Anne-Marie Lamhonwah

Cystic fibrosis transmembrane conductance regulator in human muscle: Dysfunction causes abnormal metabolic recovery in exercise.

Individuals with cystic fibrosis (CF) have exercise intolerance and skeletal muscle weakness not solely attributable to physical inactivity or pulmonary function abnormalities. CF transmembrane conductance regulator (CFTR) has been demonstrated in human bronchial smooth and cardiac muscle. Using 31P‐magnetic resonance spectroscopy of skeletal muscle, we showed CF patients to have lower resting muscle adenosine triphosphate and delayed phosphocreatine recovery times after high‐intensity exercise, suggesting abnormal muscle aerobic metabolism; and higher end‐exercise pH values, suggesting altered bicarbonate transport. Our objective was to study CFTR expression in human skeletal muscle.

A third human carnitine/organic cation transporter (OCTN3) as a candidate for the 5q31 Crohn's disease locus (IBD5).

Organic cation transporters function primarily in the elimination of cationic drugs in kidney, intestine, and liver. The murine organic cation/carnitine (Octn) transporter family, Octn1, Octn2, and Octn3 is clustered on mouse chromosome 11 (NCBI Accession No. NW_000039). The human OCTN1 and OCTN2 orthologs map to the syntenic IBD5 locus at 5q31, which has been shown to confer susceptibility to Crohns disease. We show that the human OCTN3 protein, whose corresponding gene is not yet cloned or annotated in the human reference DNA sequence, does indeed exist and is uniquely involved in carnitine-dependent transport in peroxisomes. Its functional properties and inferred chromosomal location implicate it for involvement in Crohns disease.

Characterization of organic cation/carnitine transporter family in human sperm

Spermatozoan maturation, motility, and fertility are, in part, dependent upon the progressive increase in epididymal and spermatozoal carnitine, critical for mitochondrial fatty acid oxidation, as sperm pass from the caput to the cauda of the epididymis. We demonstrate that the organic cation/carnitine transporters, OCTN1, OCTN2, and OCTN3, are expressed in sperm as three distinct proteins with an expected molecular mass of 63 kDa, using Western blot analysis and our transporter-specific antibodies. Carnitine uptake studies in normal control human sperm samples further support the presence of high-affinity (OCTN2) carnitine uptake (K(m) of 3.39+/-1.16 microM; V(max) of 0.23+/-0.14 pmol/min/mg sperm protein; and mean+/-SD; n=12), intermediate-affinity (OCTN3) carnitine uptake (K(m) of 25.9+/-14.7 microM; V(max) of 1.49+/-1.03 pmol/min/mg protein; n=26), and low-affinity (OCTN1) carnitine uptake (K(m) of 412.6+/-191 microM; V(max) of 32.7+/-20.5 pmol/min/mg protein; n=18). Identification of individuals with defective sperm carnitine transport may provide potentially treatable etiologies of male infertility, responsive to L-carnitine supplementation.

Two distinct mutations at the same site in the PCCB gene in propionic acidemia.

Propionic acidemia is an inborn error of metabolism resulting from a deficiency of propionyl-CoA carboxylase activity. The alpha- and beta-subunits of the enzyme are encoded by the PCCA and PCCB genes, respectively. Using direct sequencing and restriction digests of amplified reverse transcripts and genomic DNA, we have identified two mutations of the PCCB gene in a propionic acidemia patient from the pccC complementation subgroup (the PCCB gene contains the major complementation group pccBC and subgroups pccB and pccC). One of the proband alleles contains an inframe 3-bp deletion inherited from the father which results in the deletion of an isoleucine residue in the beta-subunit of the enzyme. The other mutant allele, inherited from the mother, has a 14-bp deletion and an addition of 12 bp of new sequence at the same site as the fathers allele. The inserted sequence is a partial duplication of a sequence just upstream of the mutation site. The net result of this mutation generates a frameshift and a downstream stop codon. Examination of fibroblast mRNA from the patient showed that it consists essentially of the fathers sequence, making it effectively the only expressed allele for the beta-protein. A survey of additional patient cell lines revealed the insertion/deletion rearrangement in three additional patients, two from the pccBC group and one unclassified. The 3-bp deletion allele was unique to the proband. The identification of two distinct alleles occurring at the same site in the PCCB gene underscores the importance of this site in enzyme function or integrity.

Vulnerability to Oxidative Stress In Vitro in Pathophysiology of Mitochondrial Short-Chain Acyl-CoA Dehydrogenase Deficiency: Response to Antioxidants

Objective To elucidate the pathophysiology of SCAD deficient patients who have a unique neurological phenotype, among fatty acid oxidation disorders, with early developmental delay, CNS malformations, intractable seizures, myopathy and clinical signs suggesting oxidative stress. Methods We studied skin fibroblast cultures from patients homozygous for ACADS common variant c.625G>A (n = 10), compound heterozygous for c.625G>A/c.319C>T (n = 3) or homozygous for pathogenic c.319C>T (n = 2) and c.1138C>T (n = 2) mutations compared to fibroblasts from patients with carnitine palmitoyltransferase 2 (CPT2) (n = 5), mitochondrial trifunctional protein (MTP)/long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) (n = 7), and medium-chain acyl-CoA dehydrogenase (MCAD) deficiencies (n = 4) and normal controls (n = 9). All were exposed to 50 µM menadione at 37°C. Additonal conditions included exposure to 39°C and/or hypoglycemia. Time to 100% cell death was confirmed with trypan blue dye exclusion. Experiments were repeated with antioxidants (Vitamins C and E or N-acetylcysteine), Bezafibrate or glucose and temperature rescue. Results The most significant risk factor for vulnerability to menadione-induced oxidative stress was the presence of a FAO defect. SCADD fibroblasts were the most vulnerable compared to other FAO disorders and controls, and were similarly affected, independent of genotype. Cell death was exacerbated by hyperthermia and/or hypoglycemia. Hyperthermia was a more significant independent risk factor than hypoglycemia. Rescue significantly prolonged survival. Incubation with antioxidants and Bezafibrate significantly increased viability of SCADD fibroblasts. Interpretation Vulnerability to oxidative stress likely contributes to neurotoxicity of SCADD regardless of ACADS genotype and is significantly exacerbated by hyperthermia. We recommend rigorous temperature control in SCADD patients during acute illness. Antioxidants and Bezafibrate may also prove instrumental in their management.

Upregulation of mammary gland OCTNs maintains carnitine homeostasis in suckling infants.

BACKGROUND Transport of L-carnitine, essential cofactor of fatty acid metabolism, into breast milk is critical for the normal growth and development of the suckling infant. OBJECTIVE To increase understanding of developmental expression of carnitine/organic cation (Octn) transporter family at different stages of murine breast development for carnitine delivery. METHODS We applied our transporter-specific antibodies to mOctn1, mOctn2 and mOctn3 to sections of mammary glands of virginal non-lactating, pregnant, late lactating and post-lactating C3H females. RESULTS We demonstrated differential expression of mOctn1, -2 and -3 in epithelial ducts, specialized myoepithelial cells and fatty stroma. There was notable upregulation of all three Octns and mRNA by RT-PCR concurrent with an increase in epithelial ducts in breasts of pregnant (15days gestation) and lactating mice (15-days post-partum) compared to virginal 6 week old females, and notable downregulation in expression of Octns 15 days after cessation of lactation. In lactating murine mammary gland at 15 days post-partum, there was a marked increase of fat globules in epithelial ducts. Octn1 and Octn2 had similar expression patterns in lactating gland cells which formed fat globules that were exocytosed into the lumen of alveoli along with transporters Octn1 and Octn2. Octn3 was primarily localized to myoepithelial cells surrounding the ducts at all stages of breast development. CONCLUSIONS There is a dynamic upregulation of the Octn family in pregnant and lactating breasts which likely provides the suckling infant with adequate carnitine for the rapid postnatal upregulation of fatty acid oxidation and ketogenesis critical for cerebral energy metabolism during fasting hypoglycemia.

The mdx mouse as a model for carnitine deficiency in the pathogenesis of Duchenne muscular dystrophy.

Introduction: Muscle and cardiac metabolism are dependent on the oxidation of fats and glucose for adenosine triphosphate production, for which L‐carnitine is an essential cofactor. Methods: We measured muscle carnitine concentrations in skeletal muscles, diaphragm, and ventricles of C57BL/10ScSn‐DMDmdx/J mice (n = 10) and compared them with wild‐type C57BL/6J (n = 3), C57BL/10 (n = 10), and C3H (n = 12) mice. Citrate synthase (CS) activity was measured in quadriceps/gluteals and ventricles of mdx and wild‐type mice. Results: We found significantly lower tissue carnitine in quadriceps/gluteus (P < 0.05) and ventricle (P < 0.05), but not diaphragm of mdx mice, when compared with controls. CS activity was increased in mdx quadriceps/gluteus (P < 0.03) and ventricle (P < 0.02). This suggests compensatory mitochondrial biogenesis. Conclusions: Decreased tissue carnitine has implications for reduced fatty acid and glucose oxidation in mdx quadriceps/gluteus and ventricle. The mdx mouse may be a useful model for studying the role of muscle carnitine deficiency in DMD bioenergetic insufficiency and providing a targeted and timed rationale for L‐carnitine therapy. Muscle Nerve 46: 767–772, 2012

Novel myophosphorylase mutation (p.Arg94Pro) with progressive exercise intolerance

We present a 16‐year‐old girl with a unique clinical phenotype characterized by rapidly progressive exercise intolerance, transient exertional weakness, and progressive muscle cramps involving all limbs and bulbar muscles, following a first myoglobinuric episode at age 15 years, arising from homozygosity for a novel missense mutation (c.281G>C) in PYGM.

Attention deficit/hyperactivity disorder as an associated feature in OCTN2 deficiency with novel deletion (p.T440-Y449)

This boy presented with ADHD at 3 years and at 8 years was hyperactive with no documented hypoglycemia and had myopathy, cardiomyopathy, and very low serum carnitine. L‐carnitine improved his exercise intolerance, cardiomyopathy, and behavior. Analysis of SLC22A5 revealed a premature stop codon (p.R282*) and a novel in‐frame deletion (p.T440‐Y449).

NDST1 Preferred Promoter Confirmation and Identification of Corresponding Transcriptional Inhibitors as Substrate Reduction Agents for Multiple Mucopolysaccharidosis Disorders.

The stepwise degradation of glycosaminoglycans (GAGs) is accomplished by twelve lysosomal enzymes. Deficiency in any of these enzymes will result in the accumulation of the intermediate substrates on the pathway to the complete turnover of GAGs. The accumulation of these undegraded substrates in almost any tissue is a hallmark of all Mucopolysaccharidoses (MPS). Present therapeutics based on enzyme replacement therapy and bone marrow transplantation have low effectiveness for the treatment of MPS with neurological complications since enzymes used in these therapies are unable to cross the blood brain barrier. Small molecule-based approaches are more promising in addressing neurological manifestations. In this report we identify a target for developing a substrate reduction therapy (SRT) for six MPS resulting from the abnormal degradation of heparan sulfate (HS). Using the minimal promoter of NDST1, one of the first modifying enzymes of HS precursors, we established a luciferase based reporter gene assay capable of identifying small molecules that could potentially reduce HS maturation and therefore lessen HS accumulation in certain MPS. From the screen of 1,200 compounds comprising the Prestwick Chemical library we identified SAHA, a histone deacetylase inhibitor, as the drug that produced the highest inhibitory effects in the reporter assay. More importantly SAHA treated fibroblasts expressed lower levels of endogenous NDST1 and accumulated less 35S GAGs in patient cells. Thus, by using our simple reporter gene assay we have demonstrated that by inhibiting the transcription of NDST1 with small molecules, identified by high throughput screening, we can also reduce the level of sulfated HS substrate in MPS patient cells, potentially leading to SRT.