptoms in 1^st 24h Symptoms after age 24h

Preterm Full Term Acidosis No acidosis

Transient

Hyperammonemia of Newborn

INBORN ERROR OF METABOLISM

(i.e., organic acidemia or PC deficiency)

ORGANIC ACIDEMIAS

UREA CYCLE DEFECTS

PLASMA AMINO ACIDS

Citrulline markedly Elevated, no ASA Citrulline moderately

elevated, ASA present Absent Citrulline

Urine Orotic Acid

Low Elevated

CPS deficiency

OTC deficiency

Arginosuccinic aciduria Citrullinemia

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7) LIVER FUNCTION TEST: The IEMs with liver involvement is exhaustive. It is ranging from small molecule disorders such as urea cycle, amino acids to mitochondrial encephalopathies and large molecule disorders such as lysosomal disorders and peroxisomaldisoeders.

8) SERUM URIC ACID: High levels of uric acid suggest disorders of carbohydrate metabolism such as glycogenolysis or gluconeogenesis.

A low level may give clue to xanthine / hypoxanthine disorders which involves purine and pyrimidine metabolism and molybdenum cofactor deficiency.

ADVANCED SCREENING TESTS:

Based on the availability of lab, patient affordability, accuracy of results, availability of treatment modalities after diagnosis all are need to be considered before planning and subjecting to advance screening tests. This screening also useful to guide for next pregnancy, chance of recurrence, sex predilection, available prenatal diagnostic screening, new born screening and genetic councelling .

These are as follows:

• LIVER BIOPSY

• BONE MARROW EXAMINATION

• ENZYME ASSAY FROM TISSUES

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1) LIVER BIOPSY: It is usually indicated in suspected cases of GSD. Typical liver biopsy of GSD shows massive glycogen accumulation which includes glycogen within the nucleus of hepatocytes. There is marked accumulation of macro vesicular fat, but no fibrosis typically, evidence of biliary obstruction or inflammation. Enzyme assay also can be done from liver biopsy specimen.

• GSD Type I: Higher amounts of normal glycogen, and fatty infiltration seen. There may be distention of hepatocytes with glycogen and lipids, fibrosis and cirrhosis do not occur.

• GSD Type III: Generalised distention of hepatocytes by glycogen and minimal periportal fibrosis and micronodularcirrhosis.

• GSD Type IV: PAS positive hepatocytes, foamy histiocytes in reticuloendothelial system, diastase resistant inclusions, and interstitial fibrosis will be there

• GSD Type VI: Distended hepatocytes in a rosette form noted which is less compact than type I and III.

2) BONE MARROW EXAMINATION: This is useful in lysosomal storage disorders. Morphologically two typical cells types identified in bone marrow examination. They are:

• GAUCHER’S CELLS: These are large cells with 20 to 100µm diameter and a characteristic cytoplasm with wrinkled-paper appearance resulting from intracytoplasmic substrate deposition.

• NIEMANN-PICK CELLS: These cells are mostly lipid-laden foam cells, which have abundant cytoplasm with

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numerous vacuoles in cytoplasm.

3) ENZYME ASSAY FROM TISSUES: Testing for activity of an enzyme requires simulating the actual enzymatic reaction outside of body using appropriate body fluids or tissues. This can be done from cultured skin fibroblasts, leucocytes, plasma/serum, and liver biopsy specimens¹⁷.

• LYSOSOMAL STORAGE DISORDERS: It includes more than 24 enzymes. Usual requirements for testing is 4 to 5 ml blood in heparin vial. Lysosomal enzymes can also be measured on a dried blood spot.

• GALACTOSEMIA: Galactose-6-phosphate uridyltransferase (GALT), Galactokinase(GALK) and Galactose- 1-phosphate uridyl epimerase (GALE) can be done from red blood cells only. Semi-quantitative testing can also be performed from dried blood spots. This also useful in newborn screening.

• SMALL MOLECULE DISORDERS: Enzyme assay for fatty acid oxidation defects, gluconeogenesis, glycogen storage disorders, urea cycle defects, or organic acidurias, tyrosinemia, cystinosis, and porphyrias used to de the standard diagnostic modalities¹⁸.

CONFIRMATORY TESTS:

The diagnosis of IEM heavily depends on

• Detection of specific metabolite analysis using methods such as TMS, GC-MS, HPLC,

46

• Molecular genetic testing involving sequencing of particular gene or group of genes.

DETECTION OF METABOLITES:

These kinds of tests includes specific metabolites through simple chemical reaction with precision. Among the specialized biochemical tests, three methods are most commonly used newer techniques to confirm the diagnosis of IEM.

1) TANDEM MASS SPECTROMETRY: TMS Also known as mass spectrometry-mass spectrometry (MS/MS). Basic principle of TMS relies on the ionization and fragmentation of each molecule or metabolite into specific ions coupled with a robust detection system which is computerized to provide results. The utility of a TMS is multi-fold and its applications are keep expanding. It measures amino acids, organic acids and fatty acids in their acyl- carnitine esters. TMS using dried blood spot is highly sensitive but not specific.

At the same time TMS does not screen for mitochondriopathies, purine and pyrimidine disorders, congenital disorders of glycosylation (CDG) and very long chain fatty acids. Next pitfall for TMS is, it is not recommended for prenatal diagnosis.

2) GC-MS URINALYSIS FOR ORGANIC ACIDS: GC-MS is gas chromatography- mass spectrometry technique to detect metabolite specific for small molecule disorders in the urine. The organic acids are volatile and tend to evaporate without preservation. Interpretation of results of urine organic acids analysis by GC-MS requires expertise and biochemical training. Hence this is

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to be done by specialized genetic labs. The modality can also measure metabolites in bile, plasma and other body fluids including post mortem samples¹⁹.

3) HIGH PERFORMANCE LIQUID CHROMATOGRAPHY: The measurements of specific amino acids in body fluids such as plasma/ serum, urine or CSF is used for diagnosing many amino acid disorders such as phenylketonuria, tyrosinemia, MSUD, homocystinuria. There are multiple methods for quantification of amino acids – chromatographic as well as electrophoresis. Most laboratories currently employ HPLC for the relative ease and higher specificity. CSF samples should be paired with plasma samples for accurate interpretation.

MOLECULAR GENETIC TESTING:

The molecules genetic tests entails testing of either single or multiple genes depending on the suspicion of the diagnosis. The disorders for which a specific diagnosis is already made using other techniques such as GALT deficiency only one gene may be tested. On the other hand, for disorders like mitochondrial disorders, where multiple genes are involved, multiple genes are to be tested together. Two methods employed for molecular genetic testing.

1) SANGER SEQUENCING

2) NEXT GENERATION SEQUENCING.

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Both methods allow for sequencing of genes, determining the sequence of base pairs in the DNA, or coding regions of a gene. However, Sanger sequencing is time consuming, expensive, performed one by one for each fragment of gene. The NCS utilises multiplexing of all the sequencing fragments of DNA simultaneously. Hence it is upcoming the standard diagnosing methodology in most of genetic laboratories.

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REVIEW OF LITRATURE

1) Ramaswamy Ganesh et al did a study in clinical profile and outcome of children with inborn errors of metabolism over a period of 1 year from June 2017 to may 2018. According to this study, 31 newly diagnosed patients were studied for their clinical, biochemical and diagnosis, treatment, 2% of hospital admissions were diagnosed to have IEM. 65% were born to consanguineous parent. of these 31 children, 51% were lysosomal storage disorders, 26% were disorders of amino acid metabolism, 6% with carbohydrate and bile acid metabolism, 3% with mitochondrial and peroxisome metabolism& fatty acid oxidation. Overall mortality rate is this duration were 10%. Aminoacidopathies and organic acidemias were successfully treated with special formulas.

2) In a study by Meow Keong Thong et al selected testing of all ill infants and children for IEM yielded 2% positive results. Out of 264 patients the spectrum of IEM included 98 were organic acidurias, 78 amino acidopathies, 54 urea cycle disorders, 12 neurotransmitter conditions, and lysosomal disorders. In this study they reviewed the epidemiology and the spectrum of IEM from 1999 to 2005 in making the diagnosis of IEMs from patients suspected to have IEM.

That was served as adirection for primary prevention of IEM in the community.

3) Suvasinisharma et al studied about inborn errors of metabolism and epilepsy.

According to this study seizures and epilepsies are frequently encountered in patients with IEM. These epilepsies are refractory to anti -epileptic drugs, often associated with developmental delay, regression of milestones, behavioural