Gerard Vockley, MD, Research Projects

Inborn errors of metabolism are genetic disorders that affect the ability of the body to carry out vital biochemical reactions. These disorders affect two to three of every 1,000 people, and their consequences range from neonatal death to chronic disease in adults.

Discovering the underlying genetic cause of a group of these disorders involved in energy metabolism and amino acid catabolism is the focus of research being conducted by Gerard Vockley, MD, PhD, chief of the Division of Genetic and Genomic Medicine at Children’s Hospital.

Current Research

ACAD9

Mitochondrial β-oxidation of long chain fatty acyl-CoAs is well recognized as a primary metabolic pathway for maintenance of energy homeostasis and body temperature. However, it also recycles carbons from many long chain fatty acids for lipid synthesis. Little is known about the mechanistic role of the latter in the pathogenesis of symptoms in genetic defects of β-oxidation, and its derangement may in part explain the features of disorders such as neurological dysfunction or acute respiratory distress syndrome that respond poorly to treatment with alternative energy sources.

Very long chain acyl-CoA dehydrogenase (VLCAD) is the dominant long chain acyl-CoA dehydrogenase (ACAD) in energy generation in human muscle and the heart. In contrast, this study provides evidence that ACAD9 and long chain acyl-CoA dehydrogenase (LCAD) more likely function in lipid recycling and synthesis in human brain and lung, respectively, supported by their unique substrate utilization and tissue distribution pattern. Furthermore, we have identified a new genetic deficiency of ACAD9 presenting with episodic liver failure and cardiomyopathy during otherwise mild illnesses, along with chronic neurologic dysfunction. This disorder represents the first in a b-oxidation enzyme primarily involved in lipid recycling or synthesis, revealing a new mechanism of pathogenesis in human disease.

Source(s) of Support

National Institutes of Health

Principal Investigator

Gerard Vockley, MD, PhD

ACAD10

In the last half of the 20th century, the incidence of type 2 diabetes mellitus (T2DM), previously unrecognized in the Pima Indians, began to rise. Multiple factors were postulated to be responsible including environmental factors, such as diet and resultant obesity, along with a number of genetic determinants. ACAD10 was one of 30 genes further examined after demonstrating a significant signal for diabetes in a genome-wide association study (GWAS). In these studies, a single-nucleotide polymorphism (SNP), rs632650, was found to map within intron 2 of ACAD10.

The hypothetical ACAD10 protein is structurally related to the ACAD family of mitochondrial flavoproteins, which consists of nine enzymes that are similar in structure and function as they catalyze the α, β-dehydrogenation of their corresponding acyl-CoA substrates. Seven of these ACADs, LCAD, medium-chain acyl-CoA dehydrogenase (MCAD), short-chain acyl-CoA dehydrogenase (SCAD), isovaleryl-CoA dehydrogenase (IVD), isobutyryl-CoA dehydrogenase, 2-methyl-branched chain acyl-CoA dehydrogenase, and glutamate dehydrogenase, are homotetramers (~400-aa per monomer) and two, VLCAD and ACAD9, are homodimers (~640-aa per monomer). In addition to their usual location in the mitochondrial matrix, some ACADs, including LCAD, MCAD, and SCAD, have been shown to be associated with cytoplasmic GLUT4-containing vesicles where they interact with two dileucine motifs on insulin-regulated aminopeptidase (IRAP). Mutation of the dileucine motif of IRAP (amino acids 55–82) eliminates this interaction. In cells, glucose equilibrium is maintained by the GLUT4 response to insulin. The GLUT4- and IRAP-containing vesicles respond to insulin stimulation by translocation to the cell surface. The dileucine motif in IRAP plays a critical role in regulating GLUT4 trafficking. While these findings connect some of the ACADs with insulin dependent transportation of glucose within cells, the physiologic role of the ACAD proteins in this setting is unclear.

Skeletal muscle patterns of fatty acid utilization during fasting conditions have been shown to be associated with obesity-related insulin resistance and altered mitochondrial energy metabolism, including fatty acid oxidation. These abnormalities have also been shown to be present in the context of T2DM. We have recently shown that the pattern of acylcarnitines (ACNs), key metabolic intermediates of fatty acid oxidation, in the blood of obese and T2DM participants fall into two distinct patterns. First, the T2DM and obese participants had a similar accumulation of long-chain ACNs that arise from activity in the initial rounds of β-oxidation, consistent with increased flux at entry into mitochondrial β-oxidation. Diabetic participants also displayed a secondary accumulation of various shorter chain ACNs suggestive of inefficient complete fatty acid oxidation or interactions between β-oxidation and ETC. They also showed an inability to efficiently switch from fat metabolism during insulin clamp, as reflected in their inability to lower their ACNs as effectively as either the lean or obese subjects. In contrast, this pattern was not present in obese adolescents, who instead showed metabolic findings suggestive of upregulation of fatty acid oxidation.

To characterize the physiologic role of ACAD10 in intermediary metabolism and its possible link to T2DM, we have characterized an ACAD10 gene trap mouse model. Aging animals become obese on a normal diet and develop insulin-resistant hyperglycemia in response to an intraperitoneal glucose challenge. Tissue and blood ACN profiles are similar to those previously described for adult humans with T2DM. Our findings identify ACAD10 deficiency as new monogenic cause of T2DM in mice, and provide valuable insight into its potential role in the development of T2DM in Pima Indians.

Principal Investigator

Gerard Vockley, MD, PhD

Genetic Disease in Amish

Characterizing the Burden of Genetic Disease in Old Order Amish

The Old Order Amish communities (Plain people) of North America have altered health risks that stem from unbalanced population sampling of European founders followed by genetic drift in derivative generations. These population effects have resulted in a high prevalence of specific genetic disorders that vary from the general population and from each other. Several characteristics of these communities facilitate genetic analysis. Most isolates keep excellent historical and genealogical records. Due to their sociologic and/or geographic isolation, there is usually little or no migration into the group, and the members of the group exhibit relatively homogeneous lifestyles. Large nuclear families are frequent, which provides adequate numbers of affected and unaffected siblings within a sibship for blood samples. The primary genetic advantage, however, results from the interaction of two overlapping phenomena: the founder effect and inbreeding.

Mitochondrial DNA mutations have not previously been reported in any Old Order Amish community. We have recently described an Amish family with the MTTL1 mitochondrial gene mutation m.3243A>G. A second patient with an m.13513G>A (D393N) mutation has also been diagnosed. These mutations classically cause mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). We identified the first mutation in a young woman from the Mercer County Amish community at age 15 years. She had a history of developmental delay, short stature, hearing loss, fatigability, and poor appetite. She presented acutely with vomiting, altered mental status, status epilepticus, and lactic acidosis. Magnetic resonance imaging (MRI) of the brain showed a small, focal, left occipital lobe infarct. She subsequently developed other stroke-like episodes in the left occipital and temporal areas. Molecular testing revealed 74 percent heteroplasmy in saliva for the MELAS 3243A>G mutation. Several members of the extended maternal pedigree exhibit variable clinical problems including developmental delay, mild hypotonia, hearing loss, renal failure, migraine headaches, adult-onset diabetes mellitus, and recurrent miscarriages, but have never had genetic evaluations.

A patient from a second Amish family was diagnosed with MELAS/Leigh overlap syndrome resulting from the mitochondrial mutation m.13513G>A(D393N) in the ND5 subunit of respiratory chain complex I, with blood heteroplasmy level of 2 percent and urine heteroplasmy level of 43 percent. The proband was diagnosed at 12 years of age with an acute stroke after a history of developmental delay. His lactate was mildly increased. He has subsequently had recurrent strokes and developed Leigh-like basal ganglia and brainstem lesions with progressive spasticity, dysphagia, and weakness. No siblings were affected, and the mother tested negative for the mutation in blood and urine.

A third patient in this Amish community has been diagnosed with an autosomal recessive respiratory chain disorder due to a homozygous deletion in the NDUFAF2 gene, which was in one of the nine areas of homozygosity detected on SNP microarray. He had a history of developmental delay, nystagmus, and hypotonia, and was subsequently admitted to the hospital with a viral illness with progressive respiratory failure. He required intubation, and his MRI revealed Leigh-like lesions. He could not be weaned from the ventilator and three weeks later, his repeat MRI showed worsening of lesions with infarction of cerebellar white matter. He remained unresponsive and fulfilled brain death criteria, ultimately dying after discontinuation of life support.

Amish populations are unique in that they represent genetic bottlenecks dating back to the 18th century, distinguishing them from the European population as a whole, as well genetic drift, which has given rise to variable distributions of pathogenic alleles among North American settlements. Our mitochondrial studies and other clinical encounters lead us to the hypothesis that many unrecognized genetic disorders are present in the Mercer County Amish. This is in keeping with studies conducted by the Clinic for Special Children in Strasburg, Pa., founded and led by Holmes Morton. The clinic is a comprehensive care facility for the Plain people in and around Lancaster County. Morton has extensively characterized this population genetically, including variant analysis through whole-genome and exome sequencing. Not surprisingly, the Old Order Amish demonstrate significant population divergence from the general European population. More surprisingly, genetic variants in the Old Order Amish isolate in Lancaster also differ from those in Big Valley, Pa., and those in Cuyahoga County, Ohio.

The Mercer County Amish are among the least genetically characterized Amish communities in the United States, with no catalogue of either genetic disorders or variants seen in the community. We have developed a new program to characterize the genetic variability between Amish Mercer County population and other Amish counties by doing whole-exome and mitochondrial DNA sequencing. This will be crucial to determining the phenotype and frequency of other known and unknown genetic disorders in this population. This project will allow us to characterize load of genetic disease in Old Order Amish of Mercer County and identify disorders that can benefit from early treatment.

Principal Investigator

Gerard Vockley, MD, PhD

Multifunctional Fatty Acid Oxidation Complex

Characterization of a Multifunctional FAO Complex

Fatty acid β-oxidation (FAO1) and oxidative phosphorylation (OXPHOS) are key pathways involved in cellular energetics. Reducing equivalents from FAO enter OXPHOS as at the level of complexes I and III. Genetic disorders of FAO and OXPHOS are among the most frequent inborn errors of metabolism. Patients with deficiencies of either FAO or OXPHOS often show clinical and/or biochemical findings indicative of a disorder of the other pathway.

In this study, the physical and functional interactions between these pathways were examined. Extracts of isolated rat liver mitochondria were subjected to blue native polyacrylamide gel electrophoresis (BNGE) to separate OXPHOS complexes and supercomplexes, followed by western blotting using antisera to various FAO enzymes. Extracts were also subjected to sucrose density centrifugation and fractions analyzed by BNGE or enzymatic assays. Several FAO enzymes co-migrated with OXPHOS supercomplexes in different patterns in the gels. When palmitoyl-CoA was added to the sucrose gradient fractions containing OXPHOS supercomplexes in the presence of potassium cyanide, cytochrome c was reduced. Cytochrome c reduction was completely blocked by myxothiazol (a complex III inhibitor) and 3-mercaptopropionate (an inhibitor of the first step of FAO) but was only partially inhibited by rotenone (a complex I inhibitor).

Although palmitoyl-CoA and octanoyl-CoA provided reducing equivalents to OXPHOS-containing supercomplex fractions, no accumulation of their intermediates was detected. In contrast, short-branched acyl-CoA substrates were not metabolized by OXPHOS-containing supercomplex fractions. These data provide evidence of a multifunctional FAO complex within mitochondria that is physically associated with OXPHOS supercomplexes and promotes metabolic channeling.

Principal Investigator

Gerard Vockley, MD, PhD

Sterol Metabolism Disorder

A New Disorder in Sterol Metabolism

Defects in cholesterol synthesis result in a wide variety of symptoms from neonatal lethality to the relatively mild dysmorphic features and developmental delay found in Smith-Lemli-Opitz syndrome (SLOS). Our investigation identified mutations in SC4MOL as the cause of a newly recognized autosomal recessive syndrome that includes psoriasiform dermatitis, arthralgias, congenital cataracts, microcephaly, and developmental delay. This gene encodes a sterol-C4-methyl-oxidase, catalyzing demethylation of C4-methylsterols in the cholesterol synthesis pathway. C4-methylsterols are members of meiosis-activating sterols (MAS). They are first found in high concentration in testis and ovary and play roles in meiosis activation.

In this study, our team found that MAS affect the cell proliferation in both skin and blood. We also found that inhibition of sterol-C4-methyl-oxidase significantly altered the immune regulation in immunocytes. MAS are ligands of the liver X receptors α and β, which are important in regulating not only lipid transport in epidermis, but also the innate and adaptive immunity. Deficiency of SC4MOL represents a new biochemical defect in the cholesterol synthesis pathway, the clinical spectrum of which remains to be defined.

Principal Investigator

Gerard Vockley, MD, PhD

Sterol Rare Disease Consortium

Gerard Vockley, MD, PhD, is the site principal investigator for the Sterol and Isoprenoid Diseases Research Consortium (STAIR), which is uniquely focused on studying a group of diseases bound by common biochemistry, impact on health, and rarity. These include:

  • Cerebrotendinous xanthomatosis (CTX)
  • Hyperimmunoglobulinemia D with periodic fever syndrome (HIDS)
  • Niemann-Pick disease type C (NPC)
  • Sitosterolemia
  • Sjögren-Larsson syndrome (SLS)
  • Smith–Lemli–Opitz syndrome (SLOS)

STAIR activities are performed by a team of investigators chosen for their clinical research strengths and resources, a long history of collaboration, a diverse geographic distribution to allow maximal access by potential research subjects, their individual motivation to improve the health of patients, and the commitment of their institutions to support the consortium.

STAIR will conduct two major clinical studies over five years – a longitudinal natural history study of NPC and a therapeutic trial to evaluate the efficacy of antioxidant therapy in SLOS – as well as six pilot research studies involving patients with SLS, SLOS, CTX, HIDS, or sitosterolemia. Together with the intramural National Institutes of Health program, the consortium will support a full-scale training program in the field of sterol and isoprenoid diseases and share its resources and data with the NIH Rare Diseases Clinical Research Network.

Participating institutions include Oregon Health and Science University (OHSU), Eunice Kennedy Shriver National Institute of Child Health and Human Development, UPMC Children’s Hospital of Pittsburgh, Cincinnati Children’s Hospital Medical Center, University of Nebraska Medical Center, and University of Manitoba (Canada). OHSU will be the administrative home of the consortium. The following patient-support organizations will participate in STAIR activities: Smith-Lemli-Opitz/RSH Foundation, Hide and Seek Foundation, Ara Parseghian Medical Research Foundation, Dana’s Angels Research Trust, Foundation for Ichthyosis and Related Skin Types, and United Leukodystrophy Foundation. In summary, STAIR will foster multidisciplinary clinical research, promote training and education, and support projects to explore promising leads in the understanding, diagnostics, and treatment of sterol and isoprenoid diseases.

Source(s) of Support

National Institutes of Health

Principal Investigator

Gerard Vockley, MD, PhD

VLCAD

More than 100 cases of VLCAD deficiency have been documented in the literature with three different disease phenotypes. A severe infant-onset form is characterized by acute metabolic decompensation with hypoketotic hypoglycemia, dicarboxylic aciduria, liver dysfunction, and cardiomyopathy. A second form of the disease presents later in infancy or childhood but has a milder phenotype without cardiac involvement. The third form is of adolescent or adult onset and is dominated by muscle dysfunction that is often exercise-induced.

Not surprisingly, children with the severe phenotype tended to have null mutations (71 percent of identified alleles in these patients), while patients with the two milder forms of the disease were more likely to have missense mutations (82 percent and 93 percent of identified alleles for the milder childhood form and the adult form, respectively). Nevertheless, a few missense mutations were clearly associated with the severe phenotype. Although these data suggested that missense mutations in VLCAD might obviate clinical symptoms due to some degree of residual activity, no correlation was seen between the mutations identified and residual VLCAD activity in fibroblasts. Moreover, the function effects of few of the known VLCAD missense mutations have been directly characterized.

We have previously used prokaryotic expression systems to express, purify and characterize the biochemical properties of several ACD enzymes. Several of these have been crystallized and studied by X-ray diffraction, yielding informative three-dimensional models. The study of VLCAD, however, has been limited due to difficulties with prokaryotic expression. These difficulties may be related in part to physical properties that distinguish VLCAD from other ACD family members. Most of the ACDs share a common homotetrameric “dimer of dimers” structure and function in the mitochondrial matrix. In contrast, VLCAD is a homodimer with an extended 180 amino acid C-terminal domain of unknown function. Additionally, VLCAD is associated with the inner mitochondrial membrane, an interaction that has long been postulated without proof to be mediated by the C-terminus.

We have used our prokaryotic expression system to study six previously missense mutations described in VLCAD deficient patients (T220M, V243A, R429W, A450P, L462P, and R573W). T220M and V243A are the most frequently reported missense mutations in VLCAD deficient patients. R429W and R573W are among the few missense mutations believed to result in the severe clinical phenotype. A450P and L462P are located in the C-terminal domain unique to VLCAD and ACAD-9. Characterization of purified wild type, A450P, and L462P VLCAD proteins confirmed the long-held assumption that the C-terminus plays a key role in mitochondrial membrane association. The prokaryotic system developed will greatly facilitate investigation of VLCAD structure-function. 

Source(s) of Support

National Institutes of Health

Principal Investigator

Gerard Vockley, MD, PhD