Inherited Metabolic Disease in the Neonatal Period : Approach to Clinical Diagnosis

This review article highlighted the need for clinicians to be alert to the possibility of an inherited metabolic disease (IMD) being the cause of a neonatal illness and provided a systematic approach to clinical diagnosis when IMD is suspected. Inherited metabolic disease (IMD) must be considered in the differential diagnosis of an ill neonate with nonspecific unexplained features, such as poor feeding, lethargy, failure to gain weight/weight loss, coma, apnoea, hyperventilation, seizures and hypotonia. Investigation for IMD should begin with simple urine and blood screening tests. For example, the urine examination includes checking for unusual odours, urinalysis (for ketones, amino acids, and organic acids), and reducing substance in urine, ferric chloride test and dinitrophenylhydrazine test. This is followed by simple blood tests e.g., full blood count, glucose, ammonia, amino acids, urea and electrolytes ( Na, K, Cl, P, Ca) levels, creatinine levels, liver function tests, serum lactate/pyruvate ratio and blood gases. In neonates, ketonuria with acidosis is a very important laboratory finding pointing to IMD. Although the prognosis for patients with IMD presenting in the neonatal period is often poor, every effort must be made to establish the diagnosis for parental counselling and in case prenatal diagnosis is possible in future pregnancies. In conclusion, when presented with an ill full-term neonate with nonspecific, unexplained/peculiar features pursue the usual bacterial septicaemia work-up, but in addition, consider IMD and evaluate, timely, for metabolic disease. This approach is very useful since the commonest mistake in the management of a neonate with IMD is a delay in diagnosis or a misdiagnosis, resulting in a delay in starting treatment with catastrophic consequences. Key words: inherited metabolic diseases; inborn errors of metabolism; clinical diagnosis; neonatal screening. DOI: http://dx.doi.org/10.3126/jnps.v32i1.4882 J. Nepal Paediatr. Soc. Vol.32(1) 2012 57-64


Introduction
I nherited metabolic disease (IMD) refers to a disorder in which single gene defects cause clinically signifi cant blocks in metabolic pathways 1 .IMD results from mutations in DNA that code for a specifi c protein, which may act as an enzyme, receptor, transport vehicle, membrane pump or structural element 2,3 .Most IMDs are inherited as autosomal recessive trait and neonates with parental consanguinity are at increased risk 4 .The incidence of individual, IMD is relatively low with an estimate of 1 in 100,000 to 1 in 200,000 live-births ,5,6 However, collectively, the estimated incidence of IMD varies from 1 in 1,000 to 1 in 1,500 live birth 7,8.The precise incidence rates of IMD are diffi cult to come by because of undiagnosed cases.The prevalence of IMD is determined by the geographical and ethnic composition of the population.For example, among live births, the estimated prevalence are 1 in 1,400 to 1 in 5,000 in the United States 9 , 1 in 2,800 in South Korea, 10 1 in 5,000 in Thailand 11 , 1 in 784 in the United Kingdom 12 and 1 in 2,077 in Germany 13 .With the ever-increasing rate at which new ones and variations of old ones are being recognized, IMD is an area of paediatrics that is assuming an increasing role in both private practice and tertiary hospitals referral 14,15 The most common error in the management of neonates with IMD is a delay in diagnosis, resulting in a delay in commencement of appropriate treatment and, sometimes, a rapidly progressive neurologic deterioration, coma and death 9,16 .In this regard, therefore, there is a need for the paediatrician to develop a system for identifying the patient who might have an IMD and, at least, for determining the general category to which the suspected IMD belongs.The key is a systematic approach that recognizes certain constellation of signs and symptoms and utilizes simple generally available and aff ordable tests.
Inherited metabolic diseases (IMD) are revealed either as a result of the mass screening of neonates or by investigation of ill neonates presenting with a constellation of clinical features.Since developing countries cannot aff ord mass screening programmes, paediatricians in such regions have to depend on evaluation based on signs and symptom complexes to reach a diagnosis, at least tentatively.Many paediatricians feel overwhelmed not only by the number and complexity of IMD, but also, by the interpretation of laboratory tests needed to diagnose these disorders.As a result, the clinician may exhibit lack of confi dence when confronted with a neonate requiring evaluation for IMD.The situation is further compounded by paucity of guidance from the literature on the subject of clinical recognition of these diseases 17 .The majority of IMD that occur in the neonatal period are characterized by non-specifi c signs and symptoms, such as poor feeding, lethargy, failure to thrive, respiratory distress, seizures and coma which in themselves are not useful in making a diagnosis but when observed in combination, without a known cause, are suggestive of such a diagnosis 8,17 .This review article sought to (i) highlight those constellation of clinical fi ndings in the neonatal period that should alert the clinician to the possibility of existence of IMD; (ii) provide a simplifi ed approach to clinical diagnosis of IMD and; (iii) provide an approach to the use of selected simple laboratory tests in neonates suspected clinically of having an IMD.

Classification of inherited metabolic diseases
Inherited metabolic diseases can be simply categorized into three 2,15 : Classifi cation based on: (i) time of onset; (ii) clinical presentation and; (iii) biochemical basis of the disease.A review of the literature revealed that subdividing IMD by clinical presentation is the most useful approach to evaluation and accurate diagnosis of IMD 9,15,17 .This approach was, therefore, followed in this article.
When to suspect IMD 17,18,19 A. Historical findings.An inherited metabolic disease should be considered if: 1.There is a high rate of consanguineous marriage in the local population since most IMDs are inherited as autosomal recessive trait.
2. There is a family history of unexplained neonatal or infant death (prior siblings or male infants on maternal side of the family).
3. Maternal illness during pregnancy e.g., acute fatty liver of pregnancy, HELLP syndrome (may occur in pregnancies with a fetus with long chain fat oxidation defects).
4. There are persistent unexplained symptoms, such as persistent or recurrent vomiting.

Increased fetal movements(in utero seizures)
6.There is family history of unexplained neuropathy or myopathy 7. History of similar illness especially in siblings.
8. The onset of signs and symptoms listed in Table 4 after a period of apparently good health.9. Symptoms accompanying changes in diet e.g., soybased formula.
10. Failure to thrive (failure to gain weight or weight loss).
11. Failure of usual therapies to alleviated the symptoms e.g., in hypoglycaemia.

Major Clinical Manifestations of IMD in the Neonatal Period
Generally speaking, IMD usually do not present immediately after birth because the defective metabolic pathway in the fetus is compensated for by the maternal placental unit.Consequently, there is an interval (hours, days or weeks) during which the neonate appears well.Symptoms appear only when the neonate begins to depend on its own metabolism.However, some IMD like glutaric acidaemia type II, pyruvate carboxylase defi ciency, Zellweger syndrome and GM 1 gangliosidosis aff ect the fetus in utero, resulting in advanced disease at birth.Disorders of carbohydrate and protein metabolism as well as disorders of energy production (gluconeogenesis and glycogenolysis) tend to present in the neonatal period and are usually unrelenting and rapidly progressive 3,9 .

C. Laboratory findings indicating a possibility of IMD
1. Severe hypoglycaemia.
3. Urine reducing substance positive but negative with clinistix strip specifi c for glucose.
7. Lactate/pyruvate ratio of less than 25 makes the possibility of lactic acidosis, organic acidurias, urea cycle defects and disorders of fatty acid metabolism very unlikely. 20 High levels of lactate and pyruvate suggest mitochondrial defects.20

Initial (simple) screening tests
Whenever a neonate is suspected of having IMD, simple initial screening tests should be performed immediately.Such an investigation should begin with simple urine metabolic screening tests and blood analysis.First and foremost, start by checking for unusual urine odour (Table 1).Common urine metabolic screening tests are critical, especially in resource poor countries, where sophisticated medical laboratory facilities are not available (Table 2).Although many of these tests are non-specifi c, a positive result can indicate which specifi c test(s) should be performed in the evaluation process.

Advanced (secondary) screening tests
Further investigation depends on the results of the initial simple screening tests.a. Tandem mass spectrometry.
Availability of tandem mass spectrometry has revolutionized neonatal screening for IMD because it can detect many several disorders of amino acid, organic acid and fatty acid metabolism using a single blood specimen and analytical technique. 21,22In addition, it may serve as a complementary to immunoassay-based methods for congenital hypothyroidism and congenital adrenal hyperplasia. 21Chase et al 23 has shown that tandem mass spectrometry is accurate, sensitive with little or no false positives.b.Biomakers.
Specialized biochemical genetic laboratories utilize some biomarkers, such as carnitine, acylcarnitine, very long chain fatty acids and lysosomal enzymes but require appropriate-age related reference intervals.c.Magnetic resonance imaging (MRI) 24 .
The nature of myelin has been reviewed by Barkovich and the eff ects of its diff erent components on MRI parameters elucidated leading to better understanding and diagnosis of IMD. 25

d. Magnetic resonance spectroscopy (MRS).
Using MRS of the brain, Barkovich et al 26 showed that patients with mitochondrial disorders have high levels lactate.Proton nuclear magnetic resonance spectroscopic studies have shown that the technique can detect N-acetylated metabolites in urine 27 .

High
Obtain blood pH and CO 2

Aminoacidopathies or galactosaemia
Obtain blood pH and CO 2

Antibiotic therapy
Inherited Metabolic Disease in the Neonatal Period: Approach to Clinical Diagnosis This schema (Figure 1) is a guide to the elucidation of some of the metabolic disorders in neonates.Although some exceptions to this schema exist, it is appropriate for most cases.Confirmatory testing principles 1 A positive screening test must be followed by specifi c clinical evaluation and laboratory testing to confi rm the disorder.Every protocol for the evaluation of an infant with an abnormal screening result must clarify which patient need to be treated and which one has had a false-positive result.Defi nitive testing must be carried out promptly and accurately.Parents need to be educated and reassured while testing proceeds because of the intense parental anxiety associated with positive screening tests.Treatment must be commenced immediately the diagnosis is confi rmed.If an IMD is excluded, parents need a full explanation and reassurance that the neonate is well.

Diagnostic specimens to be obtained in seriously ill neonates suspected of inherited metabolic disorder
If the condition of a seriously ill neonate is deteriorating and death appears imminent, it is important to gather as much information as possible about the neonate's disorder.Specific diagnostic steps to be taken 15,18 a) Blood: i) Collect 20 -25ml of whole blood and separate the plasma from the cells and freeze in 1 -2ml aliquots at -20 0 C (for quantitative amino acids, carnitine and ketone bodies).
ii) Refrigerate an erythrocyte fraction at 4 0 C (for enzyme and peroxisomal studies).In addition, refreeze an erythrocyte fraction at -20 0 C (for enzyme/DNA studies).
b) Urine: Collect 20-30ml and store in 5ml aliquots at -20 0 C (for organic, orotic and amino acid screening).Reasons for false-negative results: 1,6,29 1.In a particular enzyme defect, levels of precursor may not increase at birth until the baby starts an independent existence (metabolism-wise) and this may require commencement of normal feeding e.g. in phenylketonuria, blood phenylalanine is normal but rise to maximum during the fi rst 2 to 3 weeks of life.Screening should, therefore, not be performed before the sixth day of life.
2. The increased concentration of precursors or production of abnormal metabolites, may be dependent on regular feeds and may fl uctuate in relation to timing of feeds.It has been shown that the presence of galactose in urine, an observation used frequently to test for galactosaemia, is not a constant feature in all cases of the disease.
3. Some enzyme systems mature weeks or months after birth and if the production of the abnormal metabolites is dependent on this late maturing enzymes, the biochemical signs may be absent in the early stages of life.An example is the late maturation of liver transaminase which converts accumulated phenylalanine to phenylpyruvic acid for excretion in the urine.

Some practical aspects of newborn screening
In newborn population screening studies, the samples which are easily obtained are: 1. Blood may be obtained at birth from the umbilical cord, and later from capillary samples by pricking the heel.For ease of collection and transportation the blood samples.Many analyses are then performed on these dry blood spots.
2. Urine may be collected conveniently by leaving a fi lter paper inside the nappy.
3. Blood transfusion gives false-negative results and sample deterioration false-positive results in galactosemia.

Conclusion
It is my hope that the clinical diagnostic guidelines presented in this review article, based on a systematic approach that recognizes a constellation of clinical features and utilizes common and aff ordable screening laboratory tests will assist clinicians, especially those working in resource poor countries, in the evaluation of neonates for metabolic disorders.

Fig 1 :
Fig 1: Clinical approach to a neonate with suspected inherited metabolic disease About 3 -4mm should be taken as fullthickness sterile biopsy (cleansed with alcohol, not iodine) store in a sterile culture medium (or sterile 5% dextrose in normal saline).Transport immediately to a tissue culture laboratory for fi broblast culture and enzyme/DNA analysis.e) CSF: Store a sample at -20 0 C. f) Liver sample: May biopsy percutaneously as necessary and store at -20 0 C. g) Tissue biopsies of liver, heart, muscle and brain stored at -20 0 C. Tissue should be evaluated by light and electron microscopy (for peroxisomes, liposomes, mitochondria).h) Complete autopsy including x-ray fi lms.

Table 1 :
Inherited metabolic diseases associated with abnormal urine or body odour.
Metabolic Disease in the Neonatal Period: Approach to Clinical Diagnosis

Table 3 :
Common presenting features of IMD and common diff erential Diagnoses

Table 4 :
Signs and symptom complexes suggesting classes of inherited metabolic diseases.

Table 5 :
Major clinical manifestations of IMD in the neonatal period.

Table 6 :
Laboratory fi ndings associated with IMD in the neonatal period.

Table 7 :
Metabolic disorders that may be associated with reducing substances in the urine.

Table 8 :
Metabolic disorders associated with positive ferric chloride reaction.Inherited Metabolic Disease in the Neonatal Period: Approach to Clinical Diagnosis