Conceived and designed the experiments: JRW RIK MB MRN. Analyzed the data: JRW RIK MB BHC KFM RLM RLK MRN. Wrote the paper: JRW RIK MB BHC KFM RLM RLK MRN. Obtained research funding: MRN.
Author BHC is compensated as a member of the Speakers Board and as a member of the Scientific Advisory Board of Transgenomic, Inc., Omaha.
Previous reports indicate an association between autism spectrum disorders (ASD) and disorders of mitochondrial oxidative phosphorylation. One study suggested that children with both diagnoses are clinically indistinguishable from children with idiopathic autism. There are, however, no detailed analyses of the clinical and laboratory findings in a large cohort of these children. Therefore, we undertook a comprehensive review of patients with ASD and a mitochondrial disorder.
We reviewed medical records of 25 patients with a primary diagnosis of ASD by DSM-IV-TR criteria, later determined to have enzyme- or mutation-defined mitochondrial electron transport chain (ETC) dysfunction. Twenty-four of 25 patients had one or more major clinical abnormalities uncommon in idiopathic autism. Twenty-one patients had histories of significant non-neurological medical problems. Nineteen patients exhibited constitutional symptoms, especially excessive fatigability. Fifteen patients had abnormal neurological findings. Unusual developmental phenotypes included marked delay in early gross motor milestones (32%) and unusual patterns of regression (40%). Levels of blood lactate, plasma alanine, and serum ALT and/or AST were increased at least once in 76%, 36%, and 52% of patients, respectively. The most common ETC disorders were deficiencies of complex I (64%) and complex III (20%). Two patients had rare mtDNA mutations of likely pathogenicity.
Although all patients' initial diagnosis was idiopathic autism, careful clinical and biochemical assessment identified clinical findings that differentiated them from children with idiopathic autism. These and prior data suggest a disturbance of mitochondrial energy production as an underlying pathophysiological mechanism in a subset of individuals with autism.
Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by impaired social interaction and communication, as well as isolated interests and repetitive or stereotyped behaviors
In 1998, Lombard postulated mitochondrial dysfunction as a cause of autism
We reviewed medical records of 25 children with a diagnosis of ASD according to DSM-IV-TR criteria
Children were referred for genetic and/or metabolic evaluation of autism but not specifically for evaluation for mitochondrial disease. Children with known chromosomal or monogenic disorders were excluded. All patients met criteria for probable or definite mitochondrial disease by both the Modified Walker Criteria and the Mitochondrial Disease Criteria (MDC)
Medical records of each patient were independently reviewed by at least two members of the study team. Growth parameters were evaluated using the ABase software and North American pediatric reference values
Twenty-three of the 25 children underwent quadriceps muscle biopsies, 11 had skin biopsies, and one had a liver biopsy. Three patients had sibs with muscle biopsies. Muscle ETC determinations were performed at either Horizon Molecular Medicine, LLC, Atlanta
Sixteen of 25 children underwent analyses for selected mitochondrial mutations and 11 of 25 patients had mtDNA mutation analysis by dHPLC of the entire mitochondrial genome at Transgenomic, Inc, Omaha
Twenty-five patients–13 males and 12 females ages 2–20 years–were included in this analysis; 11 patients met DSM-IV-TR criteria for autistic disorder and 14 met DSM-IV-TR criteria for PDD-NOS. Twenty-one patients had definite mitochondrial disease and four had probable mitochondrial disease by the Modified Walker Criteria. Eleven patients met criteria for definite mitochondrial disease and 14 met criteria for probable disease using the MDC.
Number of patients | Percent of patients | |
8 | 32 | |
13 | 52 | |
7 | 28 | |
16 | 64 | |
5 | 20 | |
2 | 8 | |
2 | 8 | |
2 | 8 | |
5 | 20 | |
4 | 16 | |
4 | 16 | |
17 | 68 | |
8 | 32 | |
5 | 20 | |
2 | 8 | |
4 | 16 | |
2 | 8 | |
3 | 12 | |
3 | 12 | |
9 | 36 | |
6 | 24 | |
24 | 96 | |
21 | 84 | |
8 | 32 | |
15 | 60 |
Defined as ≥5 SD from mean age at walking
Does not include multiple gestation birth, prenatal/perinatal complications, GERD, constipation or macrocephaly.
Does not include fatigability, non-refractory seizures, macrocephaly or regression.
Twenty-one patients had histories of major non-neurological medical problems, most commonly of the gastrointestinal system, with gastroesophageal reflux affecting nine and constipation affecting eight subjects. Seven patients had structural or functional cardiovascular abnormalities. In addition, 17 patients had excessive fatigability or exercise intolerance and several children had abnormal physical exam findings including six with facial dysmorphism, four with microcephaly, four with macrocephaly, and five with growth retardation.
Twelve patients had neurological findings uncommon in ASD apart from microcephaly, including: oculomotor abnormalities (n = 4), sensorineural hearing deficit (n = 3), dysarthria (n = 3), ptosis (n = 2), movement disorders (n = 2), and hypertonia (n = 1). Five patients had seizures with one having medically refractory epilepsy.
Eight patients had markedly delayed early motor milestones. Of 14 patients with regression of previously acquired skills, nine had multiple regressions, six regressed at ages older than three years, and seven had regressions occurring with infections or other metabolic stresses. In six, gross motor skills were lost in addition to language skills. In one case, the timing of regression coincided with a recent vaccination.
Family history data were notable for likely autosomal recessive inheritance in one patient whose parents were first cousins. The family history suggested mitochondrial inheritance in three patients. Three other patients had mothers with features suggesting mitochondrial disease and four patients had sibs with either enzyme-defined mitochondrial disease and/or a clinical history consistent with mitochondrial disease.
Blood lactate was measured in all patients; 19 had at least one increased lactate level, 13 had multiple high levels and 14 had at least one lactate level greater than 3 mmol/L. Of 17 patients who had at least one blood pyruvate determination, nine (53% of those tested) had at least one increased pyruvate level. Plasma amino acids were analyzed for all patients; nine had at least one increased plasma alanine level and four had multiple high alanine levels. The level of AST and/or ALT was increased in 13. One patient had an increased ALT with a normal AST and seven had elevated AST with normal ALT. Eight patients (28%) had increased serum CK levels. Ten patients (42% of those tested) had urinary organic acid abnormalities indicative of mitochondrial dysfunction. The fibroblast lactate/pyruvate ratio was increased for three patients.
Number abnormal | Number tested | Percent of tested who were abnormal | |
19 | 25 | 76 | |
9 | 17 | 53 | |
9 | 25 | 36 | |
13 | 25 | 52 | |
8 | 25 | 32 | |
10 | 24 | 42 | |
3 | 15 | 20 | |
24 | 25 | 96 | |
10 | 21 | 48 | |
2 | 5 | 40 |
Twenty-one patients underwent cranial MRI studies; five also had cranial MRS. Ten children had abnormalities on MRI (48% of those imaged), without a single finding predominating, while two patients had increased lactate on MRS (40% of those imaged).
The most common histological abnormalities in muscle were atrophic or regenerating fibers (n = 8), reduced cytochrome oxidase (COX) staining (n = 6), variation in fiber size (n = 5), and increased myofiber lipid (n = 5). The most common ultrastructural abnormalities were abnormal mitochondrial morphology (n = 4) and increased number of mitochondria (n = 4).
Number of patients | |
5 | |
8 | |
4 | |
5 | |
1 | |
6 | |
2 | |
4 | |
4 |
Twenty patients had deficient activity of a respiratory chain complex on tissue ETC or polarographic analysis; three others had phenotypes and biochemical profiles consistent with mitochondrial disease and a sib with less than 20% activity on muscle ETC determination. Specific abnormalities included complex I defect in 16, complex III defect in five, complex II defect in two, and complex IV defect in one. Two patients had mtDNA mutations of likely pathogenicity and four had mtDNA sequence variants of unclear pathogenicity.
Number of patients | Percent of patients | Mutations | |
16 | 64 | ||
2 | 8 | ||
5 | 20 | ||
1 | 4 | ||
1 | 4 | ||
2 | 8 | 3397A>G; 4295A>G | |
4 | 16 | 3394T>C; 10394C>T; 11809T>C; 11984T>C |
The cohort of 25 patients reported here comprises the largest group of individuals with co-occurrence of ASD and defective oxidative phosphorylation reported to date. While previous case reports implicated an association of ASD and mitochondrial dysfunction, it could be argued that this was a chance occurrence in those individuals. Recent epidemiological studies indicate a population prevalence of ASD in about one in 166 children
Our results indicate diverse and complex developmental, neurological, and medical phenotypes of persons with mitochondrial autism, nearly all of which differ from those of patients with idiopathic ASD. Although many children with ASD exhibit some degree of hypotonia, most attain their early gross motor milestones on time
Recently, there has been increased concern regarding a possible causative role of vaccinations in autistic children with an underlying mitochondrial cytopathy
In addition to atypical developmental patterns, non-neurological disorders were nearly universal in our patients. Although medical co-morbidities are not uncommon in individuals with ASD, they are not reported to be present with the high frequency noted here
Along with medical co-morbidities, an increased frequency of prenatal and perinatal complications has been reported in children with ASD
Besides prenatal complications, our data indicate a high frequency of multiple gestation births. The relevance of this finding is unclear but interesting in view of previous controversy about twinning as a risk factor for autism
Also dramatically different from the general ASD population, the sex distribution of the subjects in our cohort is approximately 1∶1. Primary ASD is 3–6 times more common in males than females and this ratio is even higher in PDD-NOS
From a biochemical perspective, patients' blood and urine laboratory data revealed marked interindividual variation that did not appear to correspond with specific ETC defects or clinical phenotypes. Significant intraindividual variation was also noted for determinations of blood lactate and pyruvate levels, plasma alanine levels, serum transaminases and CPK, and urinary organic acids, with intermittent normal levels in most cases. The histological and ultrastructural abnormalities noted on muscle biopsies were also varied amongst the subjects of this cohort and similar to results noted in other cohorts of children with mitochondrial electron transport chain disorders
For most individuals with defects of oxidative phosphorylation, the diagnosis is made through ETC determination but an underlying nuclear or mitochondrial mutation usually cannot be identified
ETC complex I deficiency was the most prevalent enzyme defect, affecting 64% of our patients, followed by complex III deficiency, affecting 20%. It should be noted, however, that we cannot exclude the possibility that some patients might have biochemically mild forms of multiple ETC deficiencies. The predominance of ETC complex I deficiency is not unexpected and has been noted in cohorts of non-autistic patients with mitochondrial cytopathies
Of patients who underwent mitochondrial genomic analysis, two had rare homoplasmic DNA sequence variants of likely pathogenicity: mtDNA 3397A>G and 4295A>G. mtDNA 3397A>G, which replaces methionine with valine in a highly conserved region of the ND1 subunit of complex I, has been implicated in various neurological phenotypes and other conditions
Overall, our results demonstrate substantial clinical heterogeneity of individuals with co-occurring autism and defects of mitochondrial oxidative phosphorylation, nearly all of whom we found to be clinically distinct from children with idiopathic autism. The data do not exclude the possibility of persons with isolated autism having a disorder of oxidative phosphorylation–in fact, one of our patients did not have any major clinical features that distinguished her from typical autism. In addition, it is possible, if not likely, that a still broader clinical, biochemical and genetic spectrum of mitochondrial autism exists.
Finally, data from multiple disciplines, especially research in developmental neurobiology and genetics, point to several underlying pathophysiological mechanisms in autism, including altered neurite morphology, synaptogenesis and cell migration due to abnormalities in distinct ensembles of proteins and pathways
ASD Diagnosis and Mitochondrial Disease Criteria. PDD-NOS = Pervasive developmental disorder–not otherwise specified, MDC = mitochondrial disease criteria
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Pertinent Medical History. GERD = gastroesophageal reflux disease, POTS = postural orthostatic tachycardia syndrome, RBBB = right bundle branch block, EF = ejection fraction, LVH = left ventricular hypertrophy
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Regression History. Blank cells indicate an absence of regression of the type listed in the column
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Biochemical Data. L:P = lactate to pyruvate ratio, DCA = dicarboxylic acids, EMA = ethylmalonate, 3-MG = 3-methylglutaconate * Value is listed only if level was higher than the upper limit of the reference interval † Patient had one high level but exact value is unknown
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Neuroimaging. Blank cells indicate that patient did not undergo cranial MRI
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Enzymology and mtDNA Testing 1 Testing performed at CIDEM Lab, Cleveland 2 Testing performed at Horizon Molecular Medicine LLC, Atlanta A: selected point mutations screened B: mtDNA sequencing C: whole mitochondrial genome scan by dHPLC P: mutation of probable pathogenicity U: mutation of unclear pathogenicity OXPHOS: polarographic determination of mitochondrial oxidative phosphorylation reactions * This patient does not have mitochondrial sequence changes indicative of haplogroup K †ETC complex III, when measured separately, was normal in these patients
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We gratefully acknowledge the children and families who were a part of this project. We also thank Thomas Maloney, RN, MSN for his support of this project and care of patients in the LADDERS program.