1. Intoxication effect: IEMs in this category are the result of metabolites accumulating in the body that produce a toxic effect on different organs. The patient may become acutely ill after a symptom-free period, usually 24 to 72 hours, and concomitantly with ingestion of metabolites that are not metabolized, such as proteins or sugars. Types of IEMs that belong in this group include (1) aminoacidopathies (e.g., phenylketonuria, maple syrup urine disease, homocystinuria), (2) urea cycle defects (e.g., citrullinemia, argininosuccinic aciduria, ornithine transcarbamylase deficiency), (3) organic acidemias (e.g., propionic acidemia, methylmalonic acidemia, glutaric acidemia type I), and (4) disorders of sugar metabolism (galactosemia, hereditary fructose intolerance). Some of these disorders, such as phenylketonuria, affect primarily the brain, causing severe mental retardation but without acute decompensation. In other disorders (e.g., organic acidemias), symptoms appear shortly after protein intake (usually after the first few feedings) and include vomiting, lethargy, seizures, and coma leading rapidly to death if not recognized and treated appropriately.

2. Energy deficiency: Symptoms of disorders in this category are due to impaired energy production. In some cases, patients may be asymptomatic for a long time, until energy requirements are increased as the result of involuntary fasting, illness, infection, or strenuous exercise. A classic example of these disorders involves fatty acid oxidation (e.g., medium chain acyl–coenzyme A [CoA] dehydrogenase deficiency, very long chain acyl-CoA dehydrogenase deficiency, carnitine uptake defect, carnitine palmitoyl transferase deficiency I and II), although in this case, the block in the metabolic pathway, in addition to impairment in energy production, leads to the accumulation of intermediates that cause an intoxication effect.⁵² Other diseases in this group include glycogen storage disorders, in which hypoglycemia can occur in the presence or absence of any stress, mitochondrial disorders, and congenital lactic acidosis, in which the clinical course is progressive even in the absence of triggering conditions.⁴³

3. Disorders of complex molecules: These disorders result from defects in the synthesis or catabolism of complex molecules. They are progressive, are independent of intercurrent events, and are not related to food intake. The metabolism of complex molecules is altered in all (1) lysosomal disorders, (2) peroxisomal disorders, and (3) disorders of intracellular trafficking and processing. For some of these disorders, a therapy is now available that is usually more effective if initiated before irreversible organ damage has occurred. For this reason, some of these conditions are also being considered for newborn screening.

Prenatal Diagnosis

Despite constant progress in medical treatment, several IEMs result in severe morbidity and inevitable mortality early in life. Most of these disorders are inherited as autosomal recessive traits (Table 58-2); therefore the recurrence risk in subsequent pregnancies of the same couple is 25%. Genetic counseling of parents consists of a balanced assessment of (1) familial risk factors (parental consanguinity and ethnic origin), (2) risk of pregnancy loss as a consequence of the sampling procedure [0.5 to 1% by chorionic villus sampling (CVS), 0.5% by amniocentesis], (3) risk of maternal complications,⁵³ (4) clinical validity of the prenatal test, (5) the burden of the disease, and (6) variable phenotypic expression of the disease even within the same family.

Methods used for prenatal diagnosis of IEMs have different requirements in terms of timing, sample collection, and options for independent confirmation. CVS is performed at 10 to 13 weeks’ gestational age, has a higher risk of fetal loss as compared with classic amniocentesis, and might not provide accurate results because of possible contamination with maternal tissue. Alternatively, certain enzymes, such as those of the glycine cleavage pathway defective in glycine encephalopathy, are expressed only in cells of chorionic villi, rendering this procedure the only possibility when DNA testing is not possible.⁴ Amniocentesis is performed later in pregnancy (14 to 20 weeks) and provides both amniocytes and amniotic fluid to be used for independent and complementary diagnostic methods. Reliance on separate tests based on independent methods performed by laboratories with adequate prior experience is strongly encouraged to avoid incorrect or inconclusive results. In some IEMs (e.g., organic acidemias), amniotic fluid is tested for the presence or absence of specific metabolites, in addition to providing amniocytes for enzyme assay, DNA analysis, or both. The combination of at least two independent tests (e.g., enzyme assay + DNA; metabolite analysis + enzyme or DNA) enhances confidence in establishing a prenatal diagnosis. Before entertaining a prenatal diagnosis, one should ensure that the proband (individual first brought to medical attention in whom the diagnosis was established) related to the index case has a diagnosis confirmed by traditional methods, including enzymology when appropriate. If DNA analysis is considered, mutations of the index case should be known and confirmed as causative of the disease. Major advantages of direct metabolite analysis in amniotic fluid include independence from tissue expression and rapid turnaround time. However, analysis of direct metabolites in amniotic fluid has been reported for only a very limited number of diseases.

Newborn Screening

Newborn screening is a public health activity aimed at early identification of conditions for which timely intervention is expected to result in elimination or reduction of morbidity, mortality, and disabilities. It is an important and effective component of preventive medicine. Originally instituted in the 1960s for the early detection of phenylketonuria (PKU), the number of diseases screened for in the newborn period has dramatically increased with the introduction of MS/MS multiplex analyses of acylcarnitine and amino acid profiles.¹³ Inclusion of IEMs as a whole in newborn screening panels has been controversial, because with few exceptions, their incidence, natural history, and prospective screening experience, as well as the effectiveness of treatment, have not yet been defined.¹⁰ However, implementation of this expanded screening allows collection of these data leading to a better understanding of these diseases. The complexity of the interpretation of MS/MS newborn screening results has prompted the development of algorithms for proper confirmatory testing and differential diagnosis of all detectable IEMs (http://www.acmg.net/resources/policies/ACT/condition-analyte-links.htm/accessed September 29, 2011).

Although metabolic disorders identified by MS/MS represent the largest group of diseases identifiable by newborn screening, other IEMs and endocrine and hematologic disorders (such as galactosemia, biotinidase deficiency, cystic fibrosis, congenital hypothyroidism, congenital adrenal hyperplasia, and hemoglobinopathies) are identifiable through more traditional screening methods (e.g., enzyme assays, immunoassays, electrophoresis). Advances in therapeutic interventions for IEMs are continuously expanding the role of newborn screening. Newborn screening does not identify all metabolic disorders, and some patients can be missed by newborn screening. Therefore a symptomatic patient, at any age, should be investigated despite normal newborn screening results.

Evaluation of Symptomatic Patients

The most informative samples are collected from patients during acute metabolic decompensation. When possible, urine and blood should be collected at the same time. In several diseases, especially in fatty acid oxidation disorders, diagnostic abnormalities may not be detected when the patient has recovered from the acute episode. Urine and plasma/serum samples are stored at −20 °C until the need for specialized tests has been determined. Quantitative profiling of amino acids, carnitine, acylcarnitines, and fatty acids in plasma, and organic acids and acylglycines in urine, is the biochemical investigation necessary to diagnose these disorders. Alternatively, a blood spot on filter paper may provide enough material for one or more of the investigations described in this chapter. In case of death, collection of body fluid and tissue should be secured according to available protocols.12,54

Postmortem Screening

Among IEMs, fatty acid oxidation (FAO) disorders are those recognized more often after the diagnosis of an affected sibling or as a cause of sudden death.⁵ Early reports attributed up to 5% of cases of sudden death in children younger than 5 years to FAO,⁷ and mounting evidence indicates that some of these disorders can cause mortality in adults as well.³⁸ The postmortem evaluation of unexpected death, independent of age, especially when evidence of acute illness or infection is found, should consider FAO as a cause. This is accomplished by analysis of acylcarnitines in blood and bile spots (Figure 58-2).⁵² Reference intervals for acylcarnitines in postmortem blood and bile spots are listed in Table 58-3.

Blood and bile are collected on filter paper identical to the cards used for newborn screening; once properly dried they are shipped to the laboratory at room temperature. In cases with a higher index of suspicion, an effort should be made to collect and freeze a specimen of liver⁷ and to collect a skin biopsy for establishing the fibroblast culture to be used, if needed, to confirm a diagnosis. Although fatty infiltration of the liver and/or other organs (e.g., heart, muscle, kidneys) is a common observation in FAO disorders, the finding of macroscopic steatosis should not be used as the only criterion in deciding whether to investigate a possible underlying FAO disorder during postmortem evaluation of a case of sudden death. Sudden death from cardiac arrhythmia can be the only finding in fatty acid oxidation defects, and the absence of obvious physical findings on autopsy does not exclude this, especially in adults. In cases of sudden infant death, if parental permission to perform an autopsy is not granted, any leftover specimens or unused portions of blood spots collected for newborn screening, if still available, could be useful samples in obtaining a diagnosis.

Biochemical Genetics Tests: Analytical Considerations

In addition to clinical presentation and routine laboratory tests, the diagnosis of patients with inborn errors of metabolism relies on specific tests such as ion-exchange chromatography and liquid chromatography with tandem mass spectrometry (LC-MS/MS) for amino acid analysis, gas chromatography/mass spectrometry (GC/MS) for organic acid analysis, tandem mass spectrometry (MS/MS) with (LC-MS/MS) or without liquid chromatographic separation for acylcarnitine profiles, and LC-MS/MS or GC/MS for acylglycine profiles. The combination of these tests, with the use of different specimen types, is the key to biochemical confirmation of metabolic disorders, whose diagnosis is then definitively confirmed using DNA testing or enzyme/transporter assays.

Analysis of (1) plasma amino acids, (2) urine organic acids, and (3) plasma acylcarnitines is the mainstay for the diagnosis of most amino acidopathies, organic acidemias, and disorders of fatty acid oxidation. Because of the early identification of asymptomatic patients by newborn screening, the sensitivity and specificity of these methods need to be very high to detect even low concentrations of diagnostic metabolites. Furthermore, the availability of age-appropriate reference intervals is necessary, because the concentrations of several metabolites (e.g., acylcarnitines) change rapidly with age.

Classic Phenylketonuria and Other Hyperphenylalaninemias

Hyperphenylalaninemias result from the impaired conversion of phenylalanine to tyrosine, leading to an increased concentration of phenylalanine in body fluids. They are caused by a primary deficiency of phenylalanine hydroxylase, the enzyme that converts phenylalanine to tyrosine (Figure 58-3), or, in rare cases (<2% of total cases in the United States), by a defect in synthesis (Figure 58-4) or recycling (Figure 58-5) of the essential cofactor tetrahydrobiopterin. The combined incidence of these conditions is about 1 : 10,000 to 1 : 20,000 live births. In PKU, accumulation of phenylalanine and other metabolites such as phenyllactate and phenylpyruvate (phenylketones) occurs. Elevated phenylalanine interferes with neurotransmitter synthesis and uptake, leading to clinical symptoms of phenylketonuria.⁶³

Patients with phenylketonuria appear healthy at birth, apart from an increased incidence of gastroesophageal reflux in patients with very high phenylalanine concentrations. Delays in development, chronic eczema, and acquired microcephaly become evident after a few months of life. Abnormal brain development results in mental retardation. This and all other problems are prevented by a special diet low in phenylalanine that needs to be initiated before 3 weeks of age. Therefore, all infants are screened at birth for this condition. In utero, phenylalanine is removed from the child by the placenta. After birth, phenylalanine accumulates as the child initiates feedings and is exposed to proteins. Therefore, for optimal results, infants should be screened after 24 hours of life, when at least one feeding has occurred, to ensure an adequate rise in phenylalanine concentrations. The diagnosis of phenylketonuria is confirmed by plasma amino acid analysis. In each infant with even minimally elevated serum phenylalanine, cofactor defects need to be excluded by measuring the urine pterin profile and activity of dihydropteridine reductase (DHPR) in blood cells. Hyperphenylalaninemia due to phenylalanine hydroxylase deficiency is biochemically diagnosed when plasma phenylalanine concentrations are above the normal reference interval with a normal urine pterin profile and normal DHPR activity. Phenylalanine hydroxylase is expressed only in the liver, and confirmation of the diagnosis by enzyme assay is not routinely performed. DNA sequencing of the causative gene, however, will definitively confirm the diagnosis, if needed.

In patients with PKU due to phenylalanine hydroxylase deficiency, dietary treatment with a special formula without phenylalanine should be started as soon as possible—ideally before 3 weeks of age.⁶³ Diet needs to be continued for life. Phenylalanine concentrations are monitored periodically and should remain between 60 and 360 µmol/L (reference interval, 30 to 80 µmol/L) to ensure adequate brain development. If the concentration of phenylalanine is too low, growth of the child is compromised; if it is too high, executive functioning is impaired. High concentrations of phenylalanine in the first years of life lead to mental retardation. Phenylalanine at high concentrations is teratogenic and, depending on the concentration and the period of exposure during pregnancy, will cause (1) increased risk of spontaneous abortion, (2) congenital heart defects, (3) facial dysmorphism, (4) microcephaly, and (5) developmental delay (even in the absence of microcephaly) in the fetuses of women with PKU. Adverse pregnancy outcomes in pregnant women with PKU are minimized by maintaining phenylalanine concentrations less than 360 µmol/L.³⁷

In approximately 2% of cases, hyperphenylalaninemia is due to a deficiency of biosynthesis (see Figure 58-4) or recycling (see Figure 58-5) of the cofactor tetrahydrobiopterin (BH₄). BH₄ is also a cofactor for tyrosine and tryptophan hydroxylases (see Figure 58-5).⁴² Infants with BH₄ deficiencies show signs of neurologic involvement despite adequate dietary control of phenylalanine concentrations. Impairment of tyrosine and tryptophan hydroxylases reduces the synthesis of the neurotransmitters dopamine and serotonin, with severe neurologic consequences. BH₄ is also a cofactor for nitric oxide synthase, which catalyzes the generation of nitric oxide from arginine, although the clinical consequences of this latter impairment are not known.

Five enzyme deficiencies leading to BH₄ deficiency have been reported (see Table 58-2). One of these, sepiapterin reductase deficiency, impairs BH₄ synthesis only in the brain, because alternative pathways are available for its synthesis in the liver. As a result, patients with this latter condition have normal activity of phenylalanine hydroxylase in the liver and no hyperphenylalaninemia.²¹ Among patients with BH₄ deficiencies and elevated phenylalanine, 50% of cases are due to 6-pyruvoyltetrahydropterin synthase (6-PTPS) deficiency. These patients are clinically indistinguishable from those with classic PKU (caused by phenylalanine hydroxylase deficiency), when diagnosed through newborn screening, but they progressively deteriorate with loss of head control, truncal hypotonia with hypertonia of the extremities, drooling, swallowing difficulties, and myoclonic seizures at between 2 and 6 months of age.⁴² Treatment of these patients requires BH₄, which usually normalizes the concentration of phenylalanine (except in some cases of DHPR deficiency), and neurotransmitter precursors (L-dopa/carbidopa and 5-OH-tryptophan), which obviate the need for tyrosine and tryptophan hydroxylase.

Infants with benign hyperphenylalaninemia (phenylalanine <300 µmol/L) occasionally are identified by newborn screening because of a moderately elevated blood concentration of phenylalanine. These patients have a partial deficiency of phenylalanine hydroxylase with residual enzyme activity up to 35% of normal. Although detected by neonatal screening, they remain healthy without dietary treatment. The possibility of an underlying cofactor deficiency should be ruled out. Phenylalanine, however, needs to be monitored periodically because, depending on the diet, its concentration can increase to the point where therapy is required.