Pharmacogenetic Analysis of an 8-Year Old Girl with Reye Syndrome Associated with Use of Naproxen.

BACKGROUND Reye syndrome is a rare, yet potentially life-threatening disease characterized by acute encephalopathy and hepatic failure. This report presents the case of an 8-year-old girl with Reye syndrome and seizures after the use of naproxen. CASE REPORT An 8-year-old girl experienced a 3-day episode of fever and abdominal pain. After receiving naproxen (375 mg twice daily) starting from day -3, she exhibited hypotension, tonic seizure, and loss of consciousness (day 1). Physical examination and laboratory test results revealed acute kidney injury, metabolic acidosis, and elevated levels of lactate dehydrogenase (LDH), liver enzymes, and ferritin. On day 2, the maximum values of aspartate aminotransferase, alanine aminotransferase, LDH, creatinine, and ferritin were 955 U/L, 132 U/L, 8040 U/L, 2 mg/dL, and >40000 ug/L, respectively. She was given supportive care and recovered after 11 days (day 12), with normalization of kidney function and metabolic abnormalities. To identify possible genetic polymorphisms associated with the patient's symptoms, genotypes were tested using a drug metabolizing enzymes and transporters (DMET) gene chip. Among genes involved in the metabolism of naproxen, UGT1A6 (*1/*2) and UGT2B7 (*1/*2) resulted in possibly decreased function. Other results which may have had clinical significance included homozygote results for NAT2*6/*6 (rs1799930). CONCLUSIONS A rare case of Reye syndrome after administration of naproxen was presented in this case. A DMET gene chip was used to screen for possible genetic polymorphisms associated with Reye syndrome, but the result was inconclusive.

Dihydrolipoamide dehydrogenase deficiency: a still overlooked cause of recurrent acute liver failure and Reye-like syndrome.

The causes of Reye-like syndrome are not completely understood. Dihydrolipoamide dehydrogenase (DLD or E3) deficiency is a rare metabolic disorder causing neurological or liver impairment. Specific changes in the levels of urinary and plasma metabolites are the hallmark of the classical form of the disease. Here, we report a consanguineous family of Algerian origin with DLD deficiency presenting without suggestive clinical laboratory and anatomopathological findings. Two children died at birth from hepatic failure and three currently adult siblings had recurrent episodes of hepatic cytolysis associated with liver failure or Reye-like syndrome from infancy. Biochemical investigation (lactate, pyruvate, aminoacids in plasma, organic acids in urine) was normal. Histologic examination of liver and muscle showed mild lipid inclusions that were only visible by electron microscopy. The diagnosis of DLD deficiency was possible only after genome-wide linkage analysis, confirmed by a homozygous mutation (p.G229C) in the DLD gene, previously reported in patients with the same geographic origin. DLD and pyruvate dehydrogenase activities were respectively reduced to 25% and 70% in skin fibroblasts of patients and were unresponsive to riboflavin supplementation. In conclusion, this observation clearly supports the view that DLD deficiency should be considered in patients with Reye-like syndrome or liver failure even in the absence of suggestive biochemical findings, with the p.G229C mutation screening as a valuable test in the Arab patients because of its high frequency. It also highlights the usefulness of genome-wide linkage analysis for decisive diagnosis advance in inherited metabolic disorders.

Milder clinical course of Type IV 3-methylglutaconic aciduria due to a novel mutation in TMEM70.

Mitochondrial disorders are a large and genetically heterogeneous group of disorders posing a significant diagnostic challenge. Only approximately 10-20% of patients have identifiable alterations in their mitochondrial DNA (mtDNA). The remaining ~80-90% of affected patients likely harbor mutations in nuclear genes, most of which are still poorly characterized, and therefore not amenable to efficient screening using currently available molecular methods. Here we present a patient, who has been followed since birth after presenting with neonatal hyperammonemia, lactic acidosis, Reye-like syndrome episodes, and ventricular tachyarrhythmia. Initial biochemical work-up revealed hyperalaninemia, normal plasma glutamine, mild orotic aciduria and significant amounts of urinary 3-methylglutaconic (3-MGC) and 3-methylglutaric (3-MGA) acids. Muscle biopsy demonstrated the presence of ragged-red fibers and non-specific structural abnormalities of mitochondria. The activities of respiratory chain enzymes (complexes I-IV) showed no deficiency. Mutational analysis of the entire mitochondrial genome did not reveal deleterious point mutations or large deletions. Long-term follow-up was significant for a later-onset hypertrophic cardiomyopathy, muscle weakness, and exercise intolerance. Although she had frequent episodes of Reye-like episodes in infancy and early childhood, mostly triggered by illnesses, these symptoms improved significantly with the onset of puberty. In the light of recent reports linking cases of type IV 3-methylglutaconic aciduria (3-MGCA) and hypertrophic cardiomyopathy to mutations in TMEM70, we proceeded with sequencing analysis of this gene. We identified one previously reported splice site mutation, c.317-2A>G and a novel mutation c.494G>A (p.G165D) in an evolutionarily conserved region predicted to be deleterious. This variant was not identified in 100 chromosomes of healthy control subjects and 200 chromosomes of patients with cardiomyopathies. Western blotting using a polyclonal antibody against ATP5J, subunit F6 of ATP synthase, on patient's skin fibroblasts showed undetectable amount of the ATP5J protein. In comparison to the previously reported cases, we note that our patient had normal growth parameters and cognitive development, absence of structural heart and urinary tract defects, no dysmorphic features, improvement of symptoms with age, and persistence of hypertrophic cardiomyopathy.

A role for IRF3-dependent RXRalpha repression in hepatotoxicity associated with viral infections.

Viral infections and antiviral responses have been linked to several metabolic diseases, including Reye's syndrome, which is aspirin-induced hepatotoxicity in the context of a viral infection. We identify an interferon regulatory factor 3 (IRF3)-dependent but type I interferon-independent pathway that strongly inhibits the expression of retinoid X receptor alpha (RXRalpha) and suppresses the induction of its downstream target genes, including those involved in hepatic detoxification. Activation of IRF3 by viral infection in vivo greatly enhances bile acid- and aspirin-induced hepatotoxicity. Our results provide a critical link between the innate immune response and host metabolism, identifying IRF3-mediated down-regulation of RXRalpha as a molecular mechanism for pathogen-associated metabolic diseases.

Reye-like syndrome resulting from novel missense mutations in mitochondrial medium- and short-chain l-3-hydroxy-acyl-CoA dehydrogenase.

Medium- and short-chain l-3-hydroxy-acyl-CoA dehydrogenase (M/SCHAD) deficiency is a recessively inherited disorder of fatty acid oxidation. Currently, only four patients from three families have been reported in the literature. All these patients presented with hypoglycemia associated with hyperinsulinism (HI). This association suggests that there is a role for M/SCHAD in regulating the pancreatic secretion of insulin. We present a fifth patient whose presentation was similar to Reye syndrome, a feature in common with most of the previously recognized disorders of fatty acid oxidation but with no clinical evidence of HI. Sequencing of the HAD1 gene on chromosome 4 revealed compound heterozygosity for two novel missense mutations, 170A>G, resulting in D45G, and 676T>C, resulting in Y214H. The mutant enzymes were expressed and subjected to kinetic analysis. Y214H has no detectable activity, whilst D45G, which resides in the cofactor-binding pocket, has an altered K(m) for NADH (96 microM versus 24 microM for the wild-type). This represents the first kinetic M/SCHAD mutant, which explains the high residual activity in skin fibroblasts. The lack of obvious HI in this patient may be related to the high residual activity and indicates that HI associated with M/SCHAD deficiency may only be present with complete deficiency. The spectrum of M/SCHAD phenotype should be broadened to include acute liver disease.

Pontocerebellar hypoplasia type 2 and Reye-like syndrome.

Pontocerebellar hypoplasia is an autosomal recessive syndrome with onset during the fetal period. Two subtypes of pontocerebellar hypoplasia have been described on the basis of clinical and neuropathologic criteria. Pontocerebellar hypoplasia type 2 is characterized by progressive microcephaly, early onset of extrapyramidal dyskinesia, and near absence of motor and cognitive development, without signs of either spinal or peripheral involvement. We report a clinical observation of a patient with pontocerebellar hypoplasia type 2, a 3-year-old girl with progressive microcephaly, dystonic limb movements, and absence of motor and cognitive development. Cranial magnetic resonance imaging revealed pontocerebellar hypoplasia. At the age of 2 years, she suffered a Reye-like syndrome that worsened her condition. Differential diagnosis was established with intrauterine injuries, other malformative syndromes, and neurodegenerative or neurometabolic disorders, which can be associated with cerebellar hypoplasia.

Important role of prodromal viral infections responsible for inhibition of xenobiotic metabolizing enzymes in the pathomechanism of idiopathic Reye's syndrome, Stevens-Johnson syndrome, autoimmune hepatitis, and hepatotoxicity of the therapeutic doses of acetaminophen used in genetically predisposed persons.

Upper respiratory tract febrile illnesses caused by various viruses, mycoplasma, chlamydia infections, and/or inflammatory diseases are usually observed a few days to a few (several) weeks before the onset of Reye's syndrome, Stevens-Johnson syndrome, autoimmune hepatitis (hepatotropic virus infections), or hepatotoxicity associated with therapeutic administration of acetaminophen in persons with varying degrees of deficits of important enzymatic activity. Activation of systemic host defense mechanisms by inflammatory component(s) results in depression of various induced and constitutive isoforms of cytochrome P-450 mixed-function oxidase system superfamily enzymes in the liver and most other tissues of the body. Because several cytochrome P-450 enzymes activities important for biotransformation of many endogenous and egzogenous substances show considerable variability between individuals, in some genetically predisposed persons, even the administration of therapeutic doses of a drug may result in serious clinical mishaps, if an important concomitant risk factor (eg, acute viral infection) is involved. Several inflammatory cytokines, such as interleukins, transforming growth factor beta1, human hepatocyte growth factor, and lymphotoxin, downregulate gene expression of major cytochrome P-450 enzymes with the specific effects on mRNA levels, protein expression, and enzyme activity observed with a given cytokine varying for each P-450 studied, thus eventually leading to metabolite-mediated adverse drug reactions and immunometallic diseases which sometimes result in tissue injury beyond the site(s) where metabolic bioactivation takes place. On the other hand, it must be emphasized that inhibition of metabolism of several drugs, as well as influence on the concentration and/or ratio of various cytokines in inflamed tissues, may exert beneficial effects in patients with different diseases, thus opening new therapeutic possibilities. Clinically relevant interactions may be exemplified by the effects of some fluoroquinolone antibiotics, such as pefloxacin and ciprofloxacin, which probably have a steroid-sparing effect in some patients with frequently relapsing nephrotic syndrome, and an increased bioavailability of several drugs following concomitant intake with freshly pressed grapefruit juice, eventually caused by inhibition of their metabolism, mediated mainly by CYP3A and specifically inhibited by naturally occurring flavonoids.

Do NSAIDs contribute to acute fatty liver of pregnancy?

Acute fatty liver of pregnancy (AFLP) and the childhood encephalopathy known as Reye's syndrome are both characterised by microvesicular steatosis. Mothers with AFLP are frequently heterozygous for a mutation which reduces the activity of the trifunctional protein (TP) of fatty-acid oxidation. Several lines of evidence suggest that blockade of fatty-acid oxidation may also be the underlying cause of Reye's syndrome, and epidemiological studies have identified aspirin taken during a viral illness as a contributing factor to the development of the disease. The hypotheses are presented:* that children with Reye's syndrome may also be heterozygous for TP mutation, and* that inhibition of the residual long-chain fatty-acid oxidation by NSAIDs including aspirin precipitates the similar symptoms observed in patients with Reye's syndrome and AFLP. Identification of NSAIDs as candidates for the unidentified factor which precipitates AFLP suggests that avoidance of NSAIDs during pregnancy may lead to a reduction in the incidence of this life-threatening disease.

Diagnosis of mitochondrial trifunctional protein deficiency in a blood spot from the newborn screening card by tandem mass spectrometry and DNA analysis.

Trifunctional protein (TFP) plays a significant role in the mitochondrial beta-oxidation of long-chain fatty acids. Its deficiency impairs the energy generating function of this pathway and causes hypoketotic hypoglycemia once hepatic glycogen stores are depleted. A Reye-like syndrome, cardiomyopathy, and sudden death may follow. The diagnosis is based on demonstration of significantly decreased enzyme activity of at least two of the three involved enzymes in fibroblasts. The possibility of prospective diagnosis of TFP deficiency by newborn screening using tandem mass spectrometry (MS/MS) has not been evaluated. We report the postmortem diagnosis of a male newborn, who suffered sudden death at 2 wk of age, and his younger sister, who died of cardiomyopathy complicated by acute heart failure at the age of 6 mo, after she had acquired a common viral infection. Blood spots from the original newborn screening cards were the only remaining material from the patients. Analysis by MS/MS revealed acylcarnitine profiles consistent with either TFP or long-chain 3-hydroxyacyl coenzyme A dehydrogenase (LCHAD) deficiency. To prove the diagnosis, the alpha- and beta-subunit genes coding for TFP were examined. The patients were compound heterozygous for a 4-bp-deletion and an a-->g missense mutation, both in the same exon 3 donor consensus splice site. This is the first report of the diagnosis of TFP deficiency using blood spots obtained for newborn screening and suggests that TFP deficiency may be detectable by prospective newborn screening using MS/MS.

Visualization of mitochondria with green fluorescent protein in cultured fibroblasts from patients with mitochondrial diseases.

cDNAs for green fluorescent protein (GFP) and for a GFP fusion protein containing the presequence of human ornithine transcarbamylase (pOTC-GFP) were transfected into cultured human fibroblasts. GFP cDNA gave diffuse fluorescence throughout the cytoplasm and the nucleus, whereas pOTC-GFP cDNA gave mitochondria-associated fluorescence. Fluorescent mitochondrial structures could be classified into five patterns: thread-like mitochondria, fine thread-like ones, rod-like ones, granular ones, and granular ones with weak cytosolic fluorescence. pOTC-GFP mutants resulted in a loss of mitochondrial fluorescence and an appearance of weak fluorescence throughout the cytoplasm. pOTC-GFP cDNA was transfected into fibroblasts from patients with various mitochondrial diseases. Higher ratios of fibroblasts with granular mitochondria and those with fine thread-like ones were observed in a patient with Reye's syndrome and a patient with Kearns-Sayre syndrome. Weak cytosolic fluorescence was sometimes observed in fibroblasts from these patients. This method will be useful to analyze mitochondrial structural alterations and disorders of mitochondrial protein import.

Frequency of 985A-to-G mutation in medium-chain acyl-CoA dehydrogenase gene among patients with sudden infant death syndrome, Reye syndrome, severe motor and intellectual disabilities and healthy newborns in Japan.

The prevalence of the 985A-to-G mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene among Japanese patients with sudden infant death syndrome, Reye syndrome, unknown fatty acid oxidation disorders and severe motor and intellectual disabilities was studied using the PCR/Nco-I method for molecular diagnosis. A frequency study of this common mutation was also conducted on blood samples and left over Guthrie cards from 329 healthy newborns in Japan. Neither heterozygotes nor homozygotes for the 985A-to-G mutation were identified among both patients and controls. The result of the present study accord with previous reports that MCAD deficiency is a common disorder in Caucasians, but quite rare among Japanese. Therefore, newborn mass-screening for MCAD deficiency using this method will not be practical in Japan. However, it still seems necessary to investigate a child with fatty acid oxidation disorder for the presence of MCAD deficiency, using both biochemical and molecular genetic methods.

Rapid detection of medium chain acyl-CoA dehydrogenase gene mutations by non-radioactive, single strand conformation polymorphism minigels.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inherited metabolic disorder affecting fatty acid beta oxidation. Identification of carriers is important since the disease can be fatal and is readily treatable once diagnosed. Twelve molecular defects have been identified in the MCAD gene; however, a single highly prevalent mutation, A985G, accounts for > 90% of mutant alleles in the white population. In order to facilitate the molecular diagnosis of MCAD deficiency, oligonucleotide primers were designed to amplify the exon regions encompassing the 12 mutations enzymatically, and PCR products were then screened with a single strand conformation polymorphism (SSCP) based method. Minigels were used allowing much faster run times, and silver staining was used after gel electrophoresis to eliminate the need for radioisotopic labelling strategies. Our non-radioactive, minigel SSCP approach showed that normals can be readily distinguished from heterozygotes and homozygotes for all three of the 12 known MCAD mutations which were detected in our sampling of 48 persons. In addition, each band pattern is characteristic for a specific mutation, including those mapping in the same PCR product like A985G and T1124C. When necessary, the molecular defect was confirmed using either restriction enzyme digestion of PCR products or by direct DNA sequence analysis or both. This rapid, non-radioactive approach can become routine for molecular diagnosis of MCAD deficiency and other genetic disorders.

Molecular cloning and sequence analysis of the cDNA for human mitochondrial short-chain enoyl-CoA hydratase.

Short chain enoyl-CoA hydratase (SCEH) catalyzes the second step of the mitochondrial fatty acid beta-oxidation spiral. We isolated cDNA clones for human SCEH to facilitate investigation of the enzyme structure of the gene and to examine the genetic background of Reye's syndrome and sudden infant death. Oligo(dT)-primed and random primed human liver cDNA libraries in lambda gt11 were screened using the entire sequence of the rat SCEH cDNA as a probe. Three positive clones covered the full-length cDNA sequence with an open reading frame encoding a precursor polypeptide of 290 amino acid residues, and deduced relative molecular mass (31,280) with a putative N-terminal presequence of 29 residues, a 5'-untranslated sequence of 21 bp and a 3'-untranslated sequence of 391 bp. Comparison with the rat SCEH cDNA showed that the deduced amino acid sequence of the human SCEH precursor is 84% identical to that of the rat enzyme precursor. Northern blot analysis gave a single mRNA species of 1.6 kb in the human liver, fibroblast and muscle.

Recurrent acute fatty liver of pregnancy associated with a fatty-acid oxidation defect in the offspring.

A case of a 29-year-old woman who has had two episodes both clinically and biochemically consistent with acute fatty liver of pregnancy is described. These episodes occurred in two successive pregnancies, and liver biopsy confirmed the diagnosis in the second pregnancy. Both pregnancies were managed by prompt fetal delivery; on both occasions this led to a complete biochemical resolution of the liver function abnormalities. Two healthy babies were delivered by ceasarian sections. This case is of particular importance because a rapidly progressive and devastating illness developed in both infants, leading to death at 6 1/2 and 6 months, respectively. The illness in both babies was characterized by wide-spread fatty infiltration of several vital organs and a failure of any treatment to influence the outcome of that illness. Studies suggested that the illness in the children was caused by a still ill-defined disorder of fatty acid oxidation. The biochemical disorder evidenced in this family is discussed, in an attempt to shed light on the etiology of acute fatty liver of pregnancy.

Recurrent, familial Reye-like syndrome with a new complex amino and organic aciduria.

Five of 13 siblings from a Jewish-Ashkenazi family suffered from recurrent Reye-like episodes. During attacks, these patients excreted alpha-keto-adipic, alpha-hydroxy-adipic, and alpha-aminoadipic acids, branched-chain keto acids and saccharopine in addition to lactic, pyruvic, and dicarboxylic acids characteristic of Reye syndrome. The serum concentrations of citrulline and alpha-amino-adipic acid were elevated and carnitine was at the upper limit of the normal range. Serum acetoacetate level was 4-5 times the beta-hydroxybutyrate level, but the pyruvate/lactate ratio was normal. Notably, plasma ketone bodies were lower than expected from the degree of catabolism. When the patients were symptom-free, no abnormal amino or organic acids in serum or urine were detected. These findings might be interpreted as a functional impairment at three different biochemical sites: fatty acid beta-oxidation, dehydrogenase complexes of the pyruvic, alpha-ketoglutaric, alpha-ketoadipic, and branched-chain keto acids, and pyruvate carboxylase. We suggest that in this hereditary disorder a toxic substance, exogenously or endogenously derived, interfered at multiple sites in different metabolic pathways.

Genetic diagnosis. Implications for medical practice.

Genetic diagnostic techniques increasingly permit the detection of predisposition to illness long before the onset of the disease process itself. Medicine is on the verge of becoming a predictive science as well as a diagnostic and therapeutic one. Genetic diagnosis could have profound effects on many aspects of our health care system, including the prestige and effectiveness of preventive medicine, the competitive behavior of health care organizations and insurance companies, access to private health insurance, the ability of primary care physicians to serve as gatekeepers, and other matters. This article examines the range of potential effects of the new genetic diagnostics on the organization and financing of health care. For purposes of illustration the authors examine in detail the possible consequences of genetic tests for predisposition to two diseases: Reye's Syndrome and lung cancer in smokers.

Inborn errors of metabolism in children referred with Reye's syndrome. A changing pattern.

Genetic disorders were identified infrequently among children presenting with Reye's syndrome in the past. During a two-year period, we evaluated four consecutive patients referred for intensive care of Reye's syndrome. A standard investigation for inborn errors of metabolism revealed that two patients had enzymatic defects of fatty acid oxidation, and the other two had partial deficiencies of ornithine transcarbamoylase. None had experienced a previous episode of Reye's syndrome, and three of the four had been entirely healthy in the past. Our experience suggests that as the incidence of Reye's syndrome has decreased, patients with its clinical features are now more likely to have manageable inborn errors of metabolism (eg, disorders of ureagenesis, ketogenesis, and branched-chain amino acids).