Richard H. Haas

Unusual brain growth patterns in early life in patients with autistic disorder An MRI study

Objective: To quantify developmental abnormalities in cerebral and cerebellar volume in autism. Methods: The authors studied 60 autistic and 52 normal boys (age, 2 to 16 years) using MRI. Thirty autistic boys were diagnosed and scanned when 5 years or older. The other 30 were scanned when 2 through 4 years of age and then diagnosed with autism at least 2.5 years later, at an age when the diagnosis of autism is more reliable. Results: Neonatal head circumferences from clinical records were available for 14 of 15 autistic 2- to 5-year-olds and, on average, were normal (35.1 ± 1.3 cm versus clinical norms: 34.6 ± 1.6 cm), indicative of normal overall brain volume at birth; one measure was above the 95th percentile. By ages 2 to 4 years, 90% of autistic boys had a brain volume larger than normal average, and 37% met criteria for developmental macrencephaly. Autistic 2- to 3-year-olds had more cerebral (18%) and cerebellar (39%) white matter, and more cerebral cortical gray matter (12%) than normal, whereas older autistic children and adolescents did not have such enlarged gray and white matter volumes. In the cerebellum, autistic boys had less gray matter, smaller ratio of gray to white matter, and smaller vermis lobules VI–VII than normal controls. Conclusions: Abnormal regulation of brain growth in autism results in early overgrowth followed by abnormally slowed growth. Hyperplasia was present in cerebral gray matter and cerebral and cerebellar white matter in early life in patients with autism.

Impairment in shifting attention in autistic and cerebellar patients

MRI and autopsy evidence of early maldevelopment of cerebellar vermis and hemispheres in autism raise the question of how cerebellar maldevelopment contributes to the cognitive and social deficits characteristic of autism. Compared with normal controls, autistic patients and patients with acquired cerebellar lesions were similarly impaired in a task requiring rapid and accurate shifts of attention between auditory and visual stimuli. Neurophysiologic and behavioral evidence rules out motor dysfunction as the cause of this deficit. These findings are consistent with the proposal that in autism cerebellar maldevelopment may contribute to an inability to execute rapid attention shifts, which in turn undermines social and cognitive development, and also with the proposal that the human cerebellum is involved in the coordination of rapid attention shifts in a fashion analogous to its role in the coordination of movement.

Statin-Associated Myopathy with Normal Creatine Kinase Levels

Context Although severe myopathy can occur with statin therapy, some patients receiving statins develop muscle symptoms but have normal serum creatine kinase levels. Contribution This report documents biopsy-confirmed myopathy in four statin-treated patients with normal creatine kinase levels. When patients were challenged with placebo or statin, symptoms and histologic changes occurred only during statin use. Implications Normal creatine kinase levels do not rule out statin-associated myopathy in patients with muscle symptoms. The frequency of this disorder is unknown. The Editors 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are the mainstay of therapy for hypercholesterolemia because of their effectiveness and exceptional safety profile (1-3). Nonetheless, many patients treated with statins have muscle symptoms, and some patients develop severe muscle toxicity. Little is known about the mechanism by which statin therapy leads to muscle toxicity. The recent withdrawal of cerivastatin from the U.S. market has highlighted both our ignorance and the need for postmarketing surveillance of these therapies (4). There are credible reports of patients who have muscle symptoms and normal serum creatine kinase levels while receiving statins. One of us described 15 patients with muscle stiffness and tenderness who had normal levels of creatine kinase. Muscle biopsies in these patients revealed ragged red fibers consistent with mitochondrial myopathy (5). Other researchers have described patients with reproducible muscle pain during statin therapy despite normal creatine kinase levels (6, 7). Cell cultures and animal models have shown statin toxicity despite normal creatine kinase levels (8, 9). Thus, it appears that muscle symptoms of some patients receiving statin therapy might represent a muscle toxicity below the threshold needed to increase creatine kinase levels. We describe four such patients who had myopathy during statin therapy despite normal creatine kinase levels. Methods Four patients were identified among the first 20 patients randomly assigned in an ongoing, unfunded, double-blinded, crossover clinical trial that had been approved by an investigational review board. The objective of the trial is to determine whether patients with muscle symptoms and normal creatine kinase levels while taking statins could distinguish blinded statin therapy from placebo. We hypothesized that some patients would be able to identify blinded statin therapy and have objective findings. Patients who had muscle symptoms during statin therapy that resolved when they were not receiving statins for 2 weeks were recruited from a clinical research group. Eligible patients had to have normal creatine kinase levels while they were experiencing symptoms. End points were 1) whether patients could accurately identify blinded statin therapy and 2) standard measures of functional capacity and muscle strength. The muscular strength tests during both placebo use and statin therapy included hip abduction and flexion measured by Nicholas Manual Muscle Tester (Lafayette Instrument Co., Lafayette, Indiana) (10). The 4 patients presented here were identified during a preliminary analysis of the first 21 patients. These 4 patients correctly identified the statin therapy phase because of the reproducible muscle symptoms. Tissue samples from percutaneous muscle biopsies were fresh frozen in liquid nitrogen for metabolic and genetic analysis and were fixed for pathologic analysis according to standard techniques (11). Pathologists reading the muscle biopsy findings were not blinded to the treatment status of the patients, and requisitions read possible myopathy. Plasma concentrations of HMG-CoA reductase inhibitors were measured by using a validated enzyme inhibition assay (12). The findings of these 4 patients are presented here and summarized in the Table. Table. Patient Characteristics Some muscle specimens were analyzed for mitochondrial point mutations at an accredited mitochondrial disease laboratory by using a standard point mutation analysis panel and Southern blotting techniques. Urine specimens were analyzed for organic acids by using standard mass spectrometry techniques. Results Patient 1 Patient 1 developed muscle aches and decreased exercise tolerance during 4 years of simvastatin therapy. She found it difficult to ascend one flight of stairs without resting to relieve severe leg aching. Symptoms did not change during a brief trial of atorvastatin. After the patient entered the trial, her muscle symptoms markedly resolved during the 2-week washout period and the blinded placebo phase. Muscle aching recurred within 48 hours of initiation of the statin phase. After 2 weeks of statin therapy, the patient again found it difficult to ascend a flight of stairs because of leg pain and weakness. She repeated the protoco l and again experienced symptoms while receiving statin; the symptoms again resolved during the placebo phase. Results of serum and urine biochemical analyses are reported in the Table. Muscle biopsy revealed extensive lipid-filled vacuoles distributed within the myofibers (Figure), along with cytochrome oxidasenegative myofibers. Figure. Muscle biopsy specimens obtained while patients were receiving and not receiving statin therapy. parts A, C, and E parts B, D, and F The patients muscle symptoms and hip weakness improved 3 months after she had discontinued statin therapy. At that point, repeated muscle biopsy showed complete resolution of the pathologic abnormalities (Figure). Patient 2 Patient 2 developed muscle pains and weakness while receiving lovastatin, 40 mg/d. She could not open jars or snap her fingers. Her physician changed her therapy to niacin for 2 years. Each time simvastatin was added to improve her lipid control, the patient developed muscle aches, weakness, and shortness of breath with exertion. She entered the double-blinded trial for two separate evaluations and correctly identified the statin phases because of reproduction of her symptoms. The patient had muscle weakness and normal creatine kinase levels during each statin phase (Table). Muscle biopsy revealed accentuated lipid droplet accumulation and cytochrome oxidasenegative muscle fibers that were consistent with myopathy. Repeated muscle biopsy performed 3 months after discontinuation of statin therapy revealed complete resolution of the abnormal lipid stores. Patient 3 Patient 3 developed aching and weakness of the shoulder, hip, and thigh while taking atorvastatin, 20 mg/d, for 2 years. During a 2-week vacation, he forgot his medication and noticed a substantial resolution of his muscle symptoms. He entered the trial for two separate evaluations and correctly identified the statin arm both times after developing weakness; his creatine kinase level was normal at both evaluations (Table). A muscle biopsy performed during atorvastatin therapy revealed increased lipid droplets, ragged red fibers, and cytochrome oxidasenegative muscle fibers, all consistent with lipid myopathy. Repeated biopsy done after the patient had not received statin therapy for 5.5 months showed resolution of the increased lipid droplets. Patient 4 Patient 4 began receiving atorvastatin, 10 mg/d, for combined hyperlipidemia, and within 2 weeks he noticed leg aches when climbing stairs. His symptoms resolved 2 weeks after he had stopped taking atorvastatin. Upon enrollment in the trial, he correctly identified the blinded statin therapy and had measurable hip weakness despite a normal creatine kinase level (Table). His muscle biopsy demonstrated cytochrome oxidasenegative fibers. Electron microscopy showed abnormal lipid droplets surrounded by electron-dense membranous debris. The patient did not undergo biopsy after discontinuation of statin therapy. Testing We performed a series of tests to exclude possible causes of mitochondrial dysfunction other than statin therapy. Biochemical testing of these patients during statin therapy did not reveal any evidence of carnitine deficiency or hypothyroidism. Discussion Randomized trials and community surveillance have demonstrated an extremely low incidence of serious muscle toxicity in patients receiving statins: 1 case per 10 000 (13, 14). Despite this low incidence, many patients attribute their muscle symptoms to statin therapy. The similar clinical, pathologic, and biochemical features of our four patients suggest that the patients have the same type of myopathy. More important, these features are convincingly related to statin therapy and fulfill the accepted criteria for an adverse drug reaction: 1) Muscle symptoms occurred when the patients were receiving statins in a double-blinded fashion, 2) these symptoms normalized when the patients received placebo, 3) muscle toxicity recurred upon re-exposure to the statin, as it had occurred upon previous exposures, 4) the adverse reaction was confirmed by pathologic and biochemical findings, and 5) the pathologic abnormality reversed upon discontinuation of statin therapy (15). A limitation of our report is that the pathologists were not blinded to the treatment status of the patients at biopsy. Nonetheless, the histochemical findings in these four patients were surprisingly similar. Unusually prominent accumulations of lipid were present in the type 1 fibers on oil red O stains or electron microscopy in all four patients. Other evidence of myopathy included cytochrome oxidasenegative staining fibers and ragged red fibers. These myopathic findings reversed when three of these patients had repeated biopsy while not receiving statins. Increased lipid stores, cytochrome oxidasenegative myofibers, and ragged red fibers are features of mitochondrial respiratory chain dysfunction and suggest myopathy due to a metabolic abnormality. Although some of these findings may rarely occur in myofibers because of aging, the pattern of increased lipid is unusual. It has pre

Evidence of linkage between the serotonin transporter and autistic disorder

The serotonin transporter gene (HTT) is a primary candidate in autistic disorder based on efficacy of potent serotonin transporter inhibitors in reducing rituals and routines. We initiated a candidate gene study of HTT in trios consisting of probands with autistic disorder and both parents. Preliminary transmission/disequilibrium test (TDT) analysis with 86 families revealed no evidence for linkage or linkage disequilibrium between autistic disorder and a polymorphism in the second intron of HTT. However, preferential transmission of a short variant of the HTT promoter was found in the same 86 trios (TDT χ2 = 4.69, 1 d.f., P = 0.030). In further analyses, we considered haplotypes of the HTT promoter variant and second intron locus as alleles in a multiallelic TDT. Results confirmed the significance of the effect of this region (TDT χ2 = 11.85, 4 d.f., P = 0.018). This provides preliminary evidence of linkage and association between HTT and autistic disorder.

Linkage-Disequilibrium Mapping of Autistic Disorder, with 15q11-13 Markers

Autistic disorder is a complex genetic disease. Because of previous reports of individuals with autistic disorder with duplications of the Prader-Willi/Angelman syndrome critical region, we screened several markers across the 15q11-13 region, for linkage disequilibrium. One hundred forty families, consisting predominantly of a child with autistic disorder and both parents, were studied. Genotyping was performed by use of multiplex PCR and capillary electrophoresis. Two children were identified who had interstitial chromosome 15 duplications and were excluded from further linkage-disequilibrium analysis. Use of the multiallelic transmission-disequilibrium test (MTDT), for nine loci on 15q11-13, revealed linkage disequilibrium between autistic disorder and a marker in the gamma-aminobutyric acidA receptor subunit gene, GABRB3 155CA-2 (MTDT 28.63, 10 df, P=.0014). No evidence was found for parent-of-origin effects on allelic transmission. The convergence of GABRB3 as a positional and functional candidate along with the linkage-disequilibrium data suggests the need for further investigation of the role of GABRB3 or adjacent genes in autistic disorder.

Up-regulation of hypoxia-inducible factors HIF-1α and HIF-2α under normoxic conditions in renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function

Hypoxia induces transcription of a range of physiologically important genes including erythropoietin and vascular endothelial growth factor. The transcriptional activation is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric member of the basic helix–loop–helix PAS family, composed of α and β subunits. HIF-1α shares 48 per cent identity with the recently identified HIF-2α protein that is also stimulated by hypoxia. In a previous study of hemangioblastomas, the most frequent manifestation of hereditary von Hippel-Lindau disease (VHL), we found elevated levels of vascular endothelial growth factor and HIF-2α mRNA in stromal cells of the tumors. Mutations of the VHL tumor suppressor gene are associated with a variety of tumors such as renal clear cell carcinomas (RCC). In this study, we analysed the expression of the hypoxia-inducible factors HIF-1α and HIF-2α in a range of VHL wildtype and VHL deficient RCC cell lines. In the presence of functional VHL protein, HIF-1α mRNA levels are elevated, whereas HIF-2α mRNA expression is increased only in cells lacking a functional VHL gene product. On the protein levels, however, in VHL deficient cell lines, both HIF-α subunits are constitutively expressed, whereas re-introduction of a functional VHL gene restores the instability of HIF-1α and HIF-2α proteins under normoxic conditions. Moreover, immunohistochemical analyses of RCCs and hemangioblastomas demonstrate up-regulation of HIF-1α and HIF-2α in the tumor cells. The data presented here provide evidence for a role of the VHL protein in regulation of angiogenesis and erythropoiesis mediated by the HIF-1α and HIF-2α proteins.

The in-depth evaluation of suspected mitochondrial disease

Mitochondrial disease confirmation and establishment of a specific molecular diagnosis requires extensive clinical and laboratory evaluation. Dual genome origins of mitochondrial disease, multi-organ system manifestations, and an ever increasing spectrum of recognized phenotypes represent the main diagnostic challenges. To overcome these obstacles, compiling information from a variety of diagnostic laboratory modalities can often provide sufficient evidence to establish an etiology. These include blood and tissue histochemical and analyte measurements, neuroimaging, provocative testing, enzymatic assays of tissue samples and cultured cells, as well as DNA analysis. As interpretation of results from these multifaceted investigations can become quite complex, the Diagnostic Committee of the Mitochondrial Medicine Society developed this review to provide an overview of currently available and emerging methodologies for the diagnosis of primary mitochondrial disease, with a focus on disorders characterized by impairment of oxidative phosphorylation. The aim of this work is to facilitate the diagnosis of mitochondrial disease by geneticists, neurologists, and other metabolic specialists who face the challenge of evaluating patients of all ages with suspected mitochondrial disease.

Mitochondrial Disease: A Practical Approach for Primary Care Physicians

Notorious variability in the presentation of mitochondrial disease in the infant and young child complicates its clinical diagnosis. Mitochondrial disease is not a single entity but, rather, a heterogeneous group of disorders characterized by impaired energy production due to genetically based oxidative phosphorylation dysfunction. Together, these disorders constitute the most common neurometabolic disease of childhood with an estimated minimal risk of developing mitochondrial disease of 1 in 5000. Diagnostic difficulty results from not only the variable and often nonspecific presentation of these disorders but also from the absence of a reliable biomarker specific for the screening or diagnosis of mitochondrial disease. A simplified and standardized approach to facilitate the clinical recognition of mitochondrial disease by primary physicians is needed. With this article we aimed to improve the clinical recognition of mitochondrial disease by primary care providers and empower the generalist to initiate appropriate baseline diagnostic testing before determining the need for specialist referral. This is particularly important in light of the international shortage of metabolism specialists to comprehensively evaluate this large and complex disease population. It is hoped that greater familiarity among primary care physicians with the protean manifestations of mitochondrial disease will facilitate the proper diagnosis and management of this growing cohort of pediatric patients who present across all specialties.

Autism Associated With the Mitochondrial DNA G8363A Transfer RNALys Mutation

We report a family with a heterogeneous group of neurologic disorders associated with the mitochondrial DNA G8363A transfer ribonucleic acid (RNA)Lys mutation. The phenotype of one child in the family was consistent with autism. During his second year of life, he lost previously acquired language skills and developed marked hyperactivity with toe-walking, abnormal reciprocal social interaction, stereotyped mannerisms, restricted interests, self-injurious behavior, and seizures. Brain magnetic resonance imaging (MRI) and repeated serum lactate studies were normal. His older sister developed signs of Leigh syndrome with progressive ataxia, myoclonus, seizures, and cognitive regression. Her laboratory studies revealed increased MRI T2-weighted signal in the putamen and posterior medulla, elevated lactate in serum and cerebrospinal fluid, and absence of cytochrome c oxidase staining in muscle histochemistry. Molecular analysis in her revealed the G8363A mutation of the mitochondrial transfer RNA Lys gene in blood (82% mutant mitochondrial DNA) and muscle (86%). The proportions of mutant mitochondrial DNA from her brother with autism were lower (blood 60%, muscle 61%). It is likely that the origin of his autism phenotype is the pathogenic G8363A mitochondrial DNA mutation. This observation suggests that certain mitochondrial point mutations could be the basis for autism in some individuals. (J Child Neurol 2000;15:357-361).

Diagnosis and management of mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society.

Purpose:The purpose of this statement is to review the literature regarding mitochondrial disease and to provide recommendations for optimal diagnosis and treatment. This statement is intended for physicians who are engaged in diagnosing and treating these patients. Methods:The Writing Group members were appointed by the Mitochondrial Medicine Society. The panel included members with expertise in several different areas. The panel members utilized a comprehensive review of the literature, surveys, and the Delphi method to reach consensus. We anticipate that this statement will need to be updated as the field continues to evolve. Results:Consensus-based recommendations are provided for the diagnosis and treatment of mitochondrial disease.Conclusion:The Delphi process enabled the formation of consensus-based recommendations. We hope that these recommendations will help standardize the evaluation, diagnosis, and care of patients with suspected or demonstrated mitochondrial disease.Genet Med 17 9, 689–701.