Richard L. Boriack

Fatal Hepatic Short-Chain L-3-Hydroxyacyl-Coenzyme A Dehydrogenase Deficiency: Clinical, Biochemical, and Pathological Studies on Three Subjects with This Recently Identified Disorder of Mitochondrial β-Oxidation

ABSTRACT This report describes the clinical, biochemical, and pathological findings in three infants with hepatic short-chain L-3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) deficiency, a recently recognized disorder of the mitochondrial oxidation of straight-chain fatty acids. Candidate subjects were identified from an ongoing study of infant deaths. SCHAD analysis was performed on previously frozen liver and skeletal muscle on subjects with a characteristic urine organic acid profile. Autopsy findings were correlated with the biochemical abnormalities. Enzyme analysis in liver revealed marked deficiency in SCHAD with residual activities of 3–11%. All subjects had normal activity in skeletal muscle. However, Western blot analysis of SCHAD revealed an identical truncated protein in both liver and muscle from one patient, suggesting that SCHAD is similar in liver and muscle and that the normal activity in muscle may be due to other enzymes with C4 activity. Autopsy findings revealed marked steatosis and a muscle pattern consistent with spinal muscular atrophy in one patient. Lipid storage was less pronounced in one patient and not detected in the third patient who had a well-documented history of recurrent hypoglycemia. This is the initial pathological characterization of this enzyme defect, and our observations suggest that SCHAD deficiency is a very severe disorder contributing to early infant death.

Effect of L-Carnitine Supplementation on Cardiac Carnitine Palmitoyltransferase Activities and Plasma Carnitine Concentrations in Adriamycin-Treated Rats

Adriamycin (ADR) inhibits the carnitine palmitoyl transferase (CPT) system and consequently the transport of long-chain fatty acids across mitochondrial membranes. l-Carnitine (CARN) plays a major role in fatty acid oxidation by translocating activated long-chain fatty acids into the matrix of mitochondria. CARN has been shown to be of benefit in certain cardiac conditions including cardiomyopathy and myocardial infarction. This study was devised to investigate the effect of CARN on altered CPT I and CPT II activity in the cardiomyopathy associated with ADR therapy. We also assessed the effect of CARN on the plasma free, total, and acylcarnitine concentrations. Four groups, each consisting of four male Sprague-Dawley rats, were studied: group 1(n = 4) was not given either ADR or CARN; group 2 (n = 4) was given ADR (15 and 20 mg/kg, respectively, cumulative dose) by i.p. injections for 1 and 2 wk; group 3 (n = 4) was given the same dose of ADR with CARN (200 mg/kg); and group 4 (n = 4) was given CARN (200 mg/kg). The activities of CPT I and CPT II in heart were significantly decreased in the ADR-treated rats (p < 0.05) in a dose-dependent manner. The reduced activities of CPT I and CPT II, inhibited by ADR, were not normalized by supplementation with CARN (p < 0.05). In rats supplemented with CARN alone, the activities of CPT I and CPT II were elevated approximately 50% above those of the control rats (p < 0.05). ADR treatment resulted in elevation of plasma free and total CARN concentrations (p < 0.05). Supplementation with CARN did not effect the increased plasma CARN concentrations resulting from ADR treatment (p < 0.05). This study supports the concept that ADR toxicity results from the inhibition of both CPT I and CPT II activities and that one of the causes of ADR-induced cardiomyopathy is a result of globally impaired fatty acid oxidation.

Isocitrate Dehydrogenase 1/2 Mutational Analyses and 2-Hydroxyglutarate Measurements in Wilms Tumors

L‐2‐Hydroxyglutaric aciduria (L‐2‐HGA) is an uncommon inborn error of metabolism, in which the patients are predisposed to develop brain tumors. Elevated levels of D‐2‐hydroxyglutarate have been demonstrated with malignant gliomas and myeloid leukemias associated with somatic mutations of the genes encoding NADP(+)‐dependent isocitrate dehydrogenases (IDH1 and IDH2, respectively). Recently, we noted a Wilms tumor in a child with L‐2‐HGA. Given the accumulating evidence that both enantiomers of 2‐hydroxyglutarate are associated with cellular transformation, we investigated if sporadic Wilms tumors are associated with IDH1 or IDH2 mutations or with elevated levels of 2‐hydroxyglutarate.

Phenotypic variability among first‐degree relatives with carnitine palmitoyltransferase II deficiency

Carnitine palmitoyltransferase (CPT) II deficiency disorders are clinically very variable. To examine the cause(s) of variable symptoms in first‐degree relatives with CPT II deficiency, four sisters with various combinations of mutations and polymorphisms in the CPT2 gene were studied, together with 20 sedentary and 24 trained healthy female subjects. One sister, whose symptoms began at age 7 years, was more severely affected than her older sister, whose symptoms began at age 16 years; both were compound heterozygotes for the common S113L mutation and Q413fs, and for the common CPT2 polymorphisms, V3681 and M647V. A third sister became hypoglycemic with fasting, was heterozygous for the S113L mutation, and homozygous for the polymorphism variants. The fourth sister was asymptomatic, heterozygous for the Q413fs mutation, and homozygous for the normal polymorphisms. Residual CPT II activity in skeletal muscle and cultured skin fibroblasts from the two myopathic sisters, and palmitate oxidation in fibrobasts, were abnormally low; cellular and total body fat oxidation were also diminished. Muscle function and fat oxidation were nomal at rest, but a switch to carbohydrate utilization occurred at lower exercise intensities than in sedentary and trained individuals, respectively. Reliance on carbohydrates during stress and hormonal alterations may explain, in part, the variance in ages of onset and serverity of symptoms in myopathic patients.

Papillary thyroid carcinoma shows elevated levels of 2-hydroxyglutarate

Elevated levels of d-2-hydroxyglutarate (d-2-HG) occur in gliomas and myeloid leukemias associated with mutations of IDH1 and IDH2. l-2-Hydroxyglutaric aciduria, an inherited metabolic disorder, predisposes to brain tumors. Therefore, we asked whether sporadic cancers, without IDH1 or IDH2 hot-spot mutations, show elevated 2-hydroxyglutarate levels. We retrieved 15 pairs of frozen papillary thyroid carcinoma (PTC) and adjacent non-neoplastic thyroid, and 14 pairs of hyperplastic nodule (HN) and adjacent non-hyperplastic thyroid. In all lesions, exon 4 sequencing confirmed the absence of known mutations of IDH1 and IDH2. We measured 2-hydroxyglutarate by liquid chromatography-tandem mass spectrometry. Compared to normal thyroid, PTCs had significantly higher d-2-HG and l-2-hydroxyglutarate (l-2-HG) levels, and compared to HNs, PTCs had significantly higher d-2-HG levels. d-2-HG/l-2-HG levels were not significantly different between HNs and normal thyroid. Further studies should clarify if elevated 2-hydroxyglutarate in PTC may be useful as cancer biomarker and evaluate the role of 2-hydroxyglutarate in cancer biology.

Wilms Tumor in a Child with L-2-hydroxyglutaric Aciduria

We report a male infant with L-2-hydroxyglutaric aciduria and Wilms tumor. L-2-hydroxyglutaric aciduria is a rare, autosomal-recessive, inborn error of metabolism characterized by a variable degree of progressive encephalopathy. Of the fewer than 100 cases reported in the literature, at least 9 patients have developed tumors of the central nervous system. To our knowledge, the present case is the 1st example of an extracranial tumor associated with L-2-hydroxyglutaric aciduria. This observation potentially widens the tumor spectrum in this metabolic disorder and may lead to further insight into the relationship between L-2-hydroxyglutaric acid and cellular transformation.

Precholesterol Sterols Accumulate in Lipid Rafts of Patients with Smith-Lemli-Opitz Syndrome and X-linked Dominant Chondrodysplasia Punctata

Systemic fetal dysmorphogenesis in disorders of postsqualene cholesterol biosynthesis is thought to be caused by disruption of Hedgehog signaling. Because precholesterol sterols such as 7-dehydrocholesterol and lathosterol can replace cholesterol in the activation of Hedgehog proteins, it is currently believed that cholesterol deficiency-related Hedgehog signaling block occurs further downstream, probably at the level of Smoothened. Experimentally, such a block in Hedgehog signaling occurs at sterol levels of <40 μg/mg protein. Recently, we studied autopsy material from 2 infants with fatal cholesterol biosynthetic disorders (Smith-Lemli-Opitz syndrome and X-linked dominant chondrodysplasia punctata) in which the hepatic cholesterol levels were far greater. In this study, we demonstrate abnormal accumulation of sterol precursors of cholesterol in membrane lipid rafts (detergent resistance membranes) prepared from liver tissues of these 2 infants: 8-dehydrocholesterol and 7-dehydrocholesterol in lipid rafts of the infant with Smith-Lemli-Opitz syndrome and cholest-8(9)-ene-3β-ol in lipid rafts of the infant with X-linked dominant chondrodysplasia punctata. We suggest that such alterations in the lipid raft sterol environment may affect the biology of cells and the development of fetuses with cholesterol biosynthetic disorders.

A Severely Affected Female Infant with X-Linked Dominant Chondrodysplasia Punctata: A Case Report and a Brief Review of the Literature

We recently performed an autopsy on a premature female newborn with rhizomesoacromelic limb shortening of the upper and lower extremities, craniofacial dysmorphism, and chondrodysplasia punctata. A diagnosis of Conradi-Hunermann-Happle syndrome or X-linked dominant chondrodysplasia punctata was made based on elevated cholest-8(9)-ene-3β-ol in serum and tissues. Molecular analysis of EBP, mutations of which are responsible for this malformation syndrome, revealed a monoallelic missense mutation, c.328 G>A (R110Q). We present this case as an illustration of an unusually severe manifestation of this disorder in a female, with additional unusual features including lack of skin manifestations and apparent bilateral symmetry of the skeletal findings.

Long-Chain L 3-Hydroxyacyl-CoA Dehydrogenase (LCHAD) Deficiency Does Not Appear to Be the Primary Cause of Lipid Myopathy in Patients With Bannayan-Riley-Ruvalcaba Syndrome (BRRS)

In order to test the hypothesis that long-chain L 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) deficiency is associated with the lipid myopathy and muscle carnitine deficiency observed in Bannayan-Riley-Ruvalcaba syndrome (BRRS), we studied the enzyme activity in cultured skin fibroblasts from three generations of a family with a clear dominant inheritance of BRRS. Enzyme activities were normal while the germline PTEN missense mutation P246L segregated with BRRS in this family. No PTEN mutations were identified in the original patient with BRRS and LCHAD deficiency. These data suggest that the previously reported case of LCHAD and BRRS either represents the coincidental concurrence of two rare genetic events or that a gene other than PTEN is related to LCHAD and BRRS.

Polyunsaturated fatty acids reverse the lysosomal storage and accumulation of subunit 9 of mitochondrial F1F0-ATP synthase in cultured lymphoblasts from patients with Batten disease

Batten disease (McKusick 204200), the juvenile form of neuronal ceroid lipofuscinosis (JNCL) is a severe autosomal recessive neurodegenerative disease characterized by progressive loss of vision, seizures, degenerating motor function and ultimately death, usually in the third decade of life (Kohlschutter et al 1993). The gene CLN3, for JNCL has recently been isolated. The protein encoded by the CLN3 gene is a highly lipophilic protein of 438 amino acid residues and molecular mass 48kDa (Lerner et al 1995). The function of the protein is unknown. The characteristic storage material has been shown to be proteolipid in nature, with subunit 9 of mitochondrial F 1 F 0 -ATP synthase being a major component (Hall et al 1991; Palmer et al 1992). We recently reported upon the reversibility of induced lysosomal storage in JNCL lymphoblast cell lines when incubated with gentamycin (an agent known to damage mitochondria) and a defined mixture of polyunsaturated fatty acids (Boriack et al 1995). In the experiments reported here we demonstrate that ATP synthase subunit 9 storage can be induced in this in vitro model and that this can be reversed by polyunsaturated fatty acids. This provides a good system for testing therapeutic regimens for this disease.