Newborn Screening and Inborn Errors of Metabolism

Chapter 58


Newborn Screening and Inborn Errors of Metabolism



An inborn error of metabolism (IEM) is a genetically determined biochemical disorder that affects an individual’s ability to convert nutrients or to use them for energy production. Typically, IEMs present in the newborn period or in infancy. Some diseases, however, such as fatty acid oxidation defects or milder variants of classic metabolic disorders, may not be detected until adulthood. Despite the long asymptomatic period, their consequences can still be devastating and may lead to death. Therefore identification and treatment of these diseases before irreversible damage occurs is critical. Clinical biochemical genetics is the discipline that deals with the diagnosis and treatment of patients with inborn errors of metabolism. In contrast to other, more common diseases, treatment of IEMs is life-long and requires frequent monitoring. The biochemical diagnosis of IEMs and treatment monitoring involve analysis of (1) metabolites, (2) enzymatic activity, and/or (3) molecular structure. Because of technological advances [such as the introduction of tandem mass spectrometry (MS/MS), allowing the simultaneous detection of multiple analytes] and improved outcomes of patients with IEMs identified and treated early, many IEMs are now included in newborn screening programs.



Clinical Presentation


Inborn errors of metabolism, which are due to impaired activity of enzymes, transporters, or cofactors, result in accumulation of abnormal metabolites (substrates) proximal to the metabolic block, or lack of necessary products. Figure 58-1 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 the concentration of product D will decrease. It is then possible that high concentrations of substrate A will accumulate and become a source of substrate for enzymes not usually involved in its metabolism, thereby producing abnormal byproducts (E and F) through alternative pathways. Accumulation of specific metabolites and their byproducts within organs and tissues and/or the lack of reaction products are the chemical bases of the pathology observed in different inborn errors of metabolism. At the same time, measurement of some of these metabolites or their byproducts serves as the basis of biochemical diagnostic testing for inborn errors of metabolism and early detection by newborn screening programs.



Symptoms of inborn errors of metabolism usually appear early in infancy, although several types become symptomatic in late childhood or adulthood. Signs and symptoms include (1) failure to thrive, (2) seizures, (3) mental retardation, (4) organ failure, and even (5) death. Inborn errors of metabolism have been divided into three broad categories, based on the effects of their metabolic derangement55:



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.



Diagnosis


With few exceptions, the diagnosis of IEM is primarily a laboratory process. For example, routine laboratory tests in the symptomatic IEM patient and a high index of suspicion often point the clinician toward a specific IEM. For example, (1) hyperammonemia without metabolic acidosis suggests a defect of the urea cycle; (2) hypoketotic hypoglycemia usually with hyperammonemia to various degrees suggests fatty acid oxidation impairment; and (3) hyperammonemia with metabolic acidosis and ketosis is suggestive of an organic acidemia (Table 58-1). The diagnosis of IEMs requires specific tests that usually are performed in Biochemical Genetics Laboratories. The core group of tests necessary for the diagnosis of IEMs includes (1) amino acid analysis in plasma, urine (in few cases), and cerebrospinal fluid (in even fewer cases), (2) organic acid analysis in urine, and (3) carnitine and acylcarnitine profile in plasma.



In contrast to common chemistry tests, biochemical genetics tests are complex and require specialized personnel to perform the tests and interpret the results. For example, it is recommended that each profile 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 analytical testing. When the results are suggestive of a metabolic disorder, interpretation should include information about the disease and should suggest additional tests to confirm the diagnosis, when appropriate.


Some metabolic disorders are treated by dietary modifications usually consisting of a life-long dietary regimen in which the nutrient in question is restricted, with supplementation of vitamins, cofactors, and, in some cases, medications. Because of the excellent outcomes of metabolic patients when treatment is initiated before symptoms or any damage has occurred, a major focus of Biochemical Genetics is on early identification of IEMs, prenatally and especially in the newborn period, through universal 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 (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.



Amino Acid Analysis by Ion-Exchange Chromatography


Several methods have been used in the analysis of amino acids in biological fluids such as plasma, urine, and cerebrospinal fluid. All involve chromatographic separation of amino acids with precolumn [high-performance liquid chromatography (HPLC), GC methods] or postcolumn (ion-exchange chromatography) derivatization, followed by detection by ultraviolet (UV), fluorescence, or mass spectrometry.1,35,61,65,77,78 The gold standard for analysis of amino acids remains ion-exchange chromatography (IEC), even though recent work using tandem mass spectrometry is showing promising results. The challenge with amino acid analysis includes the need (1) to cover a wide dynamic range, (2) to have a very low detection limit, and (3) to have a high upper limit of linearity. In addition to these analytical requirements, isomers need to be separated and quantified. With ion-exchange chromatography, the sample (plasma/urine/CSF) is deproteinized and injected onto an ion-exchange column (typically a lithium cation-exchange column). Amino acids are separated on the basis of their pKa by changing the pH and ionic strength of eluting buffers and the temperature of the column. Acidic amino acids are eluted first, followed by neutral then basic amino acids. After their elution from the column, amino acids are mixed with ninhydrin at 135 °C to form a colored adduct. The intensity of their absorbance is proportional to the concentration of the amino acid. Absorbance is read at two different wavelengths: 570 nm (maximum absorbance for amino acids) and 440 nm (maximum absorbance for imino acids, such as proline and hydroxyproline). The concentration of amino acids is calculated using an internal standard and external calibration. Identification of individual amino acids relies on retention time, ratio of absorbance at the two wavelengths (440/570 nm), and, in case of doubt, spiking of the sample with a standard.


Plasma collected under fasting conditions is the specimen of choice. In infants and small children, the sample should be collected at least 2 hours after the last feeding. Collection of serum should be avoided because of artifacts deriving from the clotting process. Blood should be collected with an anticoagulant (lithium/sodium heparin) and immediately separated and frozen until the time of analysis. Storage of samples at inappropriate temperature, at room temperature, or refrigerated has been known to result in deamination of glutamine and binding of sulfur amino acids to protein. The concentration of most amino acids in red blood cells is very similar to their concentration in plasma; however, some amino acids (e.g., aspartic acid, taurine, glutamic acid) are present at higher concentrations in red blood cells; therefore, hemolysis will result in an artificially increased concentration of those amino acids. In addition, red blood cells contain the enzyme arginase, which converts arginine to ornithine and urea. Hemolysis may release this enzyme, resulting in decreased concentrations of arginine and increased concentrations of ornithine. Results of plasma amino acid analysis are usually expressed in micromoles/liter.


Urine amino acids are useful only in the investigation of disorders of amino acid transport (e.g., cystinuria, lysinuric protein intolerance, Hartnup disorder) or of prolidase deficiency. A random urine sample, without preservative, is usually sufficient. Specific reabsorption studies may require a timed (24 hour) urine collection. The sample should be collected without preservative and kept refrigerated until the end of the collection. Urine samples, like plasma, should be frozen as soon as possible and kept frozen until analysis. Results are usually normalized to the concentration of creatinine in the specimen.


Analysis of cerebrospinal fluid (CSF) amino acids is performed for very specific cases, such as in the diagnostic investigation of glycine encephalopathy (nonketotic hyperglycinemia) and in disorders of serine metabolism. CSF should be collected while avoiding blood contamination; it should be frozen immediately and kept frozen until the time of analysis. Measurements of CSF amino acids are expressed in micromoles/liter. Amino acid results, independent of specimen type, should be correlated with clinical status, diet, and medications.



Urine Organic Acid Analysis by Gas Chromatography/Mass Spectrometry


The label organic acids includes metabolites of almost all pathways of intermediary metabolism and exogenous compounds. Organic acids analyzed by gas chromatography/mass spectrometry (GC/MS) are separated on the basis of their volatility and solubility in the stationary nonpolar liquid phase of the capillary gas chromatography column. Before GC/MS analysis is performed, organic acids must be (1) extracted, usually with an organic solvent; (2) converted to volatile trimethylsilyl (TMS) derivatives; and (3) dissolved in organic solvents.40,78 With this technique, the mass spectrometer is employed as a detector, thus allowing positive identification of organic acids both by retention time and by their characteristic fragmentation spectrum. A random urine specimen is routinely used for this analysis; however, the most informative samples for the diagnosis of IEM are collected during acute metabolic decompensation. Organic acid analysis in blood or CSF usually is not informative to establish a diagnosis. Their interpretation is challenging because hundreds of compounds will be present in a specimen. Key factors in correct interpretation of results include (1) recognition of abnormal patterns and possible interferences due to dietary or medication artifacts, (2) knowledge about metabolic disorders and their presentation, and (3) information about the clinical status of patients.



Plasma Acylcarnitine Profile


Acylcarnitines derive from conjugation of carnitine with acyl-CoA. Carnitine [3-hydroxy-4-(trimethylazaniumyl)butanoate] is a water-soluble molecule that is essential in the transfer of long-chain fatty acids inside mitochondria for beta-oxidation. In addition, carnitine binds acyl residues accumulating in several organic acidemias and in fatty acid oxidation disorders, to facilitate their excretion. In the presence of a metabolic block (organic acidemia or fatty acid oxidation disorder), specific acylcarnitines, derived from conjugation of carnitine with acyl-CoA upstream of the metabolic block, accumulate, producing a pattern that is characteristic for each disease or group of diseases, so acylcarnitine analysis plays an essential role in the diagnosis of metabolic disorders (see Figure 58-2). Such an analysis is usually performed by tandem mass spectrometry (MS/MS) with or without liquid chromatographic separation prior to MS/MS detection.39,41 Plasma/serum is the biological fluid of choice, and whole blood spotted on filter paper is used for screening of newborns. Concentrations of acylcarnitines in plasma differ from concentrations in whole blood, especially for long-chain acylcarnitines. This is thought to be due to binding of long-chain acylcarnitines to the membranes of blood cells, resulting in reduced long-chain species in plasma. Plasma for analysis of acylcarnitines should be separated immediately after collection and kept frozen until analysis. Hemolysis has been known to result in elevated long-chain acylcarnitines, misleading the diagnosis. Storage of the sample at room temperature or even refrigerated may result in hydrolysis and, consequently, reduced concentrations of acylcarnitines. Urine acylcarnitine analysis is performed only in the diagnostic work-up of specific disorders, such as glutaric acidemia type I, and only if equivocal results are obtained with other tests. Quantification of acylcarnitines is typically performed using stable isotope dilution. However, some deuterated internal standards are not available for all identified acylcarnitine species. Caution should be taken when acylcarnitine results from different laboratories are compared, because the values may change depending on the internal standards used.14



Reference Intervals


Age-appropriate reference intervals should be used in the interpretation of biochemical genetics tests. Reference intervals for urine organic acids, urine acylglycines, plasma, and urine acylcarnitines are listed in Tables 58-4, 58-5, and 58-6.






Enzyme Assay and DNA Testing


Several inborn errors of metabolism produce a characteristic pattern of metabolites that is not observed in other conditions. For most, however, the diagnosis needs to be confirmed by a more specific method involving measurement of the activity of the putatively defective enzyme/transporter and/or DNA testing. This confirmation is very critical, in that for many conditions, specific therapy, if available, needs to be continued for life or is very aggressive (organ or bone marrow transplant). In addition, for some metabolic disorders, genotype-phenotype correlation with specific mutations affects the overall prognosis. For some diseases, such as phenylketonuria, the mutant enzyme is expressed only in the liver, and it is not practical and is very invasive to obtain diagnostic confirmation by enzyme assay. DNA testing (by sequencing of the whole gene) in this case is often more useful. For several other conditions, the missing enzyme is expressed in blood cells or in fibroblasts obtained by skin biopsy. It must be noted that with decreased costs of DNA testing, this technology is widely used to provide final diagnostic confirmation. The major limitation of DNA sequencing is that for certain conditions, the same biochemical abnormality is caused by deficiency of any of a number of genes (e.g., in methylmalonic acidemia), or multiple genes might be required to encode all subunits of a single enzyme (such as in maple syrup urine disease). Further, DNA sequencing (1) might not identify all mutations causing a disease, (2) can miss single exon deletions/duplications, and (3) can identify new variations whose clinical significance is unclear because they have not been reported in other affected patients. For these reasons, biochemical and molecular investigations need to be performed together to confirm or exclude the diagnosis of a metabolic disorder.



Disorders of Amino Acid Metabolism


Concentrations of individual amino acids in physiologic fluids reflect a balance between their intake, their release from the catabolism of endogenous proteins, their filtration and reabsorption by the kidney, and their utilization by the body to synthesize proteins or to produce energy. Changes in any of these processes can affect protein and amino acid metabolism through accumulation or excessive loss of one or more amino acids.


Inborn errors of amino acid metabolism can present at any time in a person’s life; most become evident in infancy and early childhood. Affected patients may have failure to thrive, neurologic symptoms, digestive problems, psychomotor retardation, and a wide spectrum of laboratory findings. If not diagnosed promptly and treated properly, these disorders can result in poor growth, mental retardation, and death.


Table 58-2 provides a summary of the most common disorders of amino acid metabolism and transport and their characteristics. Several of these disorders are discussed in the following sections.



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.

Nov 27, 2016 | Posted by in GENERAL & FAMILY MEDICINE | Comments Off on Newborn Screening and Inborn Errors of Metabolism
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