Biomarkers in Inborn Errors of Metabolism: Clinical Aspects and Laboratory Determination

book.

Preface

Uttam Garg and Laurie D. Smith

Inborn errors of metabolism are biochemical genetic disorders that result from the deficiency of enzymes, membrane transporters, or other functional proteins. Patients with these disorders may present with either acute overwhelming sickness or a prolonged, smoldering illness. For the former, rapid diagnosis is vital in the diagnosis and follow-up of these patients to limit morbidity and mortality. For the latter, diagnosis is important for the initiation of appropriate clinical care. Clinical presentation resulting from these disorders can be quite variable. Therefore, use of laboratory tests and biomarkers are indispensable in the diagnosis and follow-up of these disorders. Rapid tests are generally available for the initial presumptive diagnosis and management of acutely ill patients. Specialized tests involving laboratory biomarkers are not available in most medical centers, and are needed for the confirmation and follow-up of these disorders. Moreover, laboratory biomarkers are used in newborn screening to diagnose biochemical genetic disorders in asymptomatic patients.

In this book, Biomarkers in Inborn Errors of Metabolism, the major emphasis is on the test selection and biomarkers used in the diagnosis and follow-up. When possible, biochemical pathways and illustrative chromatograms are provided. In addition, basic information on clinical presentation, pathogenesis, treatment, and prognosis is also presented. Furthermore, confounding factors that may mimic biomarkers used in the diagnosis of inborn error of metabolisms are also covered.

We are indebted to our authors and colleagues for their excellent contributions and making this book possible. We are also thankful to our patients and families who have taught us a lot. Last but not the least, we would like to acknowledge the support of Jeffery Rossetti and Fenton Coulthurst, editorial project managers, for their support during the preparation of this book.

Chapter 1

Introduction to laboratory diagnosis and biomarkers in inborn error of metabolism

U. Garg¹,³ and L.D. Smith²,    ¹Children’s Mercy Hospitals and Clinics, Kansas City, MO, United States,    ²University of North Carolina School of Medicine, Chapel Hill, NC, United States,    ³University of Missouri School of Medicine, Kansas City, MO, United States

Abstract

Inborn errors of metabolism (IEM) are genetic disorders of intermediary metabolism that result in metabolic defects due to deficiency of enzymes, membrane transporters, or other functional proteins. Many of these disorders are detected through newborn screening or clinical suspicion. Laboratory tests and use of biomarkers are essential in the diagnosis and follow-up of patients with IEM. Specialized tests are not available in most hospitals, and need special attention in specimen collection, processing, and transport. Routine tests, although not necessarily diagnostic, are important in initial patient management and in providing guidance for further testing and management. Furthermore, there may be conditions that present with biochemical findings that mimic or confound a diagnosis. Unlike routine laboratory tests that are generally automated, most biochemical genetics tests are developed in-house. Method development and quality control can be challenging due to lack of standardized reagents and limited availability of external assessment programs. Treatment and prognosis of these disorders varies significantly, from dietary management to enzyme replacement therapies.

Keywords

Inborn errors of metabolism; inherited metabolic disorders; amino acids; organic acids; acylcarnitine; quality control; reference ranges; method evaluation

1.1 Introduction

Inborn errors of metabolism (IEM) are genetic disorders of intermediary metabolism. The majority of these disorders are due to single gene defects resulting in the deficiency of an enzyme, membrane transporter, or other functional protein. Timely biochemical genetic testing, and, in many cases, newborn screening are important in early recognition and treatment of these disorders. Often there is accumulation of toxic substrates or metabolites or deficiency of essential products. Clinical presentation resulting from IEM can be quite variable. Broadly, IEM can be divided into those that involve defects in metabolism of complex molecules, those that result in acute intoxication and those that result in energy deficiency.¹,² Patients with IEM are diagnosed through clinical suspicion or newborn screening. A family history can provide clues but does not exclude an underlying IEM. One of the scenarios that might suggest an IEM in a neonate is an acute sepsis-like illness, poor feeding, lethargy, poor growth, and so on, that does not respond to conventional treatment. In an older child, symptoms such as vomiting, metabolic acidosis, ataxia, or coma might suggest an IEM. Further testing is needed to confirm a clinical suspicion of an IEM. In addition to making a diagnosis of an IEM in a symptomatic patient, a significant number of patients are diagnosed through newborn screening programs. In recent years, newborn screening programs have expanded to include more than 50 inherited metabolic disorders, including aminoacidopathies, organic acidemias, fatty acid oxidation disorders, and lysosomal storage disorders.³–⁶ Newborn screening results are considered presumptive positive and are confirmed by more definitive laboratory tests.⁶,⁷ Many of these disorders may present later in life with chronic and progressive multisystem (skeletal muscle, liver, kidneys, gastrointestinal tract, eyes, skin, or central nervous system) symptoms. Lysosomal disorders involving the accumulation of complex molecules may present with behavioral changes, coarsening of facial features, or joint contractures. The IEM can also present with specific organ involvement such as a cardiomyopathy or hepatic dysfunction. Generally, an IEM should be considered in any patient who does not have a clear diagnosis and does not respond to conventional treatment. Laboratory tests are often needed to confirm clinical suspicion and to make a definitive diagnosis. This chapter covers the basics of laboratory tests and biomarkers in the diagnosis of IEM. A number of excellent publications are available on the clinical aspects of inherited metabolic diseases.¹,⁸–¹⁷

1.2 Laboratory Biomarkers and Tests in Diagnosis of IEM

In most IEM, clinical symptoms are nonspecific: vomiting, poor feeding, lethargy, hypotonia, seizure, poor linear growth, poor weight gain. Biochemical genetic testing is essential for the diagnosis and clinical management of patients with IEM. The purpose of testing is to either confirm or exclude the diagnosis of a suspected disorder. Once the diagnosis of a particular disorder is established, specific biomarkers are followed to monitor clinical management of the patient.

Initial laboratory tests such as blood gases, pH, glucose, electrolytes, ammonia, lactate, renal function tests, liver function tests, urinalysis, and basic hematological tests are available in most hospital laboratories. Although these tests are not diagnostic of a specific IEM, they are very helpful in managing the acutely ill patient. These tests can also provide important clues in the differential diagnosis of inherited metabolic disorders. For example, hyperammonemia, lactic acidemia, ketonuria, and hypoglycemia may point toward a metabolic condition, such as an organic acidemia.¹⁰,¹⁸ Routine laboratory tests and their association with various diseases are shown in Table 1.1.¹⁹ More specialized tests such as plasma amino acid profiles, urine organic acid profiles, plasma acylcarnitine profiles, free fatty acid profiles, pyruvate, acetoacetate, 3-hydroxybutyrate, mucopolysaccharides, oligosaccharides, enzyme activity assays, functional assays, and mutational analyses are needed to make the definitive diagnosis.⁷ Specialized tests are not available in the routine clinical laboratories, and are sent to specialized centers. The GeneTests website (https://www.genetests.org/), supported by National Institutes of Health, is an invaluable source for identifying laboratories experienced with specialized metabolic testing. Some laboratories also perform spot screening tests. These tests can provide quick, useful information when specialized tests are not available. Since these screening tests lack specificity or sensitivity, results of these tests should be interpreted with caution and in context with clinical information. Several screening tests are listed in Table 1.2.¹⁹,²⁰

Table 1.1

Routine Laboratory Tests/Biomarkers with Commonly Associated Diseases

Modified and Reprinted with permission from Garg U, Smith LD, Heese BA. Introduction to the laboratory diagnosis of inherited metabolic diseases. In: Garg U, Smith LD, Heese BA, eds. Laboratory Diagnosis of Inherited Metabolic Diseases. Washington, DC: AACC Press, 2012:1–12.

Table 1.2

Urinary Metabolic Screening Tests

Many IEM may only be apparent when a patient is under metabolic stress. When well, the disease markers may be normal or only slightly abnormal providing inconclusive information. It may be necessary to carry out functional tests to artificially create metabolic stress in these patients, although in this era of gene sequencing, functional tests are being used less frequently. In general, in functional testing, the patient is exposed to a high concentration of a specific substrate to cause metabolic stress, and specimens are collected before and after metabolic stress. Functional tests should be carried out only by an experienced care provider as they may lead to dangerously high levels of toxic metabolites and cause serious complications. In recent years, laboratory diagnostic techniques have improved significantly reducing the need for functional tests. Some functional tests are listed in Table 1.3.²¹

Table 1.3

Functional Tests

Diagnostic biomarkers for various disorders are given in Table 1.4.²,⁹,¹⁸,¹⁹,²²–²⁹

Table 1.4

Diagnostic Biomarkers for Various Disorders

B—Blood/Plasma/Serum; U—Urine; CSF—Cerebrospinal fluid; Acylcarnitine. Abbreviations: C0 =Free Carnitine; C3 =Propionylcarnitine; C4 =Isobutyryl/Butyrylcarnitine; C5:1 =Tiglylcarnitine; C5 =Isovaleryl/2-methylbutyryl; C5-OH=3-OH-Isovaleryl/2-methyl-3-OH-butyrylcarnitines; C5-DC =Glutarylcarnitine; C6 =Hexonylcarnitine; C6-DC =3-Methyl-glutarylcarnitine; C8 =Octanoylcarnitine; C10:1 =Decenoylcarnitine; C14 =Tetradecanoylcarnitine; C14:1 =Tetradecenoylcarnitine; C14:2 =Tetradecadienoylcarnitine; C16 =Hexadecanoylcarnitine; C16-OH =3-Hydroxyhexadecanoylcarnitine; C18:1 =Oleylcarnitine; C18:2 =Linoleylcarnitine; C18-OH =3-Hydroxystearoylcarnitine; C18:1-OH =3-Hydroxyoleylcarnitine; SAICAR =5-phosphoribosyl-5-amino-4-imidazole-succinocarboxamide riboside; VLCAD =Very long-chain acyl-CoA dehydrogenase deficiency.

aThese are common laboratory biomarkers/tests. Details of these biomarkers/tests are provided in individual chapters.

1.3 Specimen Types

Whole blood is collected on filter paper for newborn screening. Commonly used specimens for metabolic testing include serum or plasma, urine and cerebrospinal fluid. Amniotic fluid, fibroblasts, leukocytes, liver and muscle are also used in the diagnosis and confirmation of inherited metabolic diseases. In postmortem investigations, bile and vitreous fluids have been used in the diagnosis of IEM. For most analyses, serum/plasma is the specimen of choice since it is less variable as compared to urine. Urine is the specimen of choice for diagnosis of certain disorders such as organic acidurias and renal transport diseases. In these disorders abnormal metabolites concentrate in urine. To normalize for sample dilution and reduce variability, urine results are generally expressed relative to creatinine concentration. CSF is used in the diagnosis of neurotransmitter disorders and certain other diseases such as non-ketotic hyperglycinemia.

1.4 Specimen Collection and Processing

The importance of correct specimen collection and processing cannot be over emphasized. It is important that the right specimens are collected at the right time. Many tests need special attention during specimen collection. For example, when possible, the best specimen for the diagnosis of the aminoacidopathies is from a fasting patient. Specimens for lactate and ammonia testing should be put on ice immediately after collection. Since many medical centers do not encounter or perform specialized biochemical genetics tests, it is important that instructions for specimen collection and processing from reference laboratories be followed closely. Furthermore, for correct interpretation, it may be necessary to collect additional information such as the patient’s clinical status, medications, nutritional status, and so on.

Specimen requirements for commonly used tests are given in Table 1.5.¹⁹

Table 1.5

Specimen Collection and Handling

The table provides general information that can vary among laboratories.

Modified and Reprinted with permission from Garg U, Smith LD, Heese BA. Introduction to the laboratory diagnosis of inherited metabolic diseases. In: Garg U, Smith LD, Heese BA, eds. Laboratory Diagnosis of Inherited Metabolic Diseases. Washington, DC: AACC Press, 2012:1–12.

1.5 Specimen Analysis, Quality Control, and Quality Assurance

Specimen analysis, quality control, and quality assurance in the biochemical genetics laboratory can be quite challenging. Unlike routine chemistry laboratory studies, most biochemical genetics tests require quite extensive manual sample preparation. Most methods require specialized techniques such as gas-chromatography mass spectrometry, liquid-chromatography mass spectrometry, and tandem mass spectrometry. Specialized personnel and training are needed to properly operate and troubleshoot these instruments. Furthermore, premade commercial reagents, standards, calibrators, internal standards, and controls are not available for most of the biochemical genetics tests, and the laboratory is responsible for preparing and performing quality control of these reagents. For example, many organic acids and acylcarnitines of clinical interest are not available as pure compounds. Their estimation is based on surrogate calibrators that are semi-quantitative at best. Owing to these challenges, there is significant variability among different labs. This is evident from the College of American Pathologists (CAP) survey results shown in Table 1.6. The coefficient of variation for certain analytes is >100%. In addition, significant variability exists among laboratories in identifying the correct diagnosis (Table 1.7). For some diagnoses, 20–25% laboratories fail to identify the correct diagnosis.

Table 1.6

Variability Among Different Labs for Certain Analytes on Recent CAP Surveys (2014/2015)

a% CV (coefficient of variation) is for all methods.

Table 1.7

Laboratories Reporting Correct Diagnosis on Recent CAP Surveys (2014/2015)

Quality control is an integral part of sample analysis. At least two, and generally three, quality control samples are included in the analysis of each batch of clinical samples. Quality control samples have values in the physiological and pathological range. Patient sample results are considered acceptable if concentrations of quality control samples are within the established range. Quality control ranges are generally established by the repeat analysis of the controls and calculating the range from mean ±2 or 3 standard deviations (SDs). Mean ±2 SD range is preferred, but may result in a high rejection rate. On the other hand, mean ±3 SD range will result in high acceptance rate with the possibility of falsely low or high patient results. These quality control schemes work well for methods involving single analytes, but may not work well for the analyses involving multiple analytes such as amino acid, acylcarnitine, and organic acid profiles. For example, when the 2 SD rule is used, the chances of one quality control sample being within quality control range is 95%, and the probability of two controls being within quality control ranges is 0.95 ×0.95 =0.9025 or 90.25%. The chances of all the controls within control ranges, for a 20-component analysis, is only (0.95)^²⁰ײ = 0.128 or 12.8%. Although these calculations assume independent analyses, and therefore overestimate the failure rate, nevertheless, this does make a point that in multicomponent analysis, simple quality control schemes do not work. Therefore, subjectivity is involved in accepting quality controls in multicomponent analysis by a properly trained analyst. In addition, the presence of an analyte, regardless of concentration may be clinically important. For example, detection of a succinylacetone peak on a urine organic acid profile may be clinically significant despite the lack of an acceptable quality control for succinyl-acetone. Therefore, the analyst should be knowledgeable and be able to objectively judge the performance of a method to be acceptable for clinical use.

Another important component of quality assurance is quality assessment through internal or external quality assessment programs. In the United States, specialized biochemical genetics tests are considered high-complexity tests under Clinical Laboratory Improvement Amendments (CLIA) of 1988. Laboratories that perform these tests must meet the CLIA requirements for high-complexity testing. For accreditation and quality control and assurance purposes, laboratories have to develop internal or external quality assessment programs. External quality assessment through proficiency testing, for a limited number of analytes, is available from the CAP³⁰ and the European Research Network for Evaluation and Improvement of Screening, Diagnosis, and Treatment of Inherited Disorders of Metabolism (ERNDIM).³¹ These external proficiency-testing programs provide a glimpse into the challenges in the biochemical genetics laboratory diagnosis. As shown in Table 1.7, correct diagnosis among different labs varied from 75 to 100%.

1.6 Method Selection and Evaluation

Owing to the reasons listed above, method development and ongoing quality control for biochemical testing can be challenging. Before bringing a new method into the laboratory, several considerations including patient care needs, equipment, reagents, staffing, employee competency, quality control, and proficiency testing should be assessed. CLIA regulations and good laboratory practice require the establishment and/or verification of assay performance characteristics that include accuracy, precision, analytical sensitivity, analytical specificity, and reportable range. In addition, method validation may include method comparison with an established method, interferences, recovery, specimen stability, and verification or establishment of reference ranges.³²–³⁶

Accuracy refers to the closeness of the test result to the true values. There are various ways to determine the accuracy. It can be determined by comparing the results of the method in development with the results from a well-established reference method. In another approach, matrix-matched samples containing known amounts of the analyte are prepared, and the concentrations are compared to the known concentrations of a certified material. When possible, matrix-matched certified materials should be used. A minimum of three, ideally five to six, concentrations that cover the expected concentrations should be used. These specimens are analyzed several times, generally three to five, and the mean is calculated. Accuracy is determined by comparing the measured values with the target values, and is considered acceptable if the bias is within acceptable limits. Deviation of less than 10–20% is generally considered acceptable.

Precision of an analytical method refers to statistics of reproducibility, namely, the closeness of individual results. Precision is generally measured by evaluating imprecision, and expressed as coefficient of variation (CV) that is defined as relative standard deviation (RSD) and is calculated as SD ×100/Mean. Precision studies are generally conducted using quality control materials made from same biological matrix as the intended samples. Two to three concentrations in the expected range are generally used. Both short-term (within-run or within-day) and long-term (between-run or between-days) imprecision should be evaluated. Short-term imprecision can be evaluated by running quality control samples, ~20 times, within a run or within a day. Long-term imprecision is evaluated by analyzing quality control samples, ~20 times, at different days. Long-term imprecision is typically evaluated across users and different instruments. Imprecision of less than 10–20% is generally considered acceptable. Clinical and Laboratory Standards Institute (CLSI) guidelines are available for evaluating and verifying precision.³²,³³

Analytical sensitivity refers to lowest concentration of an analyte that can be measured with confidence. For most clinical assays, the lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) are evaluated. LLOD is the lowest concentration that a method can detect or the lowest concentration that can be differentiated from zero concentration with confidence. LLOD can be calculated by running a blank sample that does not contain an analyte of interest multiple times, and calculating the value from mean +2 or 3 SD. Since a number of biochemical genetics methods use chromatographic technique, a different approach is generally used to calculate LLOD. In chromatographic techniques, blank samples are spiked with low and varying concentrations of analyte(s) of interest. The lowest concentration that produces a clear peak is considered LLOD. LLOQ is defined as the concentration that can be measured with a defined accuracy and precision. Limit of quantitation can be calculated by analyzing samples of low analyte concentrations. CV is calculated at each concentration. The lowest concentration at which an acceptable CV is obtained is referred as LLOQ.

Analytical specificity, also frequently referred as selectivity, refers to the ability of an assay to measure an analyte of interest only and not other analytes. Ideally, a method should not measure nontarget analytes, but this is often not possible. Analytical specificity of a method is measured by spiking patient samples with potential interferents, and measuring analyte(s) of interest with and without the presence of interferents. It is generally not possible to carry out these studies in detail. Literature should be consulted for possible interferences and the laboratory should be aware of these interferences, and make sure that the patient results are not affected or reported in case of interferences. In recent years, mass spectrometry assays are increasingly being used in biochemical genetics laboratories. These methods are relatively more specific and less prone to interferences.

Reportable range, also referred as linearity, is the range of analyte concentrations that a method can measure without dilution. In multistandards assay, it is the range between the lowest and highest standards used to produce a calibration curve. Reportable range can be calculated by analyzing four to six concentrations that cover the reportable range, and calculating the measured values with the target values within allowable error. CLSI guidelines are available for evaluating linearity.³⁷ In a more complex multiple component analysis, it may not be easy or feasible to calculate reportable range for all the analytes. For example, in amino acids analysis, it is difficult to validate very high concentrations of individual amino acids by spiking very high concentrations of individual amino acids. Also, it is not known how a very high concentration of one amino acid can affect the quantitation of another amino acid. To avoid delay in result reporting, it may be acceptable to report an approximate value that is close to an actual value.

Reference intervals also called as reference values, normal values, and expected values, refer to the range of values found in a healthy or reference population. For endogenous compounds, reference intervals are customarily defined as limits covering central 95% of the values or the mean ±2 SD (if the data has Gaussian distribution). Reference ranges are either established or taken from the literature and verified. Many factors including age, sex, race, geographic location, fasting, specimen type, and so on can influence reference intervals. Reference intervals must be verified or established using well-defined