Case 1 -

The Burden of Proof in The Postmortem Diagnosis Of Mitochondrial Disease Hannes Vogel Stanford University Medical Center Stanford, California

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Clinical History: This male patient was a former 6lb 12oz product of a full term pregnancy. He had been previously diagnosed at 2-3 months of age with hypertrophic cardiomyopathy, hypotonia, lactic acidosis and poor control of oral feeds. He had gastroesophageal reflux and subsequently required nasogastric tube feeds. A gastrostomy button and fundoplication were performed as well as a left thigh muscle biopsy. The biopsy showed features "suggestive of" a mitochondrial myopathy and subsequent mitochondrial enzyme analyses revealed reduced activities in complexes I, II-III, and IV which approached statistical significance but were not quite at 2 standard deviations below the normal reference mean. Ophthalmologic exam was normal and head MRI was normal except for extra axial fluid collections. He was unable to sit alone and was not crawling. He spoke a few intelligible words. He had global hypotonia with absent deep tendon reflexes. Chest x-ray demonstrated marked cardiomegaly with normal pulmonary vascular markings. From the time of initial echocardiogram, cardiac ejection fraction had declined from 67% to 39%. At 15 months of age he developed acute severe respiratory distress and despite maximal efforts he experienced irreversible cardiopulmonary arrest. A full autopsy was performed. The brain weighed 785.7 grams. The gyral pattern was well developed. The cerebellum and brain stem were normal. There was a slight discoloration of the white matter of the temporal lobes; no lesions were seen in the basal ganglia or thalami. Serial sections of the brain stem perpendicular to its long axis show a slightly dilated aqueduct and no grossly identifiable lesion or discoloration in the midbrain, pons, or medulla. Histologic section from the pons is submitted. Diagnosis: Multisystem mitochondriopathy with Leigh disease Case 1 - Figure 1 - Ventral pons focus showing demyelination and gliosis with preservation of neurons. Case 1 - Figure 2 - Oncocytic change involving choroid plexus. Mitochondrial diseases are now known to affect all tissues, with the possible exception of metabolically quiescent types such as bone or cartilage. Therefore, the postmortem documentation of mitochondrial disease presumes careful sampling of diverse tissues, influenced as usual by clinical observations. The most metabolically active tissues, namely the CNS and muscle, are traditionally viewed as most vulnerable to disease in mitochondriopathies, but it is important to realize that an increasing diversity of non-neurological presentations are recognized; greater than 30% of pediatric mitochondriopathies do not have presenting neurological manifestations. Most mitochondriopathies involve the otherwise unexplained combination of multisystemic symptoms that is essentially pathognomonic for a respiratory chain disorder. Symptoms must include at least three of described organ system presentations, namely neurologic, muscular, cardiac, renal, nutritional, hepatic, endocrine, hematologic, otologic, ophthalmologic, dermatologic, or dysmorphic (see Table below). Insofar as the postmortem examination must confer relevance to the premortem clinical, radiological, laboratory, and even pathological observations, the following list of manifestations of mitochondrial disease is paralleled by both routine and special studies available to the autopsy pathologist. These manifestations are compiled from recently published studies attempting to formulate a more objective means of rating the likelihood that a pediatric patient has mitochondrial disease. One may expect the autopsy to reflect the premortem circumstance whereby a diversity of findings must be consolidated into a final diagnosis, and that the diagnosis of probable or possible mitochondrial disease may be as much as the data may permit one to conclude. Clinical Features Postmortem Findings Metabolic and other investigations Elevated lactate blood> 2000 µmol/l at least 3 occasions (spontaneous, postprandial or post GTT) Elevated L/P-ratio > 18 (only if lactate is elevated) Elevated alanine (blood) > 450 µmol/l Elevated CSF lactate (> 1800 µmol/l, with normal blood lactate) Elevated CSF protein Elevated CSF alanine Urine: elevated excretion of lactate or TCA cycle intermediates · elevated excretion of ethylmalonic acid or 3-methylglutaconic acid or dicarbonic acids (adipic, suberic and sebacic acid) abnormal 31P-MRS in muscle – abnormally elevated inorganic phosphate (Pi) and reduced phosphocreatine/ Pi ratio as compared to normal controls Decreased respiratory chain enzyme activity. <20% - major <30% - minor Special Postmortem Studies Most blood or CSF determinations used to diagnose mitochondrial disease are not meaningful postmortem. Rapidly frozen muscle may be used to measure respiratory chain enzyme activities, however activities in brain decrease after death, slope of regression being 9.5%/h of mean initial activity for complex I. Frozen specimens show no significant decrease of respiratory chain activities in 1 year. Meaningful results are possible if brain specimen is sampled within 8h after death. Muscular Signs and Symptoms Progressive external ophthalmoplegia Ptosis, facies myopathica Exercise intolerance Reduced muscle power or muscular hypotonia Episodes of acute rhabdomyolysis Dystonia Myalgias Spastic paraparesis Weakness, fatigue Premortem Muscle Morphology Ragged red fibers or ragged blue fibers COX-negative fibers Abnormal (reduced or patchy) SDH-staining Strongly SDH-reactive blood vessels EM: abnormal mitochondria Abnormal mitochondrial inclusions (crystalline, globular) Honeycomb or concentric pattern, paucity of cristae Postmortem Muscle Examination Random sampling of arm or leg skeletal muscle is the proper approach postmortem, emulating the premortem biopsy. Snap freeze muscle for cryotomy (H&E, modified Gomori trichrome, ORO, ATPase @ pH 9.4/4.3, SDH, cytochrome oxidase), and for possible biochemical or genetic analyses. Fix small portion in glutaraldehyde for possible EM. Ragged red fibers in extraocular muscles in KSS, CPEO. Studies of diverse non-mitochondrial conditions show mitochondrial abnormalities, therefore diagnostic specificity of extraocular muscle exam is doubtful. Other Involvement – CNS Delayed or absent psychomotor development or mental retardation (IQ < 70) Loss of acquired skills Stroke-like episodes (transient hemianopia, hemiplegia etc.) Migraine Frank seizures or abnormal EEG Myoclonus or myoclonic epilepsy Cortical blindness Signs and symptoms of pyramidal tract involvement Signs and symptoms of extrapyramidal involvement Signs and symptoms of brainstem involvement Signs and symptoms of cerebellar involvement MRI Leigh syndrome (in T2 hyperintense lesions in putamina, globi pallidi, caudate nuclei) Stroke-like picture (not confined to a vascular territory) Leukodystrophy Cerebral or cerebellar atrophy 1H-MRS brain: clearly discernible lactate peak Brain and Spinal Cord Examination Gross Weight. Separate cerebellar weight if small. Distribution of infarcts (-striatal, -non vascular territory) Spongy white matter degeneration (MELAS, MERRF) Leigh disease: symmetrical grey-brown/cystic lesions of brainstem tegmentum and basal ganglia/thalamus, involving (decreasing order): substantia nigra, inferior colliculi/periaqueductal grey, medullary tegmentum, spinal cord, cerebellar roof nuclei/adjacent white matter, pontine tegmentum, corpus striatum, inferior olives, subthalamic nuclei, thalamus. midbrain generally more affected in severe infantile cases. Microscopic Rarefaction of grey and white matter with spongy change to vacuolar change to cavitary necrosis. Gliosis Vascular prominence Neuronal sparing MELAS: "cactus-like" Purkinje cells, atrophy of cerebellar granular cells EM: abnormally shaped, enlarged mitochondria with unusual cristal morphology, crystalloid inclusions. Most EM abnormalities are nonspecific and may be seen in other metabolic diseases or due to drugs or toxins. Other Involvement – Multisystem Hematology Sideroblastic anemia Pancytopenia Gastrointestinal tract Acute or chronic hepatic dysfunction Failure to thrive Exocrine pancreatic insufficiency Intestinal pseudoobstruction Otherwise unexplained chronic diarrhea Endocrine Short stature (< - 2 SD or < 3rd %tile) Delayed puberty Diabetes mellitus type I or type II or impaired glucose tolerance Hypoparathyroidism Central diabetes insipidus Adrenal insufficiency Goiter Hypothyroidism Ovarian failure Short stature Heart Cardiomyopathy (hypertrophic or dilatative) in the absence of a vitium cordis or hypertension Conduction block Kidney Proximal tubular dysfunction Focal segmental glomerulosclerosis Barrter syndrome Eyes Cataract Retinopathy Optic atrophy Ears Sensorineural hearing loss Nerve Peripheral neuropathy Cutaneous Hair abnormalities Hypertrichosis Rashes and pigmentation disorders General Exacerbation of listed symptoms or signs with minor illness Sudden unexplained neonatal or infantile death in family history Reported Postmortem Pathology Hematology Sideroblastic anemia: mitochondrial ferritin-positive erythroblasts; iron in mitochondria by EM (In myelodysplastic syndrome, excessive apoptosis in hematopoietic and stromal cells, ringed sideroblasts with iron-laden mitochondria, increased phagocytosis reported) Gastrointestinal tract Liver: fatty change, abnl mitochondria by EM Intestine: accumulations of abnormal mitochondria in smooth muscle Endocrine Parathyroid: no reports of pathology; anti-mitochondrial staining of oxyphil cells and a weak reactivity against chief cells of normal parathyroid reported in idiopathic hypoparathyroidism Pancreas: hyaline degeneration/loss of islets, loss of A and B cells; (abnormal muscle mitochondria reported in diabetes) Heart Abundant, enlarged, morphologically abnormal mitochondria by EM. Kidney Abnormal mitochondria in tubular epithelium by EM; focal segmental glomerulosclerosis, tubular atrophy Eyes LHON cases and a rat model revealed a selective loss of retinal ganglion cells and the corresponding axons. Loss of fibers in the central part of optic nerve with variable preservation of fibers in the far periphery Nerve Axonopathy, primary demyelinating or mixed. Crystalline-like inclusions in the Schwann cell cytoplasm in MERRF with multiple mtDNA deletions. Skin Mitochondrial accumulation in glands, fragile hair shafts Pathology not yet reported Inner ear Thyroid Ovary