Brian D. Lake

A Missense Mutation of Cytochrome Oxidase Subunit II Causes Defective Assembly and Myopathy

We report the first missense mutation in the mtDNA gene for subunit II of cytochrome c oxidase (COX). The mutation was identified in a 14-year-old boy with a proximal myopathy and lactic acidosis. Muscle histochemistry and mitochondrial respiratory-chain enzymology demonstrated a marked reduction in COX activity. Immunohistochemistry and immunoblot analyses with COX subunit-specific monoclonal antibodies showed a pattern suggestive of a primary mtDNA defect, most likely involving CO II, for COX subunit II (COX II). mtDNA-sequence analysis demonstrated a novel heteroplasmic T-->A transversion at nucleotide position 7,671 in CO II. This mutation changes a methionine to a lysine residue in the middle of the first N-terminal membrane-spanning region of COX II. The immunoblot studies demonstrated a severe reduction in cross-reactivity, not only for COX II but also for the mtDNA-encoded subunit COX III and for nuclear-encoded subunits Vb, VIa, VIb, and VIc. Steady-state levels of the mtDNA-encoded subunit COX I showed a mild reduction, but spectrophotometric analysis revealed a dramatic decrease in COX I-associated heme a3 levels. These observations suggest that, in the COX protein, a structural association of COX II with COX I is necessary to stabilize the binding of heme a3 to COX I.

Liver failure associated with mitochondrial DNA depletion

BACKGROUND/AIMS Liver failure in infancy can result from several disorders of the mitochondrial respiratory chain. In some patients, levels of mitochondrial DNA are markedly reduced, a phenomenon referred to as mitochondrial DNA depletion. To facilitate diagnosis of this condition, we have reviewed the clinical and pathological features in five patients with mitochondrial DNA depletion. METHODS Cases were identified by preparing Southern blots of DNA from muscle and liver, hybridising with appropriate probes and quantifying mitochondrial DNA relative to nuclear DNA. RESULTS All our patients with mitochondrial DNA depletion died of liver failure. Other problems included hypotonia, hypoglycaemia, neurological abnormalities (including Leigh syndrome) and cataracts. Liver histology showed geographic areas of fatty change, bile duct proliferation, collapse of liver architecture and fibrosis; some cells showed decreased cytochrome oxidase activity. Muscle from three patients showed mitochondrial proliferation, with loss of cytochrome oxidase activity in some fibres but not in others; in these cases, muscle mitochondrial DNA levels were less than 5% of the median control value. The remaining two patients (from a single pedigree) had normal muscle histology and histochemistry associated with less severe depletion of mitochondrial DNA in muscle. CONCLUSIONS Liver failure is common in patients with mitochondrial DNA depletion. Associated clinical features often include neuromuscular disease. Liver and muscle histology can be helpful in making the diagnosis. Mitochondrial DNA levels should be measured whenever liver failure is thought to have resulted from respiratory chain disease.

Tay-sachs disease and related disorders: Fractionation of brain N-acetyl-β-hexosaminidase on DEAE-cellulose

Following the demonstration by Robinson and Stirling [l] of two hexosaminidase components, A and B, in human spleen, similar components have been found in all human tissues investigated [2,3] and, with one exception [4], it is now well-established that patients with early-infantile kZ -gangliosidosis* are totally deficient in hexosaminidase A [3,4,6] . However, the recognition of closely related variants has led to some confusion when the clinical term “Tay-Sachs disease” is used to designate %a -gang liosidoses; a note on their nomenclature is therefore included. In the present study, the separation of hexosaminidase components of post-mortem brain grey matter by gradient elution from DEAE-cellulose is described. The patterns obtained in five cases of GZ gangliosidosis were compared with four controls. The controls all had a very similar enzyme pattern; two major peaks of activity, corresponding to components A and B reported in previous studies [ 1 ] , were clearly separated from several smaller intermediate fractions. The pattern in each of the five cases differed from the controls, and three types could be distinguished.

Prenatal diagnosis of lysosomal storage diseases.

The prenatal diagnosis of lysosomal storage disorders can be achieved, once the diagnosis is confirmed in the index case, by a variety of techniques including analysis of amniotic fluid, asay of enzymic activity in cultured amniotic fluid cells, cultured chorionic villus cells and by direct assay of activity in chorionic villus samples. These studies can be accompanied by ultrastructural observations which give an independent means of diagnosis. In some instances molecular genetic studies for mutation detection or linkage analysis are appropriate for prenatal diagnosis. Pseudodeficiencies of some of the lysosomal enzymes, which cause no clinical problems, can complicate the initial diagnosis particularly in metachromatic leucodystrophy where the pseudodeficiency is more common than the disease itself. Mutation analysis as well as enzyme assay is necessary not only in the index case but also in the parents before the same techniques are applied to a sample for prenatal diagnosis. A large number of lysosomal storage disorders may present as fetal hydrops and the diagnosis can be established at this late stage by fetal blood sampling and examination by microscopy as well as by biochemical assay of the appropriate enzyme or metabolite in amniotic fluid. All prenatal diagnoses in which an affected fetus is indicated should have confirmation of the diagnosis as soon as possible to reassure anxious parents, and to act as audit of the laboratorys competence to undertake prenatal diagnosis. A combined approach to prenatal diagnosis involving bio‐chemical, molecular genetic and morphological studies is recommended.

Plasma bile acids in patients with peroxisomal dysfunction syndromes: analysis by capillary gas chromatography-mass spectrometry.

Six patients with disorders of peroxisomal function have been studied. Two presented in the neonatal period with the classical features of the Zellweger syndrome, two had incomplete Zellweger phenotypes, one infantile Refsums disease and one rhizomelic chondrodysplasia punctata. Plasma bile acid profiles were determined using capillary gas chromatography-mass spectrometry. In all patients, except the case of chondrodysplasia punctata, 27-carbon and 29-carbon bile acids were present. The compounds identified included trihydroxycoprostanic acid (THCA), dihydroxycoprostanic acid (DHCA), C24-, C25- and C26-hydroxylated derivatives of THCA, a 27-carbon acid with four nuclear hydroxy groups and 3α,7α,12α-trihydroxy-27a,27b-dihomo-5β-cholestan-26, 27b-dioic acid (C29-dicarboxylic acid). THCA was present at a low concentration in the patient with infantile Refsums disease; the concentration of DHCA and the C29 dicarboxylic acid were considerably higher. The presence of abnormal bile acids in patients with Zellweger syndrome and infantile Refsums disease could be explained by the absence of peroxisomes from their hepatocytes. In chondrodysplasia punctata the cause of peroxisomal dysfunction must be different, since normal bile acid synthesis is preserved.

Bile acid analyses in "pseudo-Zellweger" syndrome; clues to the defect in peroxisomal beta-oxidation.

In Zellweger syndrome, β-oxidation of very long chain fatty acids (VLCFA) and C27 bile acids is impaired because peroxisomes are absent (Schutgens et al., 1986). In 1986, Goldfischer et al. described an infant with ‘pseudo-Zellweger’ syndrome in whom peroxisomes were present but oxidation of VLCFA and C27 bile acids impaired, due to a deficiency of peroxisomal 3-oxoacyl-CoA thiolase (Schram et al., 1987). This paper describes three siblings with deficient peroxisomal β-oxidation but structurally normal peroxisomes. Immunoreactive acyl CoA-oxidase, bifunctional protein and thiolase were all present in the liver but analysis of the C27 bile acids suggested a functional deficiency of peroxisomal thiolase (or possibly of the bifunctional protein).

Gut Lesions in Fabry's Disease Without a Rash

Twin boys with Fabrys disease and 6 affected relatives were described. Limb pains and retinal vessel tortuosity were present but no patient had angiokeratomata. One boy had a severe enteropathy with small and large bowel involvement which was investigated. Thin-layer chromatography showed that excesses of ceramide di- and trihexosides were excreted in the urine. Leucocyte α-galactosidase activity was measured: hemizygous males showed very low activity, while obligate and probable heterozygous females had values intermediate between those of the patients and the normal controls.

Hepatic Mitochondrial 3-Hydroxy-3-Methylglutaryl-Coenzyme A Synthase Deficiency

There are at least two isoenzymes of 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (EC 4.1.3.5) located in the mitochondrial matrix and the cytoplasm of hepatocytes, respectively. The mitochondrial enzyme is necessary for the synthesis of ketone bodies, which are important fuels during fasting. We report a child with a deficiency of this isoenzyme. He presented at 16 mo with hypoglycemia. There was no rise in ketone bodies during fasting or after a long chain fat load but there was a small rise after a leucine load. Measurement of β-oxidation flux in fibroblasts was normal. Using antibodies specific for mitochondrial HMG-CoA synthase, no immunoreactive material could be detected on Western blotting. Total HMG-CoA synthase activity in liver homogenate was only slightly lower than in control samples. Presumably, as there was no mitochondrial HMG-CoA synthase enzyme protein, this activity arose from the cytoplasmic or other (e.g. peroxisomal) isoenzymes. With avoidance of fasting, our patient has had no problems since presentation and is developing normally at 4 y of age.

Omenn's syndrome: Differential diagnosis in infants with erythroderma and immunodeficiency

The clinical differential diagnosis of erythroderma plus immunodeficiency and failure to thrive in neonates includes graft-versus-host-disease (GVHD), Omenns syndrome (OS), and Nethertons syndrome (NS). In addition to immunological investigations, skin biopsy is an important part of the diagnostic work-up. We reviewed biopsies from 25 patients that were retrieved from the archives of the Department of Histopathology at Great Ormond Street, of which 9 were OS, 11 were GVHD, and 5 were NS. Five patients had two biopsy specimens. Both OS and GVHD show dyskeratosis and basal vacuolation. OS always shows acanthosis and almost always parakeratosis. GVHD shows a flat epidermis and rarely parakeratosis. OS and GVHD can be distinguished after immunohistochemistry for LCA and CD68 by the relative proportions of lymphocytes and macrophages in the dermal infiltrate (predominantly lymphocytes in OS, relatively more macrophages in GVHD). Skin biopsy diagnosis of OS is difficult before 6 weeks of age because the features are poorly developed. NS can be distinguished by psoriasiform acanthosis, thickening of the basement membrane, prominent dermal blood vessels, absence of dyskeratosis, and basal layer vacuolation, and a dermal infiltrate in which lymphocytes and macrophages are equally represented. Thus, the main difference between GVHD and OS is in the proportion of lymphocytes and macrophages in the infiltrate on immunohistochemical staining for LCA and CD68, while OS and NS may be distinguished on H&E morphology alone.

Inherited Ichthyoses: A Review of the Histology of the Skin

The histology of skin biopsies from 46 cases of different forms of congenital ichthyosis was reviewed. Sections were examined for hyperkeratosis, follicular keratosis, appearance of the granular layer, epidermal thickness, tonofilament clumps, epidermal vacuolation, spongiosis, bullae and dyskeratosis, appearance of the basal layer, inflammation, mitoses, and adnexae. A detailed description of the histological features of each type of ichtnyosis studied is presented. Some ichthyoses can be recognized on routine hematoxylin and eosin staining (bullous ichthyosiform erythroderma, Nethertons syndrome, and neutral lipid storage disease); some forms require frozen sections to demonstrate fat (neutral lipid storage disease) or enzyme activity (Sjögren-Larsson syndrome). Protein electrophoresis and enzymology are necessary for X-linked recessive ichthyosis. A close liaison with the clinicians is essential for the diagnosis of all types of ichthyosis, and combined studies including routine histopathology, electron microscopy, and frozen sections may be required for the diagnosis.