Newborn screening and inborn errors of metabolism

Abstract

Introduction

Inborn errors of metabolism (IEMs) affect the ability to convert nutrients into biochemical products or to use them for energy production. IEMs typically present during the newborn period or infancy. Some diseases, however, such as fatty acid oxidation defects or milder variants of classic metabolic disorders, may not be detected until adulthood. Despite potential long asymptomatic periods, consequences can be devastating and lead to death. Therefore identification and treatment of these diseases before irreversible damage occurs are critical. Clinical biochemical genetics is the discipline that deals with the diagnosis and treatment of patients with IEMs. In contrast to other, more common diseases, the treatment of IEMs is lifelong and requires frequent monitoring. The biochemical diagnosis and treatment monitoring of IEMs involve analysis of metabolites, enzymatic activity, and/or molecular structure. Because of technological advances (such as the introduction of MS/MS allowing the simultaneous detection of multiple analytes) and improved outcomes for patients with IEM who are identified and treated early, many more IEMs are now included in newborn screening programs than before.

Inborn errors of metabolism: Clinical presentation

IEMs are due to impaired activity of enzymes, transporters, or cofactors and result in the accumulation of abnormal metabolites (substrates) proximal to the metabolic block or decreased formation of essential products. Fig. 60.1 shows a hypothetical metabolic pathway in which the substrate (A) needs to be converted into the product (D), with arrows representing individual enzymes. If an enzyme is defective (vertical rectangle), the concentration of substrate A will increase, and formation of product D will decrease. The substrate A can accumulate to very high concentrations, becoming a substrate of enzymes not usually involved in its metabolism and producing abnormal by-products (E and F) through alternative pathways. The accumulation of specific metabolites and their by-products within organs and tissues and/or the lack of reaction products are the chemical bases of the pathology observed in different IEMs. At the same time, the measurement of some of these metabolites or their by-products is the basis of biochemical diagnostic testing for IEMs and early detection by newborn screening programs.

Symptoms of IEMs usually appear early in infancy, although some conditions become symptomatic only in late childhood or adulthood. Signs and symptoms include failure to thrive, seizures, mental retardation, organ failure, and even death. IEMs can be divided into three broad categories based on the effect of their metabolic derangement.

• 1.

  Intoxication effect. The metabolites accumulating in the body produce a toxic effect on different organs. The patient may become acutely ill after a symptom-free period of time (usually 24 to 72 hours) and concomitantly with the ingestion of the substrates that cannot be adequately metabolized, such as proteins or sugars. Amino acidopathies (e.g., phenylketonuria, maple syrup urine disease, homocystinuria), urea cycle defects (e.g., citrullinemia, argininosuccinic aciduria, ornithine transcarbamylase deficiency), organic acidemias (e.g., propionic acidemia, methylmalonic acidemia, glutaric acidemia type I), and disorders of sugar metabolism (e.g., galactosemia, hereditary fructose intolerance) belong to this group. Some of these disorders, such as phenylketonuria, affect primarily the brain, causing severe intellectual disabilities, 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. The symptoms in these disorders are due to impaired energy production. In some cases, patients may not be symptomatic for a long period of time, until energy requirements are increased due to fasting, illness, infection, or strenuous exercise. Disorders of fatty acid oxidation are classic examples of these disorders. They include medium-chain acyl-CoA dehydrogenase deficiency, very long-chain acyl-CoA dehydrogenase deficiency, carnitine uptake defect, and carnitine palmitoyl transferase deficiency I and II. The accumulation of fatty acids or other intermediates can also have an intoxication effect. Other diseases in this group are glycogen storage disorders, in which hypoglycemia can occur in the presence or absence of stress, mitochondrial disorders, and congenital lactic acidosis, in which the clinical course is progressive even in the absence of triggering conditions.

• 3.

  Disorders of complex molecules. These result from defects in the synthesis or catabolism of complex molecules. These disorders are progressive, independent of intercurrent events, and not related to food intake. The metabolism of complex molecules is altered in all lysosomal disorders, peroxisomal disorders, disorders of intracellular trafficking, and processing disorders. Some of these disorders have available therapy that is usually more effective if initiated before irreversible organ damage has occurred. For this reason, some of these conditions (eg, Pompe disease, mucopolysaccharidosis type 1) have been recommended for inclusion in newborn screening panels (see Chapter 61 ).

Inborn errors of metabolism: Diagnosis

The clinical symptoms of metabolic disorders, such as lethargy, failure to thrive, vomiting, and seizures, overlap with those of common conditions, such as sepsis or liver disease; however, routine laboratory tests of the symptomatic patient and a high index of suspicion can point in the right direction. For example, hyperammonemia without metabolic acidosis can suggest a defect of the urea cycle; hypoketotic hypoglycemia, usually with hyperammonemia, to various degrees suggests a fatty acid oxidation impairment; and hyperammonemia with metabolic acidosis and ketosis is more suggestive of an organic acidemia ( Table 60.1 ). The diagnosis of IEMs requires specific tests that are usually performed in biochemical genetics laboratories. Amino acid analysis in plasma, urine (in few cases), and cerebrospinal fluid (in even fewer cases); organic acid analysis in urine; and carnitine and acylcarnitine profiles in plasma represent the core group of tests necessary for the diagnosis of IEMs. In contrast to most common chemistry tests, biochemical genetics tests are complex and require specialized personnel to perform them and interpret the results. Each profile should be interpreted in the context of clinical history, physical signs, and other laboratory studies by a board-certified doctoral scientist or physician with specialized training in metabolic disease and analytic testing. When the results are suggestive of a metabolic disorder, the interpretation should also include information about the disease and suggest additional tests to confirm the diagnosis when appropriate.

Some metabolic disorders can be treated by dietary modifications that usually consist of a lifelong dietary regimen in which the nutrient that cannot be metabolized properly is restricted, often with supplementation of vitamins and other nutrients, cofactors, and in some cases, medications. Because of the excellent outcomes for many metabolic patients when treatment is initiated before symptoms or damage has occurred, a major focus of biochemical genetics has been in early identification of IEM in the newborn period through universal newborn screening.

Prenatal diagnosis

Despite constant progress in medical treatment, several IEMs result in severe morbidity and, in some cases, mortality early in life. Most of these disorders are inherited as autosomal recessive traits ( Table 60.2 ). 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 1.0% by chorionic villus sampling [CVS], 0.5% by amniocentesis); (3) risk of maternal complications; (4) the clinical validity of the prenatal test; (5) the burden of the disease; and (6) the 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 typically performed at 10 to 13 weeks of gestational age, has a higher risk of fetal loss compared to classic amniocentesis, and may not provide accurate results owing to contamination with maternal tissue, which can make this approach unreliable. On the other hand, certain enzymes, such as those of the glycine cleavage pathway defective in glycine encephalopathy, are only expressed in chorionic villi cells, rendering this procedure the best option when DNA testing is not possible. 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 experience is strongly recommended to avoid the occurrence of either incorrect or inconclusive results. In some IEM (e.g., organic acidemias), amniotic fluid can be tested for the presence or absence of specific metabolites in addition to providing amniocytes for enzyme assay and DNA analysis. The combination of DNA testing and another independent test (e.g., enzyme assay plus DNA analysis; metabolite analysis plus DNA) should give more confidence in establishing a prenatal diagnosis. Before entertaining a prenatal diagnosis, one should make sure that the proband (individual first brought to medical attention in which 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, the mutations of the index case should be known and confirmed as causative of the disease. The major advantages of direct metabolite analysis in amniotic fluid are the independence from tissue expression and a rapid turnaround time. However, direct metabolite analysis in amniotic fluid has been reported only for a very limited number of diseases.

Newborn screening

Newborn screening is a public health activity aimed at the early identification of conditions for which timely intervention can lead to the elimination or reduction of morbidity, mortality, and disability. It is an important and effective component of preventive medicine. Originally instituted in the 1960s for the early detection of phenylketonuria (PKU), newborn screening has since dramatically increased in number of diseases screened for with the introduction of MS/MS multiplex analysis of acylcarnitine and amino acid profiles (for further discussion on chromatographic separation and mass spectrometry, refer to Chapters 19 and 20 , respectively). For several IEMs, newborn screening provides the opportunity to define their incidence, natural history, prospective screening experience, and effectiveness of treatment. The complexity of the interpretation of MS/MS newborn screening results has prompted the development of algorithms for appropriate confirmatory testing and differential diagnosis of all detectable IEMs ( http://www.ncbi.nlm.nih.gov/books/NBK55827/ ).

Although the 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, storage disorders, and hemoglobinopathies) are identifiable using more traditional screening methods (e.g., enzyme assays, immunoassays, and electrophoresis). Advances in therapeutic interventions for IEM are continuously expanding the role of newborn screening. However, newborn screening does not identify all metabolic disorders and can miss some patients. Therefore a symptomatic patient of any age should be clinically investigated, including by DNA analysis, despite normal newborn screening results.

Evaluation of symptomatic patients

The most informative samples are those collected from patients during an episode of acute metabolic decompensation. When possible, urine and blood should be collected at the same time. In several diseases, especially in fatty acid oxidation disorders, the diagnostic abnormalities may not be detectable when the patient has recovered from the acute episode. Urine and plasma or serum samples can be stored at −20 °C until the need for specialized tests has been determined. Quantitative profiling of amino acids, carnitine, and acylcarnitines in plasma and organic acids and acylglycines in urine are the biochemical investigations 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 the case of death, collection of bodily fluids and tissue should be performed according to available protocols. ^,

Postmortem screening

Among IEM, fatty acid oxidation (FAO) disorders are those recognized most often after the diagnosis of an affected sibling or as a cause of sudden death. Early reports attributed up to 5% of sudden death in children younger than 5 years of age to FAO disorders, and there is mounting evidence that some of these disorders can cause mortality in adults as well. The postmortem evaluation of unexpected death, independently of age and especially when there is evidence of an acute illness or infection, should consider FAO disorders as a cause. This can be accomplished by the analysis of acylcarnitines in blood and bile spots. Reference intervals for acylcarnitines in postmortem blood and bile spots are listed in Table 60.3 .

Blood and bile can be collected on filter paper identical to the cards used for newborn screening and shipped to the laboratory at room temperature once properly dried. In cases with a higher index of suspicion, an effort should be made to collect and freeze a specimen of liver and collect a skin biopsy for establishing a fibroblast culture that can be used, if needed, to confirm a diagnosis. Although fatty infiltration of the liver and/or other organs (e.g., heart, muscle, and kidneys) is a common observation in FAO disorders, cardiac arrhythmia can occur with or without macroscopic steatosis, and a possible underlying FAO disorder should be considered in the evaluation of a case of sudden death, even in adults. In cases of sudden infant death, if parental permission to perform an autopsy is not granted, any leftover specimens or unused portion of the blood spots collected for newborn screening, if still available, may be useful samples for obtaining a diagnosis.

Biochemical genetics tests: Analytical considerations

In addition to the clinical presentation and routine laboratory tests, the diagnosis of patients with IEM relies on specific tests such as ion-exchange chromatography and LC-MS/MS for amino acids analysis, GC-MS for organic acids analysis, LC-MS/MS with or without LC separation for acylcarnitines profile, and LC-MS/MS or GC-MS for acylglycine profile. The combination of these tests, using different specimen types, is traditionally considered optimal for biochemical confirmation of metabolic disorders, whose diagnosis is then definitively confirmed using DNA testing or functional assays (enzyme/transporter activity).

Analyses of plasma amino acids, urine organic acids, and plasma acylcarnitines are the mainstay for the diagnosis of most aminoacidopathies, organic acidemias, and disorders of FAO. To allow early identification of asymptomatic patients by newborn screening, the diagnostic sensitivity and specificity of these methods need to be very high to detect even low concentrations of diagnostic metabolites. Furthermore, availability of age-appropriate reference intervals is necessary because the concentration of several metabolites (e.g., acylcarnitines) changes rapidly with age.