Achalasia as the Harbinger of a Novel Mitochondrial Disorder in Childhood

INTRODUCTION Achalasia is a neuromuscular disorder of unknown cause manifested by three abnormalities of esophageal function: increased basal lower esophageal sphincter pressure, incomplete relaxation of the lower esophageal sphincter after swallowing and absent peristalsis in the esophageal body. The condition is uncommon at any age but less than 5% of patients present during childhood or adolescence. The incidence in children has been estimated as less than 0.1 per 100,000 (1). The majority of cases are sporadic. Familial cases suggest a role for genetic factors in disease pathogenesis. Achalasia is generally isolated but may occur as part of a multisystem disorder characterized by generalized autonomic or neuromuscular dysfunction, such as Allgrove's syndrome (2) (achalasia, alacrima, adrenocorticotropic hormone insensitivity), 4A syndrome (Allgrove's with autonomic dysfunction) or, in association with vitiligo, short stature and deafness (2,3). This report describes two siblings with achalasia presenting in early childhood. In early adolescence, both siblings developed progressive dysfunction of the central, peripheral and autonomic nervous systems. The combination of gastrointestinal dysmotility, peripheral neuropathy and generalized autonomic dysfunction suggested a mitochondrial disorder, which was ultimately established. CASE REPORTS The salient gastrointestinal and neurologic features of both cases are summarized in Table 1.TABLE 1: Summary of clinical featuresPatient 1 Birth and early development of this 24-year-old German-American man were normal. Chronic regurgitation developed in the first 6 months of life. Barium contrast radiography revealed gastroesophageal reflux with normal upper gastrointestinal anatomy. At 2 years of age, failure of medical management led to Nissen fundoplication. Achalasia was diagnosed at 3 years of age. Barium contrast radiography demonstrated a dilated esophagus, with partial obstruction at the gastroesophageal junction; endoscopy excluded a stricture of the distal esophagus. Esophageal manometry demonstrated increased basal lower esophageal sphincter pressure (38 mm Hg; normal range,15-25 mm Hg), incomplete relaxation of the lower esophageal sphincter with swallowing and absence of peristalsis in the entire esophagus. At age 3.5 years, the patient underwent a transabdominal Heller myotomy. At Age 8 years he underwent transthoracic Heller myotomy. At age 9 years he required pneumatic dilatation of the lower esophageal sphincter and at age 10 underwent surgical resection of the distal two-thirds of the esophagus with gastric tube formation. At age 13, nifedipine therapy was instituted but failed to control intractable dysphagia. At age 15 he underwent the surgical removal of his remaining esophagus with gastric pull-up to thoracic inlet because of intractable dysphagia. Gastric emptying measured by scintography (before gastric pull-up) and antroduodenal manometry were normal and he had no constipation. Histologic examination of the resected esophagus demonstrated reduced myenteric ganglion cells, consistent with the diagnosis of idiopathic achalasia (4). Beginning at 13 years, the patient complained of progressive gait problems, tremor, dysarthria and fluctuating distal limb paresthesia and pain. He reported intolerance to heat and humidity, absence of sweating and intermittent urinary retention. Tears were present only in infancy. There were no cognitive problems. Neurologic examination showed a head circumference in the 50th percentile, height between the fifth and 10th percentiles and weight below the fifth percentile. Cranial nerves were normal, without external ophthalmoplegia or hearing deficit by audiologic testing. Gait ataxia, mild upper extremity intention tremor and mild dysmetria, with bilateral Hoffmann's sign and generalized hyperreflexia (except for the Achilles tendon reflexes) were present, suggesting cerebellar and upper motor neuron dysfunction. Babinski sign was absent on the left and equivocal on the right. In addition, distal muscle weakness and atrophy, reduced vibratory and temperature sensation distally and diminished reflexes in the ankles were present, consistent with a superimposed peripheral neuropathy. The plantar arches were normal. Laboratory data. Electromyography and nerve conduction studies confirmed a severe sensory-motor axonal polyradiculoneuropathy. Magnetic resonance imaging of the head and spine (cervical, thoracic, lumbosacral) and computerized tomography of the chest and abdomen were normal. The following laboratory investigations yielded normal results: complete blood count, erythrocyte sedimentation rate, serum glucose, electrolytes, blood urea nitrogen, creatinine, calcium, phosphorus, magnesium, uric acid, aminotransferases, creatinine kinase, total protein, albumin, immunoglobulins, antinuclear antibody, folate, vitamin B12, vitamin E, thyroid profile, plasma and urine porphyrins, urinary organic acid analysis, plasma and urinary carnitine analysis, quantitative plasma amino acid analysis, cerebrospinal fluid protein, lactate and pyruvate, white blood cell alpha-galactosidase activity and adrenocorticotropic hormone stimulation test. Patient 2 The younger sister of patient 1 also had a normal birth and early development except for dry eyes. Her weight followed the 10th to 25th percentile until age 7 years when weight gain decelerated. Classical achalasia was diagnosed by standard radiologic, manometric and endoscopic criteria. A transabdominal Heller myotomy was performed. Subsequently, her course was complicated by gastroesophageal reflux disease, which failed to respond to anti-secretory medication. She underwent a Nissen fundoplication. Scintigraphic and barium contrast gastric emptying studies were repeatedly normal. Weakness began at the age of 12 years. Despite continued deceleration of weight gain to below the fifth percentile by age 14 years, her height followed the 25th to 50th percentile, and her head circumference was two standard deviations above the mean. She began to complain of numbness in her feet and weakness when climbing stairs. She had a normal gait but was unable to heel walk. She was able to communicate appropriately for age and performed well in school. Absence of tears remained her only non-gastroenterologic autonomic symptom. Neurological examination demonstrated loss of sensation in a stocking-glove distribution and moderate weakness of the ankle dorsiflexors, consistent with a peripheral neuropathy or polyradiculopathy. In addition, hyperactive reflexes (including a positive jaw-jerk) suggested superimposed upper motor neuron dysfunction. Ataxia and tremor were absent. Laboratory data. Nerve conduction studies demonstrated moderately reduced motor amplitudes with sensory preservation, suggesting a polyradiculopathy or pure motor neuropathy. The same investigations as described above for patient 1, except for white blood cell alpha-galactosidase analysis (not performed), yielded normal results. METHODS Autonomic Testing The cardiac responses to deep breathing (5) and to Valsalva maneuver (phase 4) (5,6) were used to evaluate cardiac parasympathetic cholinergic function, and the tilt table test and Valsalva maneuver (phase 2) were used to evaluate cardiac and vasomotor sympathetic adrenergic systems (7,8). Two different sweat tests, the quantitative sudomotor axon reflex test (9) and the thermoregulatory sweat test (10), were used to assess sudomotor sympathetic cholinergic function. A pilocarpine induced-tearing test measured noncardiac parasympathetic cholinergic function (11). Sources of Tissue Sural and intercostal nerve biopsy specimens were obtained from patient 1 at 14 years of age. A deltoid muscle biopsy specimen was obtained from patient 1 at 17 years of age and patient 2 at 14 years. All samples underwent light microscopic, histochemical, and ultrastructural evaluation. Genetic and Biochemical Analysis Mitochondrial DNA (mtDNA) analysis used previously published methods (12). Total cellular DNA was extracted from either muscle tissue or white blood cells, digested with the restricting enzyme Pvu II to linearize mtDNA, and subjected to Southern blot analysis. The hybridization probe consisted of 32P-labeled human mtDNA. Thymidine phosphorylase activity and plasma thymidine concentration were measured as previously described, utilizing a spectrophotometric measure of the rate of conversion of thymidine to thymine in white cell buffy coats and an isocratic high-powered liquid chromatography method, respectively. RESULTS Autonomic Testing In both patients, an abnormal heart rate response to deep breathing, blunted heart rate reduction during phase IV of the Valsalva (patient 2 only; patient 1 could not perform the Valsalva) and absent tearing and pupillary supersensitivity to pilocarpine stimulation indicated parasympathetic cholinergic dysfunction (Table 2). In addition, both subjects had sympathetic adrenergic dysfunction as demonstrated by the presence of orthostatic hypotension on the passive tilt-test. Finally, abnormal sweat production after acetylcholine iontophoresis (noted by quantitative sudomotor axon reflex test) onto the skin of the feet, calves, hands, upper arms and forehead revealed postganglionic sympathetic cholinergic dysfunction. A thermoregulatory sweat test showed corroborative abnormalities.TABLE 2: Results of autonomic testingTissue Analysis Electron microscopy of the radial and intercostal nerve biopsy specimens from patient 1 revealed axonal neuropathy affecting mostly thinly myelinated fibers. The muscle biopsy specimens from both patients contained rare hypereosinophilic fibers in addition to neurogenic atrophy. Ragged-red fibers were present on modified trichrome Gomori stained sections in patient 1. Histochemical staining for succinate dehydrogenase showed focally increased sub-sarcolemmal activity. Cytochrome oxidase deficient fibers were also present. Electron microscopic examination revealed scattered pleomorphic mitochondria with irregularly arranged cristae but no mitochondrial inclusions (Fig. 1).FIG. 1: Ultrastructural examination of skeletal muscle showing scattered markedly enlarged mitochondria with disorganized cristae oriented longitudinally along A-bands (×15,000).Genetic Analysis Southern blot analysis of the DNA isolated from skeletal muscle from patient 1 demonstrated multiple mtDNA deletions (Fig. 2). No mtDNA deletions were present in white blood cells in patient 1. The thymidine concentration in plasma and the activity of thymidine phosphorylase in buffy coat were normal, excluding the diagnosis of mitochondrial-neuro-gastrointestinal encephalopathy (MNGIE). Genetic analysis was not performed for patient 2.FIG. 2: Southern blot: Total DNA was extracted from muscle or blood, digested with the restriction enzyme Pvu II and hybridized with 32P labeled human mitochondrial DNA. Lane 1: Patient 1-DNA from blood, showing only normal mtDNA (16.5kb). Lane 2: Patient 1-DNA from muscle, showing normal mtDNA and additional bands indicating multiple deletions in mtDNA. Lane 3: Control-DNA from muscle, showing only normal mtDNA.DISCUSSION This study suggests that achalasia may be the initial manifestation of a generalized nervous system disorder. Autonomic testing can provide valuable diagnostic information by characterizing sub-clinical generalized autonomic dysfunction and a mitochondrial abnormality may produce such a disorder. Idiopathic achalasia in adults has been associated with abnormalities of general autonomic function detected through autonomic testing (13-16). Idiopathic achalasia-associated autonomic dysfunction is usually not clinically prominent. In contrast, other reports suggest that some cases of achalasia reflect a generalized neurologic disorder involving different areas of the gastrointestinal tract (1), the autonomic nervous system (Allgrove syndrome) (2) or the central nervous system (3,17). The results of neurologic examination and autonomic testing in our two family members indicate the presence of such a multisystem neurologic disorder. Hyperreflexia implies involvement of the upper motor neuron, whereas atrophy of the distal muscles, distal limb sensory loss and diminished ankle reflexes indicate that they also have peripheral nervous system involvement, which was confirmed by nerve conduction studies-electromyography. Thus, both central and peripheral nervous systems were clearly affected. In this context, autonomic testing was particularly helpful in suggesting a differential diagnosis. Peripheral nervous system impairment of both branches of the autonomic nervous system, sympathetic and parasympathetic, may occur in mitochondrial disorders (18). To our knowledge, this is the first report of detailed autonomic testing of siblings with achalasia. Although sometimes labor-intensive, establishing the diagnosis of a mitochondrial disorder provides several benefits. For the patient, it averts further diagnostic testing seeking other diagnoses, and allows for logical therapeutic trials of various co-factors or drugs that may be of benefit. For the family, it forms the basis of genetic counseling. Nonetheless, diagnosing a novel mitochondrial disorder may require non-routine laboratory testing. The subject described as case 1 in this report presented with gastrointestinal motility disturbances early in life, followed later by a peripheral and central neurologic disorder with autonomic neuropathy. Besides the suggestive multisystem clinical presentation and the findings on autonomic testing, a definitive clue was provided by the muscle biopsy, which revealed histochemical and ultrastructural abnormalities of a mitochondrial disorder. However, traditional metabolic studies of oxidative function, such as blood lactate, plasma amino acids and carnitine and urine organic acids and carnitine, were unhelpful. The identification of mtDNA deletions in muscle suggested a disorder of intergenomic communication (Fig. 2). This report demonstrates the pitfalls in diagnosing a mitochondrial disorder. When there is strong suspicion, the diagnosis should be pursued beyond the traditional blood and urinary metabolite studies through careful histologic and molecular genetic analysis of skeletal muscle. The differential diagnosis of a generalized autonomic and neurologic disorder presenting with achalasia as the chief complaint is limited. Such diseases include other mitochondrial disorders, especially MNGIE, the 4A syndrome described by Allgrove, lysosomal storage diseases such as Fabry disease (19) and rare infectious or inflammatory disorders such as Chagas' disease. Diabetes is not a consideration here because the dysphagia commonly seen in diabetes is attributable to upper gastrointestinal dysmotility rather than achalasia (20). The disease reported here best fits the category of mitochondrial encephalomyelopathies (21). The diagnosis of MNGIE was excluded clinically and biochemically, biochemically because the thymidine phosphorylase activity in buffy coat and the plasma thymidine concentration were normal in patient 1 (22). The majority of mitochondrial encephalomyelopathies are associated with mtDNA mutations (23). Clinically, our patients differ clinically from individuals with MNGIE who have extraocular muscle involvement (ptosis, ophthalmoparesis or both) and leukoencephalopathy on magnetic resonance imaging. Common points in the two disorders include thin body habitus, gastrointestinal dysmotility, sensorimotor neuropathy (although this was axonal in our patients and is demyelinating in MNGIE) and ragged-red fibers and multiple deletions of mtDNA in skeletal muscle (12). These subjects could also have a variation of Allgrove's syndrome, with alacrima, achalasia and progressive neurologic dysfunction, now found to be associated with a mitochondrial defect. In conclusion, we report a novel disorder with multiple mtDNA deletions but without defined molecular basis manifesting both achalasia and widespread neurologic abnormalities. The implications of this report are twofold: 1) The diagnostic possibility of a mitochondrial disorder should be pursued aggressively when achalasia accompanies generalized autonomic or somatic nervous system dysfunction; and 2) further understanding of the molecular abnormality underlying this disorder of esophageal and autonomic dysfunction may provide insight into the pathogenesis of both achalasia and autonomic dysfunction in general.