Approach to diagnosis of metabolic diseases

1Conceptual overview

The term “inborn errors of metabolism” was first used by Sir Archibald Garrod in his Croonian Lectures in 1908 [1] and in his monograph Inborn Errors of Metabolism in 1909 [2]. He defined these inborn errors as genetically determined diseases caused by blocks in the metabolic pathways due to deficient activity of an enzyme [3]. From Sir Garrod’s observations of patients with alkaptonuria, albinism, cystinuria, and pentosuria, he developed the concept that certain diseases of lifelong duration arise because an enzyme governing a single metabolic step is reduced in activity or missing altogether [4]. Most of these disorders may be classified as rare diseases because newborn screening data show clearly that their incidence ranges from 1:4,000 for congenital hypothyroidism to >1:250,000 (many disorders such as Maple Syrup Urine Disease (branched chain ketoaciduria).

The definition of an inborn error of metabolism is arbitrary. Scriver et al. [5] include several hundred diseases with definite biochemical genetic bases. McKusick’s catalog [6] contains several thousand diseases and disease states whose genetic abnormalities are described or assumed. Scriver et al. [5] solved the problem of exclusivity by adding “molecular” before encompassing every disease of the “textbook of the future.” The 2nd Edition of Metabolic Diseases: Foundations of Clinical Management, Genetics, and Pathology [7] is limited to those diseases with either: 1) recognized biochemical abnormalities supplemented with pathologic and genetic information, and in which a mutation leads to an enzyme deficiency that causes a metabolic disease such as phenylketonuria (PKU); or 2) disorders associated with known genetic mutations that have altered cellular physiology such as the chloride channel defect in cystic fibrosis or caused severe structural, cellular, or subcellular abnormalities. Emphasis in the book has been given to the more common and/or well defined hereditary biochemical/metabolic disorders. Prototype conditions such as Lesch Nyhan Disease, an excellent example of a nucleotide (purine) metabolism disorder, are also emphasized.

Investigation of the molecular functions of genes has determined that they control the cellular metabolic function in the pathogenesis of disease far beyond that commonly understood as a metabolic disease. The genetics of variant human phenotypes is discussed by Scriver et al. [5] and Champion [8]. The relationship between gene and enzyme was described as “one gene, one enzyme” by Beadle and Tatum [9–11]. Tatum expanded this concept as follows: 1) all biochemical processes in all organisms are under genetic control; 2) these biochemical processes are resolvable into series of individual stepwise reactions; 3) each biochemical reaction is under the ultimate control of a different single gene; and 4) mutation of a single gene results only in an alteration in the ability of the cell to carry out a single primary chemical reaction.

Pauling et al. [12] discovered direct evidence that human mutations actually produce an alteration in the primary structure of proteins. Their seminal research revealed a valine substitution for glutamic acid in sickle cell hemoglobin. Ingram [13] showed that inborn errors of metabolism were caused by mutant genes that produced abnormal proteins whose functional activities were altered.

It is estimated that some 30,000 genes constitute the human genome. A variety of genome maps are available, including some that are disease specific such as the “cancer cell map” [14]. The genome map is being constantly improved, with updated gene sequences and locations of intronic elements, revised lists of benign SNPs, additional reference genomes, databases of variant frequencies that are ethnicity-specific, and associations of specific genes with specific human diseases. In addition, combinations of genes can result in a metabolic error. Thus, although ∼7000 hereditary metabolic disorders are currently known, at least the same number of significant genetic errors as the number of genes seems theoretically possible, and an undetermined but large percentage of these may cause biochemical disorders with clinical impact. An estimation of genetic disease frequencies is shown in Table 1.

2Diagnostic strategies and tactics

Clinical diagnosis of metabolic disease is made by specific tests, biochemical analyses, and histologic and genetic studies that are discussed by Leonard et al. [15] and Cleary et al. [16]. New methods such as next generation sequencing of genes [17, 18] and even whole genome sequencing [19, 20] are facilitating diagnoses and other goals such as genetic counseling. Clinicians seek diagnosis, effective treatment, an understanding of prognosis, and the capability to explain and counsel patients and their families. Parents and patients appreciate gaps in knowledge but may be left seriously disappointed or angry without a cure. Fortunately, our increased genetic knowledge and better understanding of molecular pathophysiology of the past two decades has led to dramatic improvements in therapies using novel methods such as small molecule modulators of defective proteins [e.g., CFTR modulators for cystic fibrosis [21], gene therapy [22], and stem cell infusions [23]]. Thus, investments in expedited diagnostic strategies such as newborn screening can pay great dividends when well targeted, highly effective therapies are available.

Identification of inborn errors of metabolism provides the basis for diagnosis, prognosis, genetic counseling, and, in an increasing number of patients, targeted treatments aimed directly at the fundamental defect. Many infants and children present as medical emergencies and life-preserving treatment is begun before a definite diagnosis is available. Diagnosis should be made rapidly and the implications and complexities of treatment explained in detail. Metabolic disease is usually not suspected in patients until more common diseases are considered. Those children with a history of consanguinity or those in families with a history of unexplained death, multiple spontaneous abortions, or previously diagnosed metabolic diseases have an increased likelihood of a metabolic disease. Frequently, children (and some adults) with metabolic diseases are first seen as critically ill patients with non-specific findings. Dehydration, acidosis, vomiting, ammonemia, hypoglycemia, or seizures must be managed aggressively. Inborn metabolic errors may be suspected if response to emergency treatment is not as expected.

Metabolic diseases can be divided into 3 main categories as listed in (Table 2). The majority of these disorders are inherited as autosomal recessive traits. Some such as Duchenne muscular dystrophy are X-linked. A few are inherited as dominant traits, and mitochondrial disorders form a genetically separate category. Mitochondrial enzymes are coded by both the maternal nuclear genome and by the mitochondrial DNA.

Clinical findings associated with metabolic diseases are listed in Table 3. It has been well recognized that symptoms may be quite variable, even in a group of patients with the same mutation. Thus, it has become clear that genetic modifiers may alter the expression of signs/symptoms and that gene-environment and nutrition-metabolism relationships can also influence the disease liability of pathologic genetic mutations.

The newborn with a severe metabolic abnormality may present with symptoms of apparent sepsis or asphyxia. These symptoms usually consist of irritability, failure to feed or suck, flaccidity, or coma. Previous miscarriages or sudden unexpected death in a sibling should trigger a screening investigation for metabolic disease. Sophisticated newborn screening using 4-5 blood spots on filter paper [“Guthrie cards” named for Robert Guthrie, the originator of population-based newborn screening with dried blood spots [24]], combined with tandem mass spectrometry as discussed by Ziadeh et al. [25] and Millington et al. [26] or molecular analyses [21] can identify a number of life-threatening diseases with a single, multiplex assay. The hereditary metabolic diseases that can now be screened for from filter paper blood spots in the newborn are listed in Table 4. Recently, severe combined immunodeficiency (SCID) has been added in many regions as well as “point of care” screening methods such as oxygen saturation measurements for critical congenital heart diseases.

Metabolic abnormalities may be suspected in infants with hydrops fetalis Table 3, or with placental abnormalities. Routine placental examination may disclose fetal metabolic storage disease by the presence of vacuolations of syncytiotrophoblasts, intermediate trophoblasts, and stromal Hofbauer cells. Suspected metabolic errors in the newborn are emergencies and require prompt identification and treatment. After obtaining the appropriate specimens for testing, emergency treatment must be instituted. If the patient dies, regardless of age, preparation should be made for a metabolic disease autopsy as discussed by Ernst et al. [27].

Pregnancy may occur in patients with metabolic diseases. Some of these conditions can be teratogenic. Many such pregnancies can be successfully managed with treatment. Treatment of these disorders is discussed in each chapter.

Examples of inborn errors of metabolism potentially occurring in high frequency among specific ethnic groups are listed in Table 5. However, it should be noted that some of these disorders are quite rare now in the era of prenatal diagnosis. For instance, the incidence of Tay Sachs disease in targeted Jewish populations is extremely low worldwide [28, 29].

The most common presentations of inborn errors are without gross physical anomalies and include disorders of amino acid, fatty acid, organic acid, and carbohydrate metabolism Table 6. Others present with dysmorphic features, neonatal deaths, self-mutilation, abnormal body or urine odor, hypotonia, deafness, or recurrent acidosis with or without ketosis. Failure to recognize and, when possible, treat these infants can result in irreversible neurologic damage.

Diagnostic strategy consists of meticulous attention to history and physical examination followed by appropriate screening tests, including biochemistry, radiology, or other modalities, and specific enzyme, protein, or gene analysis. Special educational services for children with inborn errors of metabolism may be available and therefore accuracy of diagnosis is essential as discussed by Powell et al. [30]. Clinical investigation is augmented by a large number of common blood and urine tests (blood, sugar, urine pH, anion gap, cytopenia, liver function tests, sweat test, electroencephalogram, and radiography of bones). Radiologic examination may include X-rays, CT scans, and MR imaging. MRI scans should be substituted for CT scans whenever possible to avoid radiation exposure. Ophthalmologic and neurophysiologic investigations are often important. Laboratory findings in inborn errors of metabolism are listed in Table 7. Abnormal metabolites are shown in Table 8.

Abnormalities in serum electrolytes can be caused by vomiting or diarrhea. Inclusion of bicarbonate and pH assessment may indicate a large anion gap due to the presence of organic acids. Electrolytes, however, may be normal with organic acidemias.

Neutropenia or pancytopenia suggests the presence of one of the organic acidemias. A urinalysis may indicate the presence of reducing substances and the presence or absence of ketones. Ketonuria is not found in normal newborns. Urine pH above 5.0 in an acidotic infant may suggest renal tubular acidosis. Absence of ketones in association with hypoglycemia may indicate an error in fatty acid metabolism. It is helpful to save a small amount of frozen urine and plasma for later evaluation of keto acids, carnitine, and organic acids.

Elevated serum ammonia levels are found in patients with liver disease and in those with errors of the urea cycle and several of the organic acidemias. Transient hyperammonemia occurs in some newborns, and this is reversible when treatment is prompt. Urea cycle errors are frequently associated with very low blood urea levels. Some metabolic disorders cause liver cirrhosis in infants and children and some liver disorders progress to stupor and coma.

Liver function tests commonly indicate primary liver diseases. However, abnormal liver function test findings result from many of the metabolic diseases, including disorders of organic and amino acid metabolism, and many of the storage diseases.

Elevated serum lactate is usually due to hypoxemia or poor perfusion and occurs with sepsis. It may be due, however, to improper technique in drawing blood without good blood flow. Lactic acid is produced in the anaerobic metabolism of glucose through pyruvate, which is then converted in the liver to glucose. Deficiency of pyruvate dehydrogenase complex is the most common error of metabolism that causes lactic acidosis.

Myopathy can be caused by a few of the glycogen storage diseases or mitochondrial disorders. Muscle biopsy or fibroblast cultures may be diagnostic. Myoglobinuria may be present, and serum free carnitine may be decreased.

Peroxisomal action is responsible for β-oxidation of fatty acids, and deficiency results in developmental retardation. Peroxisomal disorders with such manifestations include Zellweger syndrome, adrenoleukodystrophy, and oxalosis [31]. Very-long-chain fatty acids are elevated in the serum with most forms of peroxisomal disorders.

Many chemical screening programs such as determination of organic and amino acids, oligosaccharides, and glycosaminoglycans in blood and urine are available for diagnostic workup of suspected progressive degenerative metabolic diseases. Distinguishing biochemical findings of inborn errors of metabolism are shown in Table 9.

Some inherited metabolic diseases significantly increase the risk of intercurrent illness. For example, recurrent treatment-resistant otitis media is a common problem in children with mucopolysaccharide storage diseases because distortion of the Eustachian tube and production of particularly tenacious mucus combine to create a favorable environment for bacterial colonization of the middle ear. The neutropenia that is a prominent feature of glycogen storage disease type Ib, and some of the organic acidopathies, predisposes to pyogenic infections. Classic galactosemia predisposes infants to neonatal Escherichia coli sepsis [32].

Major congenital malformations, such as meningomyelocele, complex congenital heart disease, and major congenital limb deformities, are not generally considered signs of an underlying inherited metabolic disease; however, some inherited metabolic conditions occur with dysmorphism so characteristic that a strong presumptive diagnosis can be made on physical examination alone [32]. A large number of metabolic diseases present with gross abnormalities of appearance Table 10A and 10B. Many of these have lysosomal defects, and most will have hepatosplenomegaly. These include such diseases as mucopolysaccharidoses, sphingolipidoses, and other lysosomal storage diseases. These diseases are not acutely life-threatening. Children with these conditions commonly have developmental delay. Many appear normal at birth and progressively deteriorate Table 3.

An important step between the clinical findings and the biochemical findings is the anatomic pathology, including histology, histochemistry, immunochemistry, and electron microscopy (EM). For example, screening peripheral blood for cytoplasmic vacuoles in the lymphocytes as shown in Fig. 1 may be the first indication of a storage disorder.

Some metabolic disorders present as acute encephalopathy. The earliest signs of encephalopathy may be no more obvious than excessive drowsiness, unusual behavior, or some unsteadiness of gait. Acute or intermittent ataxia is a common sign of acute encephalopathy in older children with inborn errors of metabolism. A history of recurrent attacks of unsteadiness of gait or ataxia, especially when associated with vomiting or deterioration of consciousness, should stimulate investigation of a possible inherited metabolic disease Table 11 [25].

In addition to the biochemical tests of blood and urine, solid tissues and cell cultures may be needed for biochemical assays. A biopsy specimen should be divided for biochemical analysis and morphologic investigation. This is particularly important in liver, muscle, and intestinal biopsies and in skin biopsies made for the fibroblast cultures.

Hypoglycemia occurs with starvation in young infants but also may indicate an error in gluconeogenesis, errors in fat metabolism and defects in hormone metabolism. A rapid response to glucose, especially in the presence of hepatomegaly, may suggest glycogen storage disease or fructose-1,6-diphosphatase deficiency. An approach to diagnosis when a patient presents with hypoglycemia is shown in Fig. 2. This algorithm will guide clinicians toward a proper sequence of diagnostictests.

Even after death, some of the techniques used in the living can be helpful in detecting inborn errors of metabolism. For example, even small amounts of urine may remain in the bladder and should be aspirated and saved. Tissues and body fluids should be obtained and maintained in a state suitable for the tests desired. New diseases and diagnostic tests are identified at a surprisingly rapid rate. Very few laboratories perform all the tests.

3Further development of prenatal and neonatal screening

As techniques such as next generation sequencing [17, 18] and whole genome sequencing are developed further for routine use, there will be many more commercial laboratories offering comprehensive testing as has already proved helpful for newborn intensive care units [19, 20]. However, reporting and ethical issues need to be resolved before whole genome sequencing can be used widely [33].