- Email: [email protected]
Perimortem laboratory investigation of genetic metabolic disorders
Seminars in Neonatology (2004) 9, 275e280
www.elsevierhealth.com/journals/siny
Perimortem laboratory investigation of genetic metabolic disorders John Christodouloua,b,), Bridget Wilckena,b a
Western Sydney Genetics Program, The Children’s Hospital at Westmead, Locked Bag 4001, Westmead, 2145 Sydney, NSW, Australia b School of Paediatrics and Child Health, University of Sydney, NSW, Australia
KEYWORDS Acute encephalopathy; Genetics; Inborn error of metabolism; Neonate; Stillbirth; Hydrops
Summary Over 400 rare, biochemically diverse genetic metabolic disorders (inborn errors of metabolism) have been described and the list is growing by the month. Although recent advances in the diagnosis and treatment of these disorders have substantially improved the prognosis for many of them, including those presenting in the neonatal period, a proportion of affected individuals die before the diagnosis can be confirmed and, in some cases, before the diagnosis is even considered. This review will provide an outline of the range of clinical presentations seen in neonates with genetic metabolic disorders and provide a practical approach for rapid biochemical screening for these disorders. In addition, suggested guidelines are given for the collection of relevant samples in the perimortem period, the aim being to maximize the chance of identifying any underlying genetic metabolic disorder. ª 2004 Elsevier Ltd. All rights reserved.
Introduction Genetic disorders are an important cause of morbidity and mortality, affecting growth, development and all aspects of health. Symptoms may be apparent at birth or occur later in life. Up to 30% of patients in paediatric hospitals have a genetic component to their illness,1 and 15e25% of all infant deaths have a genetic disorder as a major contributing factor.2,3 Genetic metabolic disorders (inborn errors of metabolism) are inherited disorders that disrupt
) Corresponding author. Tel.: D61-2-9845-3452; fax: D61-29845-1864. E-mail address: [email protected] (J. Christodoulou).
normal metabolic function. More than 400 of these have been described and new disorders are being identified regularly. They can be divided into six broad clinical presentations (see Table 1). Most have an autosomal recessive pattern of inheritance and, thus, consanguinity is more common amongst these families. Specific and effective treatment is possible for many due to an understanding of their biochemical basis and early diagnosis and treatment may prevent permanent disability and mortality. For those for whom treatment is not available, or is ineffective, accurate diagnosis is vital so that families can receive genetic counselling about risk recurrence and the possibility of presymptomatic affected relatives. Accurate diagnosis and genetic counselling for many of these disorders requires specialist clinical genetic, metabolic and perinatal medical services.
1084-2756/$ - see front matter ª 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.siny.2003.10.004
276 Table 1
J. Christodoulou, B. Wilcken Genetic metabolic disorders presenting in the newborn period
Predominant clinical or biochemical presentation Acute encephalopathy Hypoglycaemia
Hyperammonaemia Ketosis Disorders of acidebase status Seizures as early predominant feature
Acute hepatocellular disease
Sudden death, including cardiomyopathy
Severe hypotonia
Non-immune hydrops fetalis
Facial dysmorphism, with or without congenital malformations
Important examples
Fatty acid oxidation defects; organic acidopathies; gluconeogenic disorders, glycogen storage disorders, hereditary fructose intolerance (also consider hyperinsulinism, pituitary insufficiency) Urea cycle disorders; transient hyperammonaemia of the newborn, lysinuric protein intolerance Maple syrup urine disease Mitochondrial respiratory chain disorders, defects of pyruvate metabolism; organic acidoses Non-ketotic hyperglycinaemia; pyridoxine-dependent epilepsy; peroxisomal disorders; sulphite oxidase deficiency; molybdenum cofactor defect; folinic acid responsive seizures; glucose transporter defect Galactosaemia; fatty acid oxidation defects; tyrosinaemia type I; hereditary fructose intolerance; peroxisomal disorders; a1-antitrypsin deficiency; mitochondrial respiratory chain disorders; NiemannePick disease type C; neonatal haemochromatosis; defects of bile acid metabolism; congenital defects of glycosylation Fatty acid oxidation defects; mitochondrial respiratory chain disorders; glycogen storage disorder type IV; lysosomal storage disorders; Barth syndrome; heart-specific phosphorylase kinase deficiency Peroxisomal disorders; non-ketotic hyperglycinaemia; sulphite oxidase deficiency; molybdenum cofactor defect; congenital defects of glycosylation; congenital lactic acidoses; glycogen storage disease type II Lysosomal storage disorders; haemoglobinopathies, red blood cell glycolytic defects; neonatal haemochromatosis; glycogen storage disorder type IV; mitochondrial respiratory chain disorders Lysosomal storage disorders; peroxisomal disorders; congenital defects of glycosylation; maternal phenylketonuria; pyruvate dehydrogenase deficiency; mevalonic aciduria; SmitheLemlieOpitz syndrome
However, the key to rapid diagnosis in the neonatal or general paediatric setting is to have a heightened awareness to the clinical clues, so that some basic biochemical investigations are performed in a timely manner, which may provide further pointers to the specific diagnosis.
Genetic metabolic disorders in the newborn Table 1 summarizes the ways in which inborn errors may clinically present in the neonate, with some of the more common examples being provided. Further clinical details are available from a number of reference sources.4e6 The clinical presentations that should prompt consideration of a genetic
metabolic disease in the newborn are summarized below.
Acute encephalopathy, with or without seizures This is the most common presentation in the neonate and, typically, the affected neonate is born after a normal pregnancy, at or near term, with a normal birth weight and remains well in the early hours or days of life. Such an infant may be discharged from the newborn nursery, subsequently presenting as acutely unwell with lethargy, decreased feeding, vomiting, irritability, seizures and tachypnoea.7,8 Cerebral oedema may develop, contributing to a relentless deterioration if not treated. The enzyme defect is usually severe,
Laboratory investigation of genetic metabolic disorders leading to the accumulation of a neurotoxic metabolite. Urgent treatment may be life-saving.9 Table 2 lists the investigations that should be performed in this clinical setting. A few disorders are characterized by early seizures as the predominant symptom with features reminiscent of perinatal asphyxia, but without a supportive history.9 These disorders differ from other inborn errors presenting in the neonatal period in that in the latter disorders seizures often occur later and are a less predominant part of an acute encephalopathy.
277
Acute hepatocellular disease Hepatocellular disease may present from infancy onwards. Features include hepatomegaly, jaundice, elevated transaminase levels, abnormalities of coagulation and, sometimes, hypoglycaemia.10 The renal Fanconi syndrome may accompany liver failure in some of these disorders, whilst cataracts or facial dysmorphism may be present in others.11
Sudden death, including cardiomyopathy Table 2 Screening investigations that should be performed in an acutely ill neonate suspected of having a genetic metabolic disorder Urine Odour Dipstick tests for ketones, pH, sulphitea Reducing substances (testing for both glucose and non-glucose reducing substances) Amino, organic acid screens (including acylglycines) Blood Full blood count/film Urea, electrolytes, anion gap, creatinine Glucose Calcium Blood gases Liver enzymes Uric acid Ammonium Lactate and pyruvate Amino acidsb Carnitine and acylcarnitinesb Cerebrospinal fluid Lactate and pyruvate Glucose Amino acidsb In the case of hypoglycaemia collect blood for the following when the child is hypoglycaemic Growth hormone Cortisol Insulin Free fatty acids b-Hydroxybutyrate Acylcarnitine profile Urine should always be collected at the time of hypoglycaemia a Sulphite is very labile. A negative test result does not exclude sulphite oxidase deficiency or the molybdenum cofactor defect. b These tests should only be ordered after consultation with a biochemical geneticist or metabolic physician.
Long- and medium-chain fatty acid oxidation disorders may present with cardiomyopathy, cardiac arrhythmias, low output failure and cardiac arrest during the neonatal period.12 Cardiomyopathy may be the presenting or associated feature of a number of other disorders, and may even present as non-immune hydrops fetalis (see below and see also Table 3).
Severe hypotonia Mild hypotonia is a frequent non-specific feature in neonates, whilst severe hypotonia is associated with a number of specific inborn errors, some of which may be associated with dysmorphic features or hepatic dysfunction.4,6
Non-immune hydrops fetalis Some patients with non-immune hydrops fetalis, which is often detected before birth by prenatal ultrasonography, may have a genetic metabolic disorder (see Table 3). Other clues to a metabolic aetiology may include collodion skin, hepatosplenomegaly and skeletal deformities due to the accumulation of storage material (dysostosis multiplex).13
Facial dysmorphism, with or without congenital malformations There are a number of inherited metabolic disorders that are recognized by specific facial dysmorphism and congenital malformations. These may be a consequence of either the accumulation of teratogenic metabolites, because they impair cellular bioenergetics, or possibly their interference with embryonic development in other ways.5,9
278 Table 3
J. Christodoulou, B. Wilcken Genetic causes of stillbirth
1. Dysmorphic infant with or without growth retardation Chromosomal abnormalities Skeletal dysplasias Non-metabolic dysmorphic syndromes Hypophosphatasia (clue bone not mineralized) Inherited disorders of cholesterol biosynthesis (including SmitheLemlie Opitz syndrome and mevalonic aciduria) Lysosomal storage disorders (including GM1 gangliosidosis, infantile sialic acid storage disease, sialidosis type II, galactosialidosis, Gaucher disease type II, NiemannePick type C, MPS IV and VII, mucolipidosis type II, Farber disease) Peroxisomal disorders (including generalized peroxisomal biogenesis defects and rhizomelic chondrodysplasia punctata) Fetal mitochondrial respiratory chain defects 2. Non-immune hydrops Haemoglobinopathies Red blood cell glycolytic defects Lysosomal storage disorders Neonatal haemochromatosis Glycogen storage disorder type IV Congenital myotonic dystrophy Mitochondrial respiratory chain disorders 3. Maternal causes (normal looking baby) Maternal thrombophilic disorders, especially combinations of more than one factor (e.g. plasminogen activator inhibitor-1, factor V Leiden, certain methylene tetrahydrofolate reductase mutations, certain prothrombin mutations, hyperhomocysteinaemia, protein C, protein S and antithrombin III) 11,14e30
Sources, Refs. . GM1, monosialoganglioside; MPS, mucopolysaccharidosis.
Rapid identification and treatment of genetic metabolic disorders is critical For disorders that present in the newborn period, emergency treatment is often available that can significantly reduce or even prevent the development of permanent physical or neurological damage.7 Since the symptoms of metabolic disorders are usually non-specific in nature, rapid and accurate diagnosis will only be made if screening tests, in consultation with the clinical genetic and metabolic specialists, are considered at the outset in an undiagnosed, sick child. Specific information that should be obtained includes whether there is any
significant family history (similar illnesses in siblings, unexplained deaths, stillbirths etc.), consanguinity, whether there was an intercurrent illness and the dietary history (especially any recent changes to the diet, or periods of poor feeding). Physical examination is not usually helpful in determining the precise diagnosis although for some disorders there are specific clinical clues. For instance, hepatomegaly associated with hypoglycaemia should raise the possibility of a glycogen storage disorder or fatty acid oxidation defect, amongst other disorders.
Screening tests The clinical presentations for many of these disorders overlap and specific sets of investigations, based on the broad clinical presentation, will screen for most genetic metabolic disorders.8 These screening tests are summarized in Table 2. To obtain meaningful results, many of these samples require special handling (particularly blood collected for lactate, pyruvate, ammonium and amino acids) and, where doubt exists, discussions should be held with either a metabolic physician or biochemical geneticist before the samples are collected. These specialists can also advise with regard to specific testing that will yield a definitive diagnosis and whether there are any general or specific treatments that should be implemented.
Perimortem evaluation of a neonate suspected of having an inborn error of metabolism The unexpected death of a child, including one with multiple congenital anomalies or suspected bony dysplasia, mandates careful consideration of the possibility of a genetic disorder. Whilst this period is possibly the most distressing time parents of a dying child will ever have to face, the precise diagnosis may not be made without timely and sensitive discussion on the need for perimortem sample collection. This has important implications for the parents in terms of discussion regarding future genetic risks. There are a number of genetic disorders (including inborn errors of metabolism) that should be considered as possible causes of fetal death in utero and stillbirth.14,15 They fall into a relatively small number of clinical presentations and are summarized in Table 3.
Laboratory investigation of genetic metabolic disorders Table 4
Components of the genetic autopsy
Careful family history, including threegeneration pedigree Invite a clinical geneticist with expertise in dysmorphic syndromes to inspect the infant Clinical photographs Full skeletal survey Parental investigations for a haemoglobinopathy Maternal investigations for a thrombophilic disorder Samples to collect from the baby Blood Dried blood spots on filter paper (newborn screening cards, at least two to three cards stored at room temperature but NOT in a plastic bag (for acylcarnitine profile analysis and is a source of DNA)) Whole blood (5 ml in lithium heparin tube (for carnitine, quantitative amino acids, very long chain fatty acids; separated within 20 min of collection and stored at 70 (C); AND 5 ml in EDTA tube (for DNA extraction; can be stored at 4 (C for 48 h) AND 5 ml in lithium heparin (for chromosome analysis; must be commenced within 4 h of sample collection)) Urine Freeze and store (5 ml or more if possible, stored at 70 (C (for amino acid and organic acid profiles, acylglycines, orotic acid)) Cerebrospinal fluid Freeze and store (1 ml stored at 70 (C (for amino acid profile)) Skin Biopsy (3!2 mm full thickness collected under sterile conditions (DO NOT use iodine-containing preparations) into culture or viral transport medium, or saline soaked gauze. Store at 4 (C. Best collected within 12 h of death. Cartilage may be taken for culture if there has been a prolonged period after death before biopsies can be taken. Send as soon as possible to a cytogenetics laboratory. To be cultured for archiving in liquid nitrogen) Other biopsies Liver and muscle biopsies (for electron microscopy, histopathology and enzymology (for the latter wrap in aluminium foil, snap freeze and store at 70 (C). Collect within 4 h (preferably 2 h) of death. Consult metabolic physician or histopathologist before collection of samples) Other tissue biopsies if specific diagnoses are under consideration
Table 4 provides a schema as to what might be considered a genetic autopsy. Only certain components of this guideline may need to be followed in certain circumstances. Most important of all is
279
the need to consult with a clinical or biochemical geneticist as soon as possible, so that direction on appropriate additional investigations can be given.
Conclusions Whilst genetic metabolic disorders are individually rare, collectively they are not so uncommon. Recent advances in diagnosis and treatment of these disorders have substantially improved the prognosis for a growing number of these disorders. Early diagnosis and intervention is crucial if neurological sequelae and death are to be avoided and the recent introduction of tandem mass spectrometry to newborn screening programmes will increase the likelihood of more of these disorders being detected even before symptoms develop. No screening test, however, is a substitute for maintaining a high index of suspicion. Notwithstanding these advances, a proportion of infants will die before the diagnosis is made or even before the possibility of a genetic metabolic disorder is suspected and, in this setting, it is crucial to obtain appropriate diagnostic samples, often within a very narrow timeframe after death. Establishing the diagnosis is critical since these disorders are genetic and missing a diagnosis could have future catastrophic consequences. Early consultation with specialists in clinical genetics and metabolic medicine must be considered mandatory.
Practice points Despite the vast array of potential biochemical defects, most disorders can be categorized into a handful of clinical presentations. The judicious use of simple screening measures can often yield a specific diagnosis quickly. In cases suspected of having a genetic metabolic disorder, collection and processing of a number of biological samples must be considered to be an emergency procedure. Early consultation with specialists in clinical genetics and biochemical genetics should be considered in stillbirths, particularly in the presence of facial dysmorphism, congenital malformations, hydrops or hepatosplenomegaly.
280
J. Christodoulou, B. Wilcken
Research directions Improvements are needed in the diagnosis of inborn errors, including an expansion of newborn screening programmes. The development of normal ranges for an expanded list of tissue analytes in postmortem samples is needed. The examination of the molecular defects causing inborn errors and the exploration of phenotypeegenotype correlations should be carried out. The identification of new classes of inborn errors should be possible on the basis of new-found knowledge from the Human Genome Project. Targeted and more effective therapies need to be developed.
12.
13. 14. 15. 16.
17. 18.
19.
References 1. Weatherall DJ. The new genetics and clinical practice. Oxford: Oxford University Press; 1991. p. 32e7. 2. Verma IC, Singh B. Ethics and medical genetics in India. In: Wertz DC, Fletcher JC, editors. Ethics and human genetics: a cross-cultural perspective. Berlin: Springer-Verlag; 1989. p. 250e70. 3. Penchaszadeh VB. Reproductive genetic testing from an international perspective: impact on women in underdeveloped countries. Fetal Diagn Ther 1993;8:202e9. 4. Saudubray JM, Charpentier C. Clinical phenotypes: diagnosis/algorithms. In: Scriver C, Beaudet A, Sly W, Valle D, Childs B, Kinzler KW, et al, editors. The metabolic and molecular bases of inherited disease, vol. 1. New York: McGraw-Hill; 2001. p. 1327e403. 5. Christodoulou J. A clinical approach to inborn errors of metabolism. In: Rudolph AM, Kamei R, Overby KJ, editors. Rudolph’s fundamentals of paediatrics. New York: McGrawHill; 2002. p. 221e52. 6. Saudubray JM, Nassogne MC, de Lonlay P, Touati G. Clinical approach to inherited metabolic disorders in neonates: an overview. Semin Neonatol 2002;7:3e15. 7. Burlina AB, Bonafe L, Zacchello F. Clinical and biochemical approach to the neonate with a suspected inborn error of amino acid and organic acid metabolism. Semin Perinatol 1999;23:162e73. 8. Greene CL, Goodman SI. Catastrophic metabolic encephalopathies in the newborn period. Evaluation and management. Clin Perinatol 1997;24:773e86. 9. Burton BK. Inborn errors of metabolism in infancy: a guide to diagnosis. Pediatrics 1998;102:E69 [electronic version]. 10. Clayton PT. Inborn errors presenting with liver dysfunction. Semin Neonatol 2002;7:49e63. 11. Baumgartner MR, Poll-The BT, Verhoeven NM, Jakobs C, Espeel M, Roels F, et al. Clinical approach to inherited
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peroxisomal disorders: a series of 27 patients. Ann Neurol 1998;44:720e30. Roe CR, Ding J. Mitochondrial fatty acid oxidation disorders. In: Scriver C, Beaudet A, Sly W, Valle D, Childs B, Kinzler KW, et al, editors. The metabolic and molecular bases of inherited disease, vol. 2. New York: McGraw-Hill; 2001. p. 2297e326. Stone DL, Sidransky E. Hydrops fetalis: lysosomal storage disorders in extremis. Adv Pediatr 1999;46:409e40. Curry CJ. Pregnancy loss, stillbirth and neonatal death. A guide for the pediatrician. Pediatr Clin North Am 1992;39:157e92. Schauer GM, Kalousek DK, Magee JF. Genetic causes of stillbirth. Semin Perinatol 1992;16:341e51. Mornet E. Hypophosphatasia: the mutations in the tissuenonspecific alkaline phosphatase gene. Hum Mutat 2000; 15:309e15. Haas D, Kelley RI, Hoffmann GF. Inherited disorders of cholesterol biosynthesis. Neuropediatrics 2001;32:113e22. Clarke JTR. Storage syndrome and dysmorphism. In: Clarke JTR, editor. A clinical guide to inherited metabolic diseases. Cambridge: Cambridge University Press; 1996. p. 143e75. Cormier-Daire V, Rustin P, Rotig A, Chretien D, Le Merrer M, Belli D, et al. Craniofacial anomalies and malformations in respiratory chain deficiency. Am J Med Genet 1996;66: 457e63. Carlton DP, McGillivray BC, Schreiber MD. Nonimmune hydrops fetalis: a multidisciplinary approach. Clin Perinatol 1989;16:839e51. Steiner RD. Hydrops fetalis: role of the geneticist. Semin Perinatol 1995;19:516e24. Fischel-Ghodsian N. Prenatal diagnosis of hemoglobinopathies. Clin Perinatol 1990;17:811e28. Machin GA. Hydrops revisited: literature review of 1414 cases published in the 1980s. Am J Med Genet 1989; 34:366e90. Wisser J, Schreiner M, Diem H, Roithmeier A. Neonatal hemochromatosis: a rare cause of nonimmune hydrops fetalis and fetal anemia. Fetal Diagn Ther 1993;8:273e8. Ben-Haroush A, Yogev Y, Levit O, Hod M. Isolated fetal ascites caused by Wolman disease. Ultrasound Obstet Gynecol 2003;21:297e8. Afifi AM, Bhatia AR, Eyal F. Hydrops fetalis associated with congenital myotonic dystrophy. Am J Obstet Gynecol 1992;166:929e30. Arnon S, Aviram R, Dolfin T, Regev R, Litmanovits I, Tepper R, et al. Mitochondrial DNA depletion presenting prenatally with skin edema and multisystem disease immediately after birth. Prenat Diagn 2002;22:34e7. Kupferminc MJ, Eldor A, Steinman N, Many A, Bar-Am A, Jaffa A, et al. Increased frequency of genetic thrombophilia in women with complications of pregnancy. N Engl J Med 1999;340:9e13. Aubard Y, Darodes N, Cantaloube M. Hyperhomocysteinemia and pregnancydreview of our present understanding and therapeutic implications. Eur J Obstet Gynecol Reprod Biol 2000;93:157e65. Glueck CJ, Kupferminc MJ, Fontaine RN, Wang P, Weksler BB, Eldor A. Genetic hypofibrinolysis in complicated pregnancies. Obstet Gynecol 2001;97:44e8.
www.elsevierhealth.com/journals/siny
Perimortem laboratory investigation of genetic metabolic disorders John Christodouloua,b,), Bridget Wilckena,b a
Western Sydney Genetics Program, The Children’s Hospital at Westmead, Locked Bag 4001, Westmead, 2145 Sydney, NSW, Australia b School of Paediatrics and Child Health, University of Sydney, NSW, Australia
KEYWORDS Acute encephalopathy; Genetics; Inborn error of metabolism; Neonate; Stillbirth; Hydrops
Summary Over 400 rare, biochemically diverse genetic metabolic disorders (inborn errors of metabolism) have been described and the list is growing by the month. Although recent advances in the diagnosis and treatment of these disorders have substantially improved the prognosis for many of them, including those presenting in the neonatal period, a proportion of affected individuals die before the diagnosis can be confirmed and, in some cases, before the diagnosis is even considered. This review will provide an outline of the range of clinical presentations seen in neonates with genetic metabolic disorders and provide a practical approach for rapid biochemical screening for these disorders. In addition, suggested guidelines are given for the collection of relevant samples in the perimortem period, the aim being to maximize the chance of identifying any underlying genetic metabolic disorder. ª 2004 Elsevier Ltd. All rights reserved.
Introduction Genetic disorders are an important cause of morbidity and mortality, affecting growth, development and all aspects of health. Symptoms may be apparent at birth or occur later in life. Up to 30% of patients in paediatric hospitals have a genetic component to their illness,1 and 15e25% of all infant deaths have a genetic disorder as a major contributing factor.2,3 Genetic metabolic disorders (inborn errors of metabolism) are inherited disorders that disrupt
) Corresponding author. Tel.: D61-2-9845-3452; fax: D61-29845-1864. E-mail address: [email protected] (J. Christodoulou).
normal metabolic function. More than 400 of these have been described and new disorders are being identified regularly. They can be divided into six broad clinical presentations (see Table 1). Most have an autosomal recessive pattern of inheritance and, thus, consanguinity is more common amongst these families. Specific and effective treatment is possible for many due to an understanding of their biochemical basis and early diagnosis and treatment may prevent permanent disability and mortality. For those for whom treatment is not available, or is ineffective, accurate diagnosis is vital so that families can receive genetic counselling about risk recurrence and the possibility of presymptomatic affected relatives. Accurate diagnosis and genetic counselling for many of these disorders requires specialist clinical genetic, metabolic and perinatal medical services.
1084-2756/$ - see front matter ª 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.siny.2003.10.004
276 Table 1
J. Christodoulou, B. Wilcken Genetic metabolic disorders presenting in the newborn period
Predominant clinical or biochemical presentation Acute encephalopathy Hypoglycaemia
Hyperammonaemia Ketosis Disorders of acidebase status Seizures as early predominant feature
Acute hepatocellular disease
Sudden death, including cardiomyopathy
Severe hypotonia
Non-immune hydrops fetalis
Facial dysmorphism, with or without congenital malformations
Important examples
Fatty acid oxidation defects; organic acidopathies; gluconeogenic disorders, glycogen storage disorders, hereditary fructose intolerance (also consider hyperinsulinism, pituitary insufficiency) Urea cycle disorders; transient hyperammonaemia of the newborn, lysinuric protein intolerance Maple syrup urine disease Mitochondrial respiratory chain disorders, defects of pyruvate metabolism; organic acidoses Non-ketotic hyperglycinaemia; pyridoxine-dependent epilepsy; peroxisomal disorders; sulphite oxidase deficiency; molybdenum cofactor defect; folinic acid responsive seizures; glucose transporter defect Galactosaemia; fatty acid oxidation defects; tyrosinaemia type I; hereditary fructose intolerance; peroxisomal disorders; a1-antitrypsin deficiency; mitochondrial respiratory chain disorders; NiemannePick disease type C; neonatal haemochromatosis; defects of bile acid metabolism; congenital defects of glycosylation Fatty acid oxidation defects; mitochondrial respiratory chain disorders; glycogen storage disorder type IV; lysosomal storage disorders; Barth syndrome; heart-specific phosphorylase kinase deficiency Peroxisomal disorders; non-ketotic hyperglycinaemia; sulphite oxidase deficiency; molybdenum cofactor defect; congenital defects of glycosylation; congenital lactic acidoses; glycogen storage disease type II Lysosomal storage disorders; haemoglobinopathies, red blood cell glycolytic defects; neonatal haemochromatosis; glycogen storage disorder type IV; mitochondrial respiratory chain disorders Lysosomal storage disorders; peroxisomal disorders; congenital defects of glycosylation; maternal phenylketonuria; pyruvate dehydrogenase deficiency; mevalonic aciduria; SmitheLemlieOpitz syndrome
However, the key to rapid diagnosis in the neonatal or general paediatric setting is to have a heightened awareness to the clinical clues, so that some basic biochemical investigations are performed in a timely manner, which may provide further pointers to the specific diagnosis.
Genetic metabolic disorders in the newborn Table 1 summarizes the ways in which inborn errors may clinically present in the neonate, with some of the more common examples being provided. Further clinical details are available from a number of reference sources.4e6 The clinical presentations that should prompt consideration of a genetic
metabolic disease in the newborn are summarized below.
Acute encephalopathy, with or without seizures This is the most common presentation in the neonate and, typically, the affected neonate is born after a normal pregnancy, at or near term, with a normal birth weight and remains well in the early hours or days of life. Such an infant may be discharged from the newborn nursery, subsequently presenting as acutely unwell with lethargy, decreased feeding, vomiting, irritability, seizures and tachypnoea.7,8 Cerebral oedema may develop, contributing to a relentless deterioration if not treated. The enzyme defect is usually severe,
Laboratory investigation of genetic metabolic disorders leading to the accumulation of a neurotoxic metabolite. Urgent treatment may be life-saving.9 Table 2 lists the investigations that should be performed in this clinical setting. A few disorders are characterized by early seizures as the predominant symptom with features reminiscent of perinatal asphyxia, but without a supportive history.9 These disorders differ from other inborn errors presenting in the neonatal period in that in the latter disorders seizures often occur later and are a less predominant part of an acute encephalopathy.
277
Acute hepatocellular disease Hepatocellular disease may present from infancy onwards. Features include hepatomegaly, jaundice, elevated transaminase levels, abnormalities of coagulation and, sometimes, hypoglycaemia.10 The renal Fanconi syndrome may accompany liver failure in some of these disorders, whilst cataracts or facial dysmorphism may be present in others.11
Sudden death, including cardiomyopathy Table 2 Screening investigations that should be performed in an acutely ill neonate suspected of having a genetic metabolic disorder Urine Odour Dipstick tests for ketones, pH, sulphitea Reducing substances (testing for both glucose and non-glucose reducing substances) Amino, organic acid screens (including acylglycines) Blood Full blood count/film Urea, electrolytes, anion gap, creatinine Glucose Calcium Blood gases Liver enzymes Uric acid Ammonium Lactate and pyruvate Amino acidsb Carnitine and acylcarnitinesb Cerebrospinal fluid Lactate and pyruvate Glucose Amino acidsb In the case of hypoglycaemia collect blood for the following when the child is hypoglycaemic Growth hormone Cortisol Insulin Free fatty acids b-Hydroxybutyrate Acylcarnitine profile Urine should always be collected at the time of hypoglycaemia a Sulphite is very labile. A negative test result does not exclude sulphite oxidase deficiency or the molybdenum cofactor defect. b These tests should only be ordered after consultation with a biochemical geneticist or metabolic physician.
Long- and medium-chain fatty acid oxidation disorders may present with cardiomyopathy, cardiac arrhythmias, low output failure and cardiac arrest during the neonatal period.12 Cardiomyopathy may be the presenting or associated feature of a number of other disorders, and may even present as non-immune hydrops fetalis (see below and see also Table 3).
Severe hypotonia Mild hypotonia is a frequent non-specific feature in neonates, whilst severe hypotonia is associated with a number of specific inborn errors, some of which may be associated with dysmorphic features or hepatic dysfunction.4,6
Non-immune hydrops fetalis Some patients with non-immune hydrops fetalis, which is often detected before birth by prenatal ultrasonography, may have a genetic metabolic disorder (see Table 3). Other clues to a metabolic aetiology may include collodion skin, hepatosplenomegaly and skeletal deformities due to the accumulation of storage material (dysostosis multiplex).13
Facial dysmorphism, with or without congenital malformations There are a number of inherited metabolic disorders that are recognized by specific facial dysmorphism and congenital malformations. These may be a consequence of either the accumulation of teratogenic metabolites, because they impair cellular bioenergetics, or possibly their interference with embryonic development in other ways.5,9
278 Table 3
J. Christodoulou, B. Wilcken Genetic causes of stillbirth
1. Dysmorphic infant with or without growth retardation Chromosomal abnormalities Skeletal dysplasias Non-metabolic dysmorphic syndromes Hypophosphatasia (clue bone not mineralized) Inherited disorders of cholesterol biosynthesis (including SmitheLemlie Opitz syndrome and mevalonic aciduria) Lysosomal storage disorders (including GM1 gangliosidosis, infantile sialic acid storage disease, sialidosis type II, galactosialidosis, Gaucher disease type II, NiemannePick type C, MPS IV and VII, mucolipidosis type II, Farber disease) Peroxisomal disorders (including generalized peroxisomal biogenesis defects and rhizomelic chondrodysplasia punctata) Fetal mitochondrial respiratory chain defects 2. Non-immune hydrops Haemoglobinopathies Red blood cell glycolytic defects Lysosomal storage disorders Neonatal haemochromatosis Glycogen storage disorder type IV Congenital myotonic dystrophy Mitochondrial respiratory chain disorders 3. Maternal causes (normal looking baby) Maternal thrombophilic disorders, especially combinations of more than one factor (e.g. plasminogen activator inhibitor-1, factor V Leiden, certain methylene tetrahydrofolate reductase mutations, certain prothrombin mutations, hyperhomocysteinaemia, protein C, protein S and antithrombin III) 11,14e30
Sources, Refs. . GM1, monosialoganglioside; MPS, mucopolysaccharidosis.
Rapid identification and treatment of genetic metabolic disorders is critical For disorders that present in the newborn period, emergency treatment is often available that can significantly reduce or even prevent the development of permanent physical or neurological damage.7 Since the symptoms of metabolic disorders are usually non-specific in nature, rapid and accurate diagnosis will only be made if screening tests, in consultation with the clinical genetic and metabolic specialists, are considered at the outset in an undiagnosed, sick child. Specific information that should be obtained includes whether there is any
significant family history (similar illnesses in siblings, unexplained deaths, stillbirths etc.), consanguinity, whether there was an intercurrent illness and the dietary history (especially any recent changes to the diet, or periods of poor feeding). Physical examination is not usually helpful in determining the precise diagnosis although for some disorders there are specific clinical clues. For instance, hepatomegaly associated with hypoglycaemia should raise the possibility of a glycogen storage disorder or fatty acid oxidation defect, amongst other disorders.
Screening tests The clinical presentations for many of these disorders overlap and specific sets of investigations, based on the broad clinical presentation, will screen for most genetic metabolic disorders.8 These screening tests are summarized in Table 2. To obtain meaningful results, many of these samples require special handling (particularly blood collected for lactate, pyruvate, ammonium and amino acids) and, where doubt exists, discussions should be held with either a metabolic physician or biochemical geneticist before the samples are collected. These specialists can also advise with regard to specific testing that will yield a definitive diagnosis and whether there are any general or specific treatments that should be implemented.
Perimortem evaluation of a neonate suspected of having an inborn error of metabolism The unexpected death of a child, including one with multiple congenital anomalies or suspected bony dysplasia, mandates careful consideration of the possibility of a genetic disorder. Whilst this period is possibly the most distressing time parents of a dying child will ever have to face, the precise diagnosis may not be made without timely and sensitive discussion on the need for perimortem sample collection. This has important implications for the parents in terms of discussion regarding future genetic risks. There are a number of genetic disorders (including inborn errors of metabolism) that should be considered as possible causes of fetal death in utero and stillbirth.14,15 They fall into a relatively small number of clinical presentations and are summarized in Table 3.
Laboratory investigation of genetic metabolic disorders Table 4
Components of the genetic autopsy
Careful family history, including threegeneration pedigree Invite a clinical geneticist with expertise in dysmorphic syndromes to inspect the infant Clinical photographs Full skeletal survey Parental investigations for a haemoglobinopathy Maternal investigations for a thrombophilic disorder Samples to collect from the baby Blood Dried blood spots on filter paper (newborn screening cards, at least two to three cards stored at room temperature but NOT in a plastic bag (for acylcarnitine profile analysis and is a source of DNA)) Whole blood (5 ml in lithium heparin tube (for carnitine, quantitative amino acids, very long chain fatty acids; separated within 20 min of collection and stored at 70 (C); AND 5 ml in EDTA tube (for DNA extraction; can be stored at 4 (C for 48 h) AND 5 ml in lithium heparin (for chromosome analysis; must be commenced within 4 h of sample collection)) Urine Freeze and store (5 ml or more if possible, stored at 70 (C (for amino acid and organic acid profiles, acylglycines, orotic acid)) Cerebrospinal fluid Freeze and store (1 ml stored at 70 (C (for amino acid profile)) Skin Biopsy (3!2 mm full thickness collected under sterile conditions (DO NOT use iodine-containing preparations) into culture or viral transport medium, or saline soaked gauze. Store at 4 (C. Best collected within 12 h of death. Cartilage may be taken for culture if there has been a prolonged period after death before biopsies can be taken. Send as soon as possible to a cytogenetics laboratory. To be cultured for archiving in liquid nitrogen) Other biopsies Liver and muscle biopsies (for electron microscopy, histopathology and enzymology (for the latter wrap in aluminium foil, snap freeze and store at 70 (C). Collect within 4 h (preferably 2 h) of death. Consult metabolic physician or histopathologist before collection of samples) Other tissue biopsies if specific diagnoses are under consideration
Table 4 provides a schema as to what might be considered a genetic autopsy. Only certain components of this guideline may need to be followed in certain circumstances. Most important of all is
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the need to consult with a clinical or biochemical geneticist as soon as possible, so that direction on appropriate additional investigations can be given.
Conclusions Whilst genetic metabolic disorders are individually rare, collectively they are not so uncommon. Recent advances in diagnosis and treatment of these disorders have substantially improved the prognosis for a growing number of these disorders. Early diagnosis and intervention is crucial if neurological sequelae and death are to be avoided and the recent introduction of tandem mass spectrometry to newborn screening programmes will increase the likelihood of more of these disorders being detected even before symptoms develop. No screening test, however, is a substitute for maintaining a high index of suspicion. Notwithstanding these advances, a proportion of infants will die before the diagnosis is made or even before the possibility of a genetic metabolic disorder is suspected and, in this setting, it is crucial to obtain appropriate diagnostic samples, often within a very narrow timeframe after death. Establishing the diagnosis is critical since these disorders are genetic and missing a diagnosis could have future catastrophic consequences. Early consultation with specialists in clinical genetics and metabolic medicine must be considered mandatory.
Practice points Despite the vast array of potential biochemical defects, most disorders can be categorized into a handful of clinical presentations. The judicious use of simple screening measures can often yield a specific diagnosis quickly. In cases suspected of having a genetic metabolic disorder, collection and processing of a number of biological samples must be considered to be an emergency procedure. Early consultation with specialists in clinical genetics and biochemical genetics should be considered in stillbirths, particularly in the presence of facial dysmorphism, congenital malformations, hydrops or hepatosplenomegaly.
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Research directions Improvements are needed in the diagnosis of inborn errors, including an expansion of newborn screening programmes. The development of normal ranges for an expanded list of tissue analytes in postmortem samples is needed. The examination of the molecular defects causing inborn errors and the exploration of phenotypeegenotype correlations should be carried out. The identification of new classes of inborn errors should be possible on the basis of new-found knowledge from the Human Genome Project. Targeted and more effective therapies need to be developed.
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