Medical Genetics and Biochemistry in Diagnosis and Management

Clinical Approach

The evaluation of patients with inborn errors of metabolism begins with obtaining a comprehensive history from parents, caregivers, spouses, or other individuals familiar with the patient. Diagnostic clues may be obtained from prenatal and birth history, history of recurrent illnesses in the past, diet history with dietary preferences, and history of exposure and response to pharmaceutical agents. A family history of prior affected individuals, early deaths, or significant medical events in relatives, including unusual symptoms or signs, are invaluable to the diagnosis of metabolic diseases. The developmental history provides insight into neurologic maturation, which may be affected by the presence or lack of liver-based metabolites; recurrent episodes of hyperammonemia from urea cycle disorders (eg, ornithine transcarbamylase deficiency) or hypoglycemia (medium chain acyl-CoA dehydrogenase [MCAD] deficiency) may significantly affect neurologic function. Liver disease in children may manifest with symptoms that are distinct from those in adults and may include refusal to eat, early satiety, avoidance of foods, and onset of vomiting/lethargy with certain foods. Children affected by disorders of protein catabolism (ie, urea cycle defects) may often have a dietary history of avoiding meat, cheese, or milk. Individuals who have hereditary fructose intolerance often avoid sweet beverages or fruits because of nausea or illness induced by liver dysfunction and hypoglycemia.

Physical examination may provide clues through alterations in growth patterns or deviation from normal in weight, length, head circumference, and other parameters. Examination of the abdomen may be difficult in infants and children, and abdominal distention, pain, or tenderness may be the only available results. Neurologic findings such as acute or chronic hypotonia, hyperreflexia or hyporeflexia, muscle atrophy, and altered mental status may provide support to diagnostic considerations.

Integration of patient history with clinical signs and symptoms often helps the examiner decide what tests need to be carried out; the emphasis always remains on using noninvasive methods before turning to major tissue sampling. Some of the common findings for well-known inborn errors of metabolism are shown in Table 6.1 . Unusual inborn errors that are not associated with acute illness may often signal their presence through multiple subtle signs and may have a wide range of clinical manifestations. As an example, congenital disorders of glycosylation (CDG) are a group of disorders of abnormal glycosylation of N-linked oligosaccharide-containing proteins caused by a deficiency in 1 of at least 42 different enzymes. Manifestations may appear in infancy and may range from severe developmental delay and hypotonia with multiple organ system involvement to hypoglycemia and protein-losing enteropathy with normal development. The clinical course is also highly variable, ranging from death in infancy to normal life expectancy with minimal functional compromise. Patients affected by mitochondrial or other neuromuscular disorders may manifest histories similar to those with CDG and even show similar results on standard screening tests, in the face of completely different underlying pathophysiologic processes. The means of differentiating CDG from mitochondrial/neuromuscular disease lies within the realm of biochemical and/or molecular genetic testing.

Approach to Biochemical and Genetic Investigation

The investigation of acutely ill patients with potential inborn errors is greatly facilitated by determining the most proximal cause of symptoms (eg, acidosis, hyperammonemia, hypoglycemia). A simplified diagnostic algorithm with this approach is shown in Fig. 6.1 . Such biochemical clues may not be present in a chronically ill patient with episodic illness, and diagnostic hints have to be obtained by screening tests. Because of the multitude of disorders with similar symptoms, it is often necessary to evaluate for a long list of diseases, proceeding from the more common to the exceedingly rare. The use of screening panels for metabolites, beyond those of standard chemistry testing, is often necessary. Some of the commonly used testing panels are shown in Table 6.2 . Although positive results may be obtained in asymptomatic patients, sampling may have to be carried out on multiple occasions, especially when the patient is symptomatic. The timing of the sampling may be critical because some metabolites such as organic acids and amino acids may be influenced by recent diet and oral intake or by infusion of fluids. Analysis of the metabolites may take several days to weeks, depending on the reference laboratory. At times, treatment for a suspected disorder may need to be initiated before laboratory results become available.

Once a critical metabolite or group of compounds has been identified, specific investigations can be undertaken to define the underlying biochemical and/or genetic error precisely; these investigations may require obtaining tissue for microscopic evaluation as well as biochemical and/or genetic testing. Tissue sampling may also be required if an abnormal metabolite is not detected by the initial screening panels. It is at this point that the surgical pathologist becomes an important part of the investigating team. Because a large number of disorders involve the liver, liver biopsies are often sought for diagnosis of metabolic diseases.

Collection, Storage, and Shipping of Specimens

The diagnosis of metabolic disorders often involves the use of sophisticated methodologies, which may be offered only in specialized referral laboratories; thus, the collection, preparation, storage, and shipping of body fluids and tissue samples is a critical step in the care of patients suspected to have inborn errors of metabolism. Biologic material for biochemical and genetic analysis requires more stringent procedures for procurement, handling, and transportation to the testing facility to prevent degradation of the often labile and sometimes unstable metabolites and enzymes. Table 6.3 lists the optimal methods for collection, storage, and shipping of the most commonly procured biologic specimens.

The most common specimens used for biochemical and genetic analysis include whole blood, plasma, red or white blood cells, urine, spinal fluid, and tissue biopsy samples of the skin or other organs such as the kidney, liver, heart or muscle. In the past, tissue biopsies were usually obtained to confirm the diagnosis suspected from initial screening panels. With the advent of lower cost and rapid DNA sequencing technology offering single or multiple gene testing, tissue biopsies can often be avoided unless gene testing results are not positive. In the case of potentially affected females with X-linked or mitochondrially inherited disorders, DNA testing results may not be positive, and enzymatic diagnosis may be needed for confirmation. In the setting of mitochondrial or neuromuscular disorders, confirmation of altered protein expression needed to initiate therapy or conclude the process of evaluation may not yield definitive results and genetic testing may be the only diagnostic modality. In other cases, initial indirect testing may have failed to detect or identify an abnormal metabolite, and tissue from a symptomatic organ, such as the liver, may be needed for further testing. Diagnostic information may be derived not only from histologic findings but also from electron microscopy, enzyme assays, nucleic acid analysis, immune blots, metabolite testing, or even transient cell culture. Depending on the needs of the assay, the samples may have to be obtained by interventionally trained physicians such as surgeons, radiologists, or gastroenterologists. Pathologists may participate in tissue procurement at autopsy, which in cases of suspected metabolic disorders, should be conducted expediently to prevent autolysis and preserve metabolites of interest.

Tissue samples for diagnostic investigations may often be obtained at the same time as feeding tubes are placed in severely ill patients. Unfortunately, infants and small children with muscle disease or poor growth often have small muscle mass, which makes it difficult to obtain sufficient material for adequate enzyme or nucleic acid analysis. Tissue sampling under these circumstances may have to be directed to other sites, including the liver or skin (to obtain cells for culture), to obtain sufficient material for diagnostic testing.

Sample preparation in the operating room may be necessary before storage and/or freezing. Common sample preparation may include washing in defined media such as saline or other solutions to remove blood and other materials followed by blotting to remove moisture. Specimens destined for long-term storage or shipping to a distant facility are frozen in specific agents such as dry ice, liquid nitrogen, or other supercooled organic solvents because simple freezing at −20°C is often insufficient for long-distance travel or prolonged storage. Samples may need to be placed in specific containers that are resistant to fracture and contamination.

Skin biopsies are often obtained at the time of procurement of other tissues for alternate types of testing as well as for storage. Skin fibroblasts may be extracted from 2- to 5-mm skin punch biopsies obtained by sterile techniques from various body sites. Cultures of skin fibroblasts are usually available within 2 to 3 weeks of procurement and may be used for enzyme, DNA/RNA, or immune protein analysis. It should be remembered that not all biochemical pathways are sufficiently expressed in skin fibroblasts to give meaningful results in all assays. Although skin fibroblasts may provide clues to disorders resulting from abnormalities in nuclear DNA, they may not be helpful for disorders resulting from abnormalities of mitochondrial DNA (mtDNA). The reason is that differential assortment of mtDNA during early embryonic development precludes even transfer of pathogenic mutations to all tissues of the body. Hence, inherited disorders of mtDNA are not necessarily manifested in all tissues, and detectable abnormalities may be found only in those tissues manifesting the metabolic defect(s).

Methodologies Involved in Biochemical and Genetic Testing

The technology used for investigation of metabolic diseases ranges from time-honored methods such as Southern blotting, polymerase chain reaction, and chromatography to the extremely sophisticated state-of-the art technique of tandem mass spectrometry (MS/MS).

The mass spectrometer is a device that ionizes compounds to generate charged ions that are separated and quantified based on their mass-to-charge ratios. The fragmentation pattern displayed is compared with patterns of known compounds in a “library” that helps identify and quantify abnormal compounds. Although all mass spectrometers follow this basic principle, they may differ from each other in the method used for ionization of the analyte and the method used for separation of the ions. Electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) are commonly used for laboratory analysis of biologic compounds because they are “softer” on macromolecules; the former method causes ionization by dispersing the analyte into a fine aerosol, whereas the latter uses a laser beam to create ionization. MALDI received its name because a matrix is used to protect the molecule from being destroyed by the laser.

The two commonly used methods for separation of ions are time of flight (TOF) and quadrupole analyzers. TOF analyzers use an electric field to accelerate ions through the same potential and measure the time taken by the ions to reach the detector. Quadrupole analyzers use oscillating electric fields to alter the paths of ions passing through a radiofrequency quadrupole field.