Yield of newborn screening in Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) deficiency
Jeannette C. Bleeker, MD 1,2 *, Sacha Ferdinandusse, PhD 2, Riekelt H. Houtkooper, PhD 2, Ronald J.A. Wanders, PhD 2, Frits. A. Wijburg, MD, PhD 2, Ludo van der Pol, MD, PhD 3,
Gepke Visser, MD, PhD 1,2
1 Department of Metabolic Diseases, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands
2 Laboratory Genetic Metabolic Diseases, Departments of Clinical Chemistry and Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
3 Department of Neurology and Neurosurgery, Rudolf Magnus Institute of Neuroscience, Spieren voor Spieren Kindercentrum, University Medical Center, Utrecht, The Netherlands
* contact information: firstname.lastname@example.org, +31 20 5663827/ +31 88 7554042
Background: With the introduction of newborn screening (NBS) programs, the number
of patients diagnosed with Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) deficiency
has rapidly increased. However, many infants are asymptomatic at time of diagnosis.
We determined the effect of NBS on clinical outcome in these patients.
Method: Long term follow-up study. Dutch VLCAD deficient patients, diagnosed before
(pre-NBS) and through NBS were evaluated by a standardized protocol in the Dutch Expertise Center for Fatty Acid Oxidation Disorders. Data were collected on development, complaints, medical history, physical abnormalities and treatment during regular clinical visits.
Results: Data on outcome after 9 years are presented. Of 57 registered patients (26 pre-NBS, 31 NBS), 6 died (4 pre-NBS, 2 NBS). Of the living patients, 36 were seen during regular follow-up (17 pre-NBS, 19 NBS). Hypoglycemia was detected in 10/17 pre-NBS patients with a median age of 10 months (0-2 years). One NBS patient developed hypoglycemia shortly after birth. Cardiomyopathy was present in 3/17 pre-NBS patients and in none of the NBS patients. Myopathy was present in 15/17 pre-NBS patients, in contrast to 2/19 NBS patients. Neurological examination during regular clinical visit was abnormal in 4/17 pre-NBS patients and in one NBS patient.
Conclusion: Thus far the outcome of patients diagnosed through NBS seems favorable compared to those diagnosed pre-NBS. This could be an effect of timely treatment,
but detection of milder mutations and the natural disease course can play a role as well.
To understand the clinical spectrum in later life and the effect of different treatment strategies, we will continue to monitor the cohort.
DILATED CARDIYOMYOPATHY DUE TO PRIMARY CARNITINE DEFICIENCY: A REAL LIFE CASE STUDY
Ali Kanık, MD 1 , Marta Frigeni, MD 2 , Kayı Eliaçık, MD 1 , Ali Rahmi Bakiler, MD1,
Tijen Tanyalcin, MD PhD 3, Mei Baker,MD FACMG 4 , Nicola Longo, MD PhD 2
1 Izmir Tepecik Teaching and Research Hospital, Department of Pediatrics, Izmir, Turkey
2 Pediatric Medical Genetics, University of Utah, Salt Lake City, Utah, USA
3 Tanyalcin Medical Laboratory Selective Screening and Metabolism Unit, Izmir, Turkey
4 Co-Director, Newborn Screening Laboratory Wisconsin State Laboratory of Hygiene, Madison, Wisconsin, USA
Corresponding Author Contact Information: email@example.com, +90 53 26579230
Introduction: Dilated cardiomyopathy is characterized by left ventricular enlargement and
a reduction in the myocardial force of contraction. A specific cause cannot be identified
in about 50% of cases and many patients require a heart transplant to survive. In children,
5-10% of cardiomyopathies are caused by an inborn errors of metabolism. Primary carnitine deficiency (OMIM 212140, carnitine uptake defect, carnitine transporter deficiency
or systemic carnitine deficiency) is caused by defective activity of the high-affinity carnitine transporter (OCTN2), encoded by the SLC22A5 gene and can result in impaired fatty acid oxidation. This report describes the symptomatic diagnosis of a 8 year-old girl with primary carnitine deficiency and dramatic response of her cardiac function to carnitine supplementation.
Case: A-8- year-old girl was referred for a history of dilated cardiomyopathy first diagnosed
at 9 months of age. She presented with fatigability, abdominal pain and respiratory distress. Her parents were first cousins. Physical examination indicated failure to thrive (weight at the 3rd centile) with normal height (50th centile). Cardiac exam was significant for a 3/6 systolic murmur best heard at the lower left sternal border together with cardiac failure. Echocardiogram indicated an enlarged left ventricle end-diastolic diameter (LVED=56 mm, normal 33-47 mm) with a decreased left ventriculer ejection fraction (LVEF= 44%, normal
56-78%) and reduced shortening fraction (SF=20%, normal 28-44%). A plasma acyylcarnitine profile indicated very low free carnitine (C0=0.57 uM, normal 8-50 uM) with very low levels
to absent long-chain acylcarnitines (C16= 0.02 uM, C18=0 uM). The patient was treated with L- carnitine 100 mg/kg/day divided into 3 doses and after 12-month the cardiomyopathy resolved with normalization LVED at 46 mm, LVEF at 57%, and SF at 30% with mild mitral valve insuffiency (present before therapy). Failure to thrive also improved with weight above the 5th centile. Sequencing of the SLC22A5 gene indicated homozygosity for c.614T>G, p.M205R
in exon 3. This amino acid is highly conserved and in silico analysis with Polyphen2 and SIFT suggested a pathogenic role of this change. Expression studies in Chinese Hamster Ovary cells confirmed that the M205R mutation completely abolished carnitine transport
(0% of normal OCTN2 activity). Homozygosity for this mutation was consistent with parental consanguinity.
Conclusion: Identification of the underlying causes of cardiomyopathy with subsequent disease-specific treatments may lead to improved outcomes. Primary carnitine deficiency
is one of the treatable causes of cardiomyopathy since complete resolution of the heart disease is possible with carnitine supplementation. We hope that with the advent of national expanded newborn screening in Turkey by tandem mass spectrometry (MS/MS) more patients will be diagnosed, before the onset of cardiomyopathy or irreversible complications.
* EXPRESSION OF NOVEL MUTATIONS IN THE CARNITNE TRANSPORTERFROM PATIENTS WITH PRIMARY CARNITINE DEFICIENCY.
Frigeni M, Balakrishnan B 1 , Yin X 1 , Pasquali M 1,2,3 , Longo N 1,2,3
1 Dept Pediatrics, 2Pathology and 3ARUP Laboratories, University of Utah, Salt Lake City, UT, USA.
Email: Marta.Frigeni@hsc.utah.edu Phone: 801-585- 9558
Primary carnitine deficiency is a recessive disorder of the carnitine cycle caused by mutations in the SLC22A5 gene. Patients can present with an acute metabolic decompensation early
in life, or later in life with skeletal and cardiac myopathy or sudden death from arrhythmia. The diagnosis, suspected because of clinical presentation or low levels of free carnitine in the newborn screening, can be confirmed by functional studies in fibroblasts or by SLC22A5 gene sequencing.
Here we compare functional studies to gene sequencing for the diagnostic confirmation
of primary carnitine deficiency.
Carnitine transport was measured in skin fibroblasts of 382 patients suspected of having primary carnitine deficiency. SLC22A5 gene sequencing was performed in 95 patients with reduced carnitine transport. Missense variants identified were expressed in CHO cells and tested for carnitine transport.
Carnitine transport was reduced to ≤20% of normal in fibroblasts of 140/382 subjects. SLC22A5 gene sequencing in 95/140 subjects identified variants in 84% of the alleles. Expression of 88 missense changes (33 novel) in CHO cells demonstrated reduced carnitine transport in 73/88. Prediction algorithms (SIFT, Polyphen-2) correctly predicted the functional effects of missense variants in 77% of cases.
These results indicate that functional studies remain the best strategy to confirm or exclude
a diagnosis of primary carnitine deficiency, since DNA sequencing of the SLC22A5 gene fails to identify causative variants in about 16% of the alleles and existing software can correctly predict the pathogenicity of missense variants in only 77% of cases
Clinical outcome per age
DIFFERENT CLINICAL PHENOTYPES WARRANT TAILOR MADE DIETSIN PATIENTS WITH LONG CHAIN FATTY ACID OXIDATION DISORDERS
Irene L. Kok, RD 1,2 , Jeannette C. Bleeker, MD 2,3 , Sacha F. Ferdinandusse, PhD 3,
epke Visser, MD PhD 2
1 Department of Internal Medicine and Dermatology, Dietetics, Wilhelmina Children’s Hospital, UMCU, Utrecht
2 Department of Metabolic Diseases, Wilhelmina Children’s Hospital, UMCU, Utrecht;
3 Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam,
Corresponding author contact information: E-mail: I.L.Kokfirstname.lastname@example.org Phone: 0031651835664
Introduction: Patients with long chain fatty acid oxidation disorders can be treated with
a fat-restricted, MCT-supplemented diet. However, patients diagnosed by newborn screening and with mild phenotypes may not use a diet at all.
Aim: To evaluate current dietary strategies in patients with long chain fatty acid oxidation disorders. Methods: Dietary intake was evaluated by a three day food record or dietary history. In addition, a questionnaire regarding use of supplements and medical nutrition, changes
in the diet during illness or sports activities and the maximal feeding pause was scored
by a research dietician.
Results: Forty-two patients were included (28 VLCADD, 6 LCHADD, 3 MTPD, 5 CPT2D).
All patients had initially a prescribed maximum feeding pause. MCT supplementation was used by 33% of the VLCADD patients, 75% of the CPT2D and 100% of the LCHADD and MTPD patients. The use of MCT supplements did not correlate with the presence of complaints
(e.g. hypoglycemia, fatigue, exercise intolerance, myoglobinuria, and/or cardiomyopathy).
No difference was observed in BMI of patients < 18 years compared to their healthy peers. However patients >18 years were more often overweight.
Conclusion: Different dietary strategies are used especially by patients with VLCADD, most only use a diet during illness. Further investigations are needed to determine the optimal individual treatment strategies. Furthermore, caution is needed in the dietary advice to prevent overweight.
Table 1. Newborn Screen Result (specimen collected on day of life 3)
ASSESSMENT OF GLYCEROL PHENYLBUTYRATE AS A CHAPERONE IN THE TREATMENT OF PATIENTSWITH MCAD DEFICIENCY CAUSED BY THE COMMON 985 A>G (K304E) MUTATION
RaeLynn Forsyth, BA, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
Gerard Vockley, MD, PhD, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA
Corresponding Author Contact Information: email@example.com Phone: 724-984- 2529
Background: MCAD deficiency (MCADD) is the most common fatty acid oxidation disorder, and historically, caused a spectrum of sequelae including sudden death. Addition of MCADD to the newborn screening panel allowed preventative therapy from birth through dietary modifications. However, there is no pharmacological treatment for MCADD that can protect patients when fasting is unavoidable. We have previously shown that phenylbutyrate can act as a chaperonin to the misfolded MCAD protein in cells with the most common MCADD mutation (c.985A>G, p.K304E).
Methods: Four adult patients with MCADD and at least one copy of the common mutation were enrolled. Blood and urine was obtained after a twelve hour fast for metabolic and pharmacokinetic studies. After the first fast, subjects were started on study drug, and after the second and third fasts, their dose was increased pending the pharmacokinetic studies performed at the prior visit.
Results: All four subjects had improvement in one or more metabolic parameters while
on phenylbutyrate. Three subjects had a decrease in acylglycine intermediates that are specific for MCADD. Other parameters were more subtly improved. Subjects were not sufficiently stressed metabolically to observe change in glucose and ketone levels.
Two subjects did reach “toxic” levels of phenylbutyrate, but had no symptoms of toxicity.
Conclusions: This pilot study suggests that phenylbutyrate can improve metabolic flux through fatty acid oxidation in patients with MCADD due to the c.985A>G mutation.
A phase 2 trial is currently being planned.
* Travel Grant Awardees Selected for an oral presentation at INFORM 2016
OUTCOMES OF INFANTS WITH POSITIVE NEWBORN SCREENSFOR VLCAD DEFICIENCY IN SOUTHERN ALBERTA
Marisa Charda c , Karen Saboa, Aneal Khan a,b,c , Rebecca Sparkes a,b,c
Alberta Children’s Hospital, Calgary, Alberta, Canada a
Dept. of Pediatrics, University of Calgary, Alberta, Canada b
Dept. of Medical Genetics, University of Calgary, Alberta, Canada c
Objectives: Very long chain fatty acyl-CoA dehydrogenase (VLCAD) deficiency is a fatty acid oxidation disorder (FAOD) with a spectrum of severity ranging from a potentially fatal, neonatal cardiomyopathic form to mild late-onset variants with minimal or no symptoms. After a positive newborn screen it can be difficult to accurately predict the phenotype with
the diagnostic testing available. The aim of this study was to determine the outcomes
of infants with screen positive results for VLCAD deficiency.
Methods: In this retrospective cohort study, the medical files of all patients who screened positive for VLCAD deficiency since expanded newborn screening began in Southern Alberta on April 1, 2007 were reviewed. We determined the false positive and true positive rates
of screening, newborn screen results, diagnostic testing (acylcarnitines, fatty acid oxidation studies, ACADVL genotype), dietary and illness management, follow up tests, and clinical features.
Results: Seventeen (17) patients screened positive for VLCAD deficiency. Of those, one was symptomatic with severe neonatal-onset disease. Another five patients likely had mild VLCAD deficiency, and only one of these patients has been symptomatic to date. The remaining
11 patients had indeterminate results, and of these five were likely false positive due to heterozygosity for one pathogenic ACADVL variant.
Conclusions: Newborn screening for VLCAD can be lifesaving. However, the majority who screen positive will be heterozygous ACADVL mutation carriers, have mild or asymptomatic VLCAD deficiency or have an indeterminate diagnosis which may lead to family anxiety
and possibly to over-medicalization of some children.
INTERIM RESULTS FROM AN OPEN-LABEL PHASE 2 STUDY TO ASSESS SAFETY AND CLINICAL EFFECTS OF INVESTIGATIONAL UX007 (TRIHEPTANOIN) IN SUBJECTS WITH LONG- CHAINFATTY ACID OXIDATION DISORDERS (LC-FAOD)
Vockley J, MD, PhD 1; Burton B, MD 2; Berry G, MD 3; Longo N, MD 4 ; Phillips J, MD 5;
Sanchez-Valle A, MD 6; Tanpaiboon P, MD 7; Grunewald S, MD, PhD 8; Murphy E, MD 9;
Bowden A, PhD 10; Cataldo J, DO 10; Marsden D, MD 10; Kakkis E, MD, PhD 10
1 University of Pittsburgh, Pittsburgh, PA, USA;
2 Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, Illinois, USA;
3 Boston Children’s Hospital, Boston, MA, USA;
4 University of Utah, Salt Lake City, UT, USA;
5 Vanderbilt Medical Center, Nashville, TN, USA;
6 USF Health, Morsani College of Medicine, Tampa, FL, USA;
7 Children’s National Medical Center, Washington DC, USA;
8 Great Ormond Street, UCL Institute of Child Health, London, UK;
9 National Hospital for Neurology and Neurosurgery, London, UK;
10 Ultragenyx Pharmaceutical Inc., Novato, CA, USA
LC-FAOD are a group of autosomal recessive genetic disorders characterized by metabolic deficiencies in which the body is unable to convert long-chain fatty acids into energy causing serious liver, muscle, and heart disease. This single-arm, open-label Phase 2 study evaluated 29 patients (aged 10 months to 58 years) with LC-FAOD demonstrating severe ongoing disease despite standard-of- care treatment. The majority of patients qualified based on severe musculoskeletal disease. After a four-week run-in period on current regimen (27 of 29 patients on MCT oil), UX007 (triheptanoin), a medium odd-chain fatty acid, was titrated to a target dose of 25-35% of total daily caloric intake. Patients were evaluated over 24 weeks on several endpoints, including cycle ergometry, 12-minute walk test (12MWT), and quality of life (QoL). Patients performed only the assessments appropriate for their age and clinical status.
At week 24 cycle ergometry testing, the seven age- and condition-eligible patients showed
a mean 60% (+446.8 watts) increase (median: +127.5 watts; min, max: -388, +2438) over
a baseline of 744.6 watts. In the last assessment for 12MWT (Week 18), the eight eligible patients showed a mean increase of +188 meters (median: 93.5; min, max: - 80, 880)
from a baseline mean of 673.4 meters, an increase of 28%. Additionally, adult self-reported, health-related QoL (SF-12v2) physical component summary scores were significantly improved, mean increase 8.9 points, supporting the clinical meaningfulness of the observed changes
in exercise tolerance tests. No difference was seen in parent-reported scores (SF-10) for the pediatric patients. Overall, 18 patients (62%) had treatment-related adverse events, predominantly gastrointestinal (55%), which were mild-to- moderate in severity.
In patients suffering with ongoing severe LC-FAOD, treatment with UX007 showed improvements in exercise tolerance and muscle function, and were associated with positive changes in self-reported QoL. Further investigation in a controlled confirmatory study
CHARACTERIZING THE MOLECULAR ARCHITECTURE OF MITOCHONDRIAL ENERGYMETABOLISM APPARATUS AND ITS IMPORTANCE TO CLINICAL PATHOPHYSIOLOGY
Yudong Wang 1, Johan Palmfeldt 2 , Niels Gregersen 2, Aexander M. Makhov 3,
James F. Conway 3, Amy C Goldstein 1, Lina Ghaloul-Gonzalez 1, Hana Alharbi1, Meicheng Wang 4, Xuemei Zeng 5,Nathan A Yates 5, and Jerry Vockley 1,6
1 Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA
2 Research Unit for Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark
3 Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA
4 Department of Pharmacology, University of Pittsburgh School of Pharmacy, Pittsburgh, PA
5 Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA
6 Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA
Mitochondrial energy metabolism is comprised of three major biochemical pathways: oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and the tricarboxylic acid cycle. OXPHOS is organized into functional enzymatic complexes that in turn assume higher order structures called super complexes. We have previously demonstrated functional and physical interactions between OXPHOS and FAO, forming a mitochondrial energy super-structure that promotes metabolic channeling, potentially allowing multiple enzymatic reactions to occur without release of intermediates, and thus optimizing metabolic efficiency.
We now report the use of 2-dimension blue native/SDS-PAGE western blots, mass spectrometry proteomic techniques, and immune-electron microscopy studies to characterize the physical interaction of fatty acid oxidation proteins with each other and the OXPHOS super complexes.
We find that the mitochondrial trifunctional protein, an NADH+ generating enzyme, directly interacts with complex I NADH+ binding domain, as well as with very long chain acyl-CoA dehydrogenase of FAO. Electron transfer flavoprotein dehydrogenase, which funnels reducing equivalents from acyl-CoA dehydrogenases in FAO to OXPHOS, interacts with the co-enzyme
Q reduction site of complex III and complex I, consistent with the known interaction of these two enzymes in super complexes. Two-dimension blue native/SDS-PAGE also identifies short chain 3-hydroxyacyl- CoA dehydrogenase, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase extensively interacting with OXPHOS
complex I and the super complexes.
Patients with deficiencies of either FAO or OXPHOS often show clinical and/or biochemical findings indicative of a disorder of the other pathway. In a series of explanted hearts from transplant patients with a variety of disorders, interactions.
NUTRITIONAL MANAGEMENT OF VERY LONG CHAINACYL-COA DEHYDROGENASE DEFICIENCY (VLCAD)
Ashleigh Mitchell 1 , Sue Thompson 1 , Sarah Slack 1 , Kaustuv Bhattacharya 2
1 Department of Nutrition and Dietetics, The Children’s Hospital Westmead, Sydney, Australia
2 Genetic Metabolic Disorders Service, The Children’s Hospital Westmead, Sydney, Australia
Corresponding author contact information: Ashleigh.firstname.lastname@example.org
Mob: +614 04 982 921
Expanded newborn screening (NBS) has resulted in identification of patients with VLCAD that are asymptomatic at time of diagnosis. The variability of phenotypes within the condition
and limited genotype, phenotype correlation has complicated nutritional management. Furthermore, the rarity of the condition has made evidenced based studies difficult
and prompted consensus workshops to guide therapy. Our aim was to examine nutritional management of children with VLCAD using a retrospective audit conducted January 2016. Eighteen patients were identified (15 diagnosed by NBS/symptomatic at birth, 3 diagnosed between 1 and 11 years old). In infancy, medium chain triglyceride (MCT) based formula was recommended for 13 patients, providing ~30% (n=8), 50% (n=4) or 70% (n=1) of energy intake, depending on initial feeding method and clinical assessment. Two received long chain triglyceride (LCT) based formula alone. Introduction to food education aimed to achieve <10% (n=11), 10-20% (n= 4) or <30% (n=3) energy from LCTs with MCT medical foods as an energy source. Triheptanoin was used by 2 patients. Maximum fasting duration was a median
of 4 hours at diagnosis, 6 hours at 6 months and 8 hours at 12 months of age. Unwell recommendations increased proportion of MCT formula/ medical food with addition
of a carbohydrate source in most cases (n=12). Two patients required gastrostomy placement. Essential fatty acid supplements were prescribed only with biochemically evident deficiency (n=7). Further studies quantifying LCT intake compared to prescribed diet and subsequent correlation with phenotype expression are required.
* DISRUPTION OF BRAIN AND LIVER MITOCHONDRIAL FUNCTION CAUSED BY CIS-4- DECANOYLCARNITINE: IS L-CARNITINESUPPLEMENTATION PROTECTIVE TO MCAD DEFICIENT PATIENTS?
Moacir Wajner 1,2 *, Cristiane Cecatto 1 , Janaína Camacho da Silva 1,
Alessandro Wajner 1 ,Kálita dos Santos Godoy 1 ,Rafael Teixeira Ribeiro 1,
Alexandre Umpierrez Amaral 1
1 Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
2 Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
*email@example.com Tel: +55 51 99777161
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is biochemically characterized
by predominant tissue accumulation of octanoic (OA), decanoic (DA) and cis-4- decenoic (cDA) acids, as well as by their carnitine by- products. Untreated patients present episodic encephalopathic crises with coma and seizures and biochemical liver alterations, whose pathophysiology is poorly known. We investigated the in vitro effects of OA, DA, cDA, octanoylcarnitine and decanoylcarnitine on important mitochondrial functions in rat brain and liver mitochondria. DA and particularly cDA increased state 4 respiration and diminished state 3 and uncoupled respiration, as well as RCR in both tissues, suggesting that these fatty acids behave as uncouplers and as metabolic inhibitors of oxidative phosphorylation. DA and cDA also markedly decreased mitochondrial membrane potential (Δψm), NAD(P)H content and capacity to retain Ca2+ in brain and liver, with the most pronounced effects of cDA. These biochemical alterations were totally prevented by the classical inhibitors of mitochondrial permeability transition (mPT) CsA and by ruthenium red in Ca2+-loaded mitochondria, indicating the involvement of mPT and the importance of mitochondrial Ca2+ concentrations, respectively. cDA also induced lipid peroxidation in brain and liver and increased hydrogen peroxide formation in brain mitochondria, indicating that lipid oxidative damage/oxidative stress may underlie some of the cDA-induced observed effects. Interestingly, octanoylcarnitine and decanoylcarnitine did not alter any of the tested parameters, suggesting no toxicity of these compounds. Our results suggest that DA and more prominently cDA impair important brain and liver mitochondrial functions, possibly contributing to the neuropathology and liver alterations of MCAD deficiency and that carnitine supplementation inducing medium chain acylcarnitine formation may be beneficial to the patients.
Financial support: We thank PROPESQ/UFRGS, FAPERGS and CNPq.
IDENTIFIYING PATIENTS WITH VLCAD DEFICIENCY PRESENTING WITH AN ABNORMAL NBS USING INTEGRATED METHODS
Rossana Sanchez MD, Rani H. Singh PhD, Michael J. Gambello MD, PhD,
Angela Wittenauer RN, Kristen Narlow RD, Patricia Hall PhD, Hong Li MD, PhD
Emory University Department of Human Genetics, Atlanta, GA
Corresponding Author: Hong Li, MD PhD Hong.firstname.lastname@example.org 4047788529
Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is the most common disorder of longchain fatty acid oxidation. It may present from the newborn period to adulthood
with hypoketotic hypoglycemia, cardiomyopathy or rhabdomyolysis.
After a positive newborn screen (NBS) suggestive of VLCCADD, the current follow
up algorithm recommends doing an acylcarnitine profile (ACP). ACP can be normalized
in the well fed state and may be uninformative. The aim of this study is to evaluate
the utility of using integrated methods including biochemical, molecular and functional studies to help determine true positives.
Methods: We retrospectively reviewed the medical records of 56 cases with a positive NBS consistent with VLCADD, who were seen in Metabolism Clinic at Emory, starting on January 1st of 2007 to date in Georgia.
Results: 32/56 patients had abnormal ACP. All patients had gene sequencing
and 53 variants were identified. However, 33 variants were classified as VOUS. 30/56 patients had deletion/duplication analysis and none were positive. Most patients did
not have parental studies done. After biochemical and molecular studies, 10/56 patients still had inconclusive results. Of these 3 had oxidation probe assay in fibroblasts, two were normal and the other had a mild abnormality.
Conclusion: We suggest that if ACP is not conclusive of a diagnosis of VLCADD, a systematic workup should be performed and the cumulative data including biochemical, molecular
and functional studies should be conditionally essential to confirm the diagnosis.
DELINEATING THE PATIENT POPULATION WITH MEDIUM CHAIN ACYL-COA DEHYDROGENASE DEFICIENCY (MCADD):BRITISH COLUMBIA CHILDREN’S HOSPITAL (BCCH) CLINIC EXPERIENCE
Kathleen Duddy RN, MSN, CPNP (P) 1 , Ramona Salvarinova MD, FRCPC, FCCMG 1* , Graham Sinclair PhD FCCMG 2 , Sylvia Stockler MD, PhD, MBA, FRCPC 1
1 Division of Biochemical Diseases, Department of Pediatrics BC Children’s Hospital, Vancouver Canada
2 Department of Pathology and Laboratory Medicine, BC Children’s Hospital, Vancouver, Canada
Keywords: Medium chain acyl-CoA dehydrogenase deficiency, l-carnitine, free carnitine, newborn screening, variants.
Background: Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is a rare genetic disorder, and the most common fatty acid oxidation disorder occurring 1 in 12000 births
in British Columbia (BC). MCADD has been part of the new born screening (NBS) program
in BC since 2002. Following confirmation of the newborn screening for MCADD, targeted genetic testing for the common two mutations in the ACADM gene is performed. Full gene sequencing is undertaken for patients where one or no common mutation is detected.
We currently follow 50 patients with MCADD, age 4 weeks to 14 years. While initially
all MCADD patients were started on L-Carnitine at birth, the practice was changed
in 2013 supplementing only patients with below the normal free carnitine levels.
Methods: Chart review was performed and information on several parameters was obtained: molecular results, patients started on L-carnitine at birth, carnitine supplementation including dosage and free carnitine levels.
Results: The clinic follows 50 patients; 28 are homozygous for the common c.985A>G variant, and 4 patients are compound heterozygous for the c.985A>G and c.199T>G variant.
Currently 37 patients are supplemented with L-Carnitine; dose ranging from 13 to 105 milligram/kilogram/day (mean of 43 milligram/kilogram/day). 7 patients were not started
on L-carnitine at birth and maintained normal free carnitine level on follow up. With the change in practice 18 patients stopped L-carnitine, 11 of which required restarting supplementation.
Summary: The MCADD patients differ in their need for carnitine supplementation.
Further delineation of factors influencing this, including genotype correlation will
* ASSESSMENT OF MITOCHONDRIAL BIOENERGETICS INFATTY ACID OXIDATION DEFICIENT FIBROBLASTS
Bianca Seminotti 1*, PhD, Al-Walid Mohsen 1 , PhD, Guilhian Leipnitz 1 , PhD, Anuradha Karunanidhi 1 , MSc, Vera Y. Roginskaya 2 , MSc, Bennett Van Houten 2 , PhD, Peter Wipf 3 , PhD, Jerry Vockley 1 , MD, PhD.
1 Division Medical Genetics, Department of Pediatrics, University of Pittsburgh,
Pittsburgh, PA, USA
2 Department of Pharmacology and Chemical Biology, University of Pittsburgh,
Pittsburgh, PA, USA
3 Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA
email@example.com, +1 (412) 692-9133
The mitochondrial fatty acid β-oxidation (FAO) pathway is an essential supplier of acetyl- CoA, the major source of reducing equivalents that drives ATP synthesis. Inherited defects have been described for most of the genes of FAO proteins, including medium-chain acyl- CoA dehydrogenase (MCAD) and very long-chain acyl-CoA dehydrogenase (VLCAD) deficiencies. These are characterized by hypoglycemia in both disorders, and rhabdomyolysis and cardiomyopathy in VLCAD deficiency. However, cellular pathophysiology leading to these problems remains unclear.
In this study, MCAD and VLCAD deficient fibroblasts were cultured in the absence
of glucosefor 48-72 h to shift cellular metabolism to fatty acids as their energy source. Oxygen consumption rate was monitored by a Seahorse XF analyzer. Mitochondrial mass
and superoxide production were examined by flow cytometry. ATP production was measured by luminometry and protein immunocontent by western blotting.
MCAD and VLCAD deficient cells showed decreased basal respiration, reserve capacity
and ATP production. We also observed absence of VLCAD protein, increased VDAC1 protein and a decrease of the complex I subunit ND6 in VLCAD deficient fibroblasts. Superoxide production was increased in VLCAD deficient fibroblasts, which was prevented by the mitochondrially targeted ROS scavenger JP4-039, but not by bezafibrate and N- acetylcysteine. Mitochondrial mass was increased in both cell lines.
Our findings identify a secondary decrease respiratory chain function in MCAD and VLCAD deficient fibroblasts that may play a role in increasing cellular pathophysiology beyond that expected on the basis of reduction of fatty acid oxidation. These pathophysiological manifestations suggest new possibilities for therapeutic strategies for FAO disorders.
TRIHEPTANOIN (UX007) TREATMENT IN PATIENTS WITH LONG-CHAIN FATTY ACID OXIDATION DISORDERS (LC- FAOD) AND CARDIOMYOPATHY
J Vockley, J Charrow, J Ganesh, M Eswara, GA Diaz, E McCracken, R Conway,GM Enns,
R Wang, DL Marsden
Objectives: The most severe LC-FAOD phenotype may present in infancy, when energy demands are high. Symptoms include cardiomyopathy, arrhythmia, hypoglycemia, and hepatic dysfunction. Morbidity and mortality are high despite detection by newborn screening (NBS) and early intervention with standard treatment, including supplementation with even medium chain triglycerides (MCT). Triheptanoin (UX007) is an investigational 7-carbon odd medium chain triglyceride, shown in animal studies to be both ketogenic and gluconeogenic,
by providing anaplerotic substrate for the depleted TCA cycle.
Design and Methods: Triheptanoin was provided through expanded access programs
for 8 infants and 2 older patients with cardiomyopathy. All were previously treated
with MCT. We reviewed data provided by the treating physicians.
Results: Ten patients with LC-FAODs presented with cardiomyopathy in infancy or childhood, despite treatment with MCT. Eight were detected by NBS. Seven were treated with triheptanoin (4g/kg) on emergency protocols (FDA eINDs) and 3 in existing Early Access programs. Improvement occurred within 48 hours in 2 patients near death. Seven were successfully weaned from support and remain stable; a moderately severe patient with declining ventricular function did not require acute support. Two patients died: 1 developed pericardial effusion, bradycardia and cardiac arrest after treatment for almost 4 weeks; 1 died of sepsis,
with normal heart function. Adverse events were primarily GI distress; 7 patients remain clinically well; 1 withdrew from treatment due to GI distress.
Conclusion: These data demonstrate potential therapeutic benefit of triheptanoin treatment
in patients with LC- FAOD cardiomyopathy. Further studies are warranted to confirm these initial promising findings.
*10 YEAR-FOLLOW- UP IN PATIENTS WITH LONG-CHAIN 3-HYDROXY ACYL COADEHYDROGENASE DEFICIENCY (LCHADD)
TREATED WITH HEPTANOATE (C7)
Manuela Zlamy1 , Karin Pichler 1 , Miriam Michel 1 , Sabine Scholl-Bürgi 1, Daniela Karall 1
Department of Pediatrics I, Inherited Metabolic Disorders, Medical University of Innsbruck, Austria
Corresponding author: Manuela, Zlamy, MD PhD, Department of Pediatrics I, Inherited Metabolic Disorders Medical University of Innsbruck, Innsbruck, Austria
Email: firstname.lastname@example.org Phone: +0043 512 504 23600
Long-chain 3-hydroxy acyl CoA dehydrogenase deficiency (LCHADD) is an autosomal recessive disorder. In patients with LCHADD the accumulation of toxic intermediates
of β- oxidation causes either immediate symptoms likehypoketotic hypoglycaemia as well
as long- term complications. Therapy with heptanoate (C7) utilizes the anaplerotic effect
of this substance, wherethe allocation of an odd-carbon numbered substrate for the citric acid cycle and the electron transport chain enhances ATP production.
We retrospectively evaluated clinical and biochemical parameters of 3 patients treated with heptanoate and a fat- defined diet over a time period of almost 10 years. All patient have confirmed LCHADD and are homozygous for the common mutation c.1528G>C.
The treatment dose of heptanoate equates 0.6-0.8 g/kg/d. Patients treated with heptanoate appeared clinically more stable than before therapy. Hospitalization rates and days
of hospitalisations decreased in all 3 patients after initialization of therapy. Patient 1 showed no more CK peaks after heptanoate was started. Patient 2 still showed episodes
of rhabdomyolysis but the associated CK concentrations were lower than before.
Patient 3 showed laboratory signs of hepatopathy during infections, without any significant changes of the CK concentrations.
Our data shows, that with ongoing age patients treated with heptanoate clinically stabilize. However, it is unclear if this stabilization is due to the anaplerotic effect of heptanoate or less infection rates with increasing age. Furtherprospective placebo-controlled studies as well
as development of in-vitro and in-vivo test models are needed to evaluate the exact impact
of heptanoate supplementation and to clarify the cellular mechanisms in the use of heptanoate.
Reference: Karall D, et al. Clinical outcome, biochemical and therapeutic follow-up
in 14 Austrian patients with Long- Chain 3-Hydroxy Acyl CoA Dehydrogenase Deficiency (LCHADD). Orphanet Journal of Rare Diseases. 2015; 10:21.
Travel Grants have been awarded see the Home page and the updated Agenda.
2017 INFORM Symposium Call for Abstracts
The INFORM conference planning committee welcomes your abstracts for display and presentation at this year's 2017 INFORM symposium.
The submission period is February 1, 2017 - June 7, 2017.
Abstracts are not to exceed 550 words,(excluding title, authors affiliations,charts,graphs and tables) spaces are not counted as a word or character. Please submit your abstract by forwarding the PDF to email@example.com . Place the Name of your abstract in the Subject line of the email.
Abstract Template (please format your PDF accordingly)
Notification: 8 Abstracts will be chosen to be presented at INFORM 2017, Travel Grant recipients will be notified by June 27, 2017.
Junior Investigators will receive first consideration for travel grant awards.
THE USE OF MASSIVE ANABOLIC THERAPY TO RESCUE AN INFANTWITH HEART FAILURE AND VLCAD DEFICIENCY
Amy Kritzer, MD, Ed Neilan, MD PhD, Anne O’Donnell, MD PhD, Monica Wojcik, MD,
Francis Rohr, RD, Stacey Tarrant, RD, Wen-Hann Tan, BMBS and Gerard Berry, MD Boston Children’s Hospital, Boston, MA
Email: firstname.lastname@example.org Phone: 978-877- 6573
This patient was born at 34 weeks with birth weight of 1835 grams. Newborn screen
showed C14:1 level of 2.99 μmol/L (cutoff 0.8 μmol/L). ACADVL gene sequencing revealed
2 mutations confirming VLCADD diagnosis. Physical examination, CK and EKG/echocardiograms were normal. The infant was placed on Enfaport®. At 2½ months,
she had emesis requiring hospitalization and intravenous glucose. Due to persistent intermittent emesis at 4 months she was switched to Lipistart®, but emesis persisted.
At six months she was admitted to the ICU because of increased emesis and poor feeding. She received D10NS at 1.5X maintenance. The physical examination revealed lethargy and poor perfusion. An echocardiogram showed LV dilatation, systolic dysfunction and mitral regurgitation. The LV ejection fraction was 0.45 but by six days was 0.30. She was treated
for heart failure with furosemide and milrinone. High concentrations of intravenous glucose (GIR>15) were administered. Innovative therapy with triheptanoin at 2 grams/kg/day on was started on day 6. By 17 days the LV-ejection fraction was 0.56. Attempts to wean off the high glucose infusion were associated with elevations of CK. G-J tube was placed due to persistent emesis on triheptanoin. Consequently, the parents requested that the triheptanoin
be discontinued. The patient was discharged at 9 months of age on MCT oil. She was readmitted to the ICU at 13 months due to RSV. On admission she had a normal LV ejection fraction (0.54). A foot wound developed secondary to IV infiltrate, and her condition deteriorated. She subsequently died following a cardiopulmonary arrest.
*IMPAIRED ANAPLEROSIS AFTER EXERCISE IN MURINEVERY LONG-CHAIN ACYLCOA DEHYDROGENASE DEFICIENCY (VLCAD-/- )IS IMPROVED WITH TRIHEPTANOIN CHOW
Garen Gaston, MS 1, Jon A. Gangoiti, MS 2, Bruce A. Barshop, MD, PhD 2 ,
Shelley Winn, PhD 1 , Cary O. Harding, MD 1 , Melanie B. Gillingham 1
1 Department of Molecular & Medical Genetics at Oregon Health & Science University, Portland, Oregon, USA,2Department of Pediatrics, Genetics Division, University of California San Diego, La Jolla, California, USA.
Odd-chain fatty acid supplementation has been suggested as a way to increase citric acid cycle intermediate (CACi) pools and energy metabolism in subjects with long-chain FAO disorders but the evidence for CACi depletion in these disorders has not been conclusively demonstrated. We exercised murine VLCAD-/- at 60% VO2 max to exhaustion on a treadmill and exercised wild type (WT) animals similarly and measured CACi in cardiac tissue by stable-isotope dilution targeted metabolomics after homogenization and extraction with ice-cold methanol. (AB Sciex API 4000 tandem mass spectrometer). WT mice increased cardiac malate and citrate concentrations with exercise but VLCAD-/- animals did not suggest impaired anaplerosis with high-energy demands of exercise. WT and VLCAD-/- animals chow were then fed MCT or Triheptanoin at 30% of total energy for 4 weeks. Animals were exercised at 60% VO2 max for 60 minutes on a treadmill and CACi was measured in cardiac tissue as described above. There was no difference in citrate concentrations but malate concentration in cardiac tissue was higher in both WT and VLCAD-/- animals fed Triheptanoin compared to animals
`fed MCT. These observations suggest that a Triheptanoin diet likely induces an anaplerotic effect. Funded by Ultragenyx Inc.
*RIBOFLAVIN-RESPONSIVE AND NON-RESPONSIVE MUTATIONS IN THEFAD SYNTHASE GENE CAUSE MULTIPLE ACYL-COA DEHYDROGENASE AND COMBINED RESPIRATORY CHAIN DEFICIENCY
Signe Mosegaard, BSc, Aarhus, Denmark, Rikke Olsen, Eliska Holzerova,
Teresa A. Giancaspero, Veronika Boczonadi, Maria Barile and Holger Prokisch
Email: email@example.com Phone: +45 20835507
Riboflavin is an essential vitamin and precursor to FMN and FAD, essential cofactors
to numerous dehydrogenases involved within cellular metabolism, antioxidant defense, protein folding and apoptosis. Riboflavin is transformed to FMN by riboflavin kinase (RFK)
and to FAD by FAD synthase (FADS). The FADS protein, encoded by FLAD1, consists
of a molydopterin-binding domain (MPTb) in the N-terminus and a phosphoadenosine
5’-phosphosuldate (PAPS) reductase domain responsible for the synthesis of FAD in the
The biochemical profile of riboflavin deficiency is elevated multiple acyl carnitines and organic aciduria that resembles the biochemistry of Multiple Acyl-CoA Dehydrogenation Deficiency (MADD) caused by defects in the electron transfer flavoprotein genes that support mitochondrial acyl-CoA dehydrogenation reactions. In recent years the number of MADD associated genes have increased by the identification of defects in genes responsible for transport of riboflavin or mitochondrial FAD transport, yet the genetic basis of many cases remains unexplained.
In this multicenter study we present patients from seven unrelated families suffering from MADD and respiratory chain deficiencies, caused by mutations in FLAD1. In four families
we identified biallelic frameshift variants in the MPTb domain and we would expect
a non-functional PAPS domain and thereby no FAD synthesis, but surprisingly we found FAD synthesis. By using protein mass spectrometry and RNAseq analysis we identified isoforms
of FLAD1, only encoding the PAPS domain and with the ability to produce FAD. Three patients with riboflavin-responsive disease all harbored missense mutations within the PAPS domain. In vitro reconstitution with FAD rescued the stability of one of these mutant proteins.
Please note that this research project has been accepted as an articlein American Journal
of Human Genetics.
* NORMAL INSULIN SENSITIVITY IN A PATIENT WITH VERY LONG-CHAIN ACYLCOADEHYDROGENASE (VLCAD) DEFICIENCY DURING A HYPERINSULINEMIC-EUGLYCEMICCLAMP (HEC) WITH AN INTRALIPID INFUSION
Melanie B. Gillingham 1 , PhD, Julie M. Martin 1 , MS, Eric Baetscher, BS 2 ,
William Rooney PhD 2 , Cary O. Harding, MD 1 , Jonathan Q. Purnell, MD 3
1 Department of Molecular & Medical Genetics,
2 The Advanced Imaging Research Center, & 3The Knight Cardiovascular Institute at Oregon Health & Science University, Portland, Oregon, USA
Email: firstname.lastname@example.org Phone: 503-319- 2404
Controversy exists as to whether insulin resistance is due to impaired mitochondrial fatty acid oxidation (FAO) and subsequent buildup up of lipids and lipid byproducts (lipotoxicity),
or the result of excess FAO flux and intra-cellular accumulation of intermediate byproducts
of metabolism. To address this controversy, we have initiated a randomized, crossover study of subjects with VLCAD, long-chain 3- hydroxyacylCoAdehydrogenase (LCHAD) or medium-chain acylCoA dehydrogenase (MCAD) deficiencies and a matched healthy control group
who will receive infusions of either intralipids or a control solution of glycerol during
a hyperinsulinemic-euglycemic clamp (HEC) to measure insulin sensitivity. We have completed both glycerol-control and intralipid HECs in a control subject and an intralipid HEC in a subject with VLCAD deficiency. Insulin sensitivity and respiratory quotient (RQ = 0.7) was markedly lower in the control subject during intralipid infusion compared to glycerol-control infusion (RQ =0.87) in association with a 31% increase in soleus lipid content. In contrast, the subject with VLCAD deficiency remained insulin sensitive during the intralipid co-infused HEC with
a RQ =0.85 in association with a 15% increase in soleus muscle lipid content. These data are consistent with the hypothesis that a block in long-chain FAO protects against intralipid-induced insulin resistance. However, free fatty acid uptake may be down regulated in FAO subjects compared to controls preventing lipotoxicity. Additional subjects will be studied
to test the relationships between mitochondrial dysfunction, impaired FAO, accumulation
of intracellular lipid, and insulin resistance in humans.
Funded by R01DK102813.
INTEROGATING THE EXOME FOR METABOLICALLY RELEVANT VARIANTS IN THESETTING OF UNDIAGNOSED DISEASE
Dennis Johnson 1, Christopher Adams 1, Katherine Chao 1, Ariane Soldatos 1, Lynne Wolfe 1, Hsu-Tso Ho 1, Charles Hoppel 2, William Gahl 1, David Adams 1
1 NHGRI, Undiagnosed Diseases Program, National Institutes of Health, Bethesda, MD,
2 Case Western Reserve University, Cleveland, OH.
The NIH Undiagnosed Diseases Program (UDP) evaluates patients with illnesses that remain undiagnosed despite extensive medical investigations. Exome and genome analysis are undertaken on UDP patients with the objective of identifying rare Mendelian variants consistent with the patient’s primary underlying phenotype.
Commonly, several plausible variants are found associated with varying levels of bioinformatic evidence. In these cases, ancillary data sets including metabolic data such
as acylcarnitines may assist in variant prioritization by examining pathway associations,
even when metabolic diseases are not explicitly part of the differential diagnosis; most participants suspected of having a well described metabolic disease would likely have been identified prior to presenting to the UDP.
By analyzing exome data in the context of metabolic data and, conversely, examining metabolic data in the setting of comprehensive sequencing, we pursued more precise clinical correlations to assist us in recognizing primary and secondary disease associations.
The exome provided a complete metabolic genotype by which to assess the genetic contribution of a given metabolic profile, and metabolic profiles provided a functional phenotype through which pathway hypothesis could be generated and candidate genes identified.
We hypothesize that potentially revealing metabolic variants and subtle metabolic abnormalities may be identified using a combination of family-based controls, candidates from genomic studies and detailed patient phenotyping. We present a working strategy including disease segregation analyses for metabolic disease genes, patient-specific gene panels for non- segregation disease association analyses, and metabolic familial profile segregation analyses aimed at identifying primary, secondary and ancillary disease associated metabolic parameters.
Our case demonstrates that in CACT deficiency, hydroxylated acylcarnitines can be elevated, along with long chain acylcarnitines.
HYDROXYLATED ACYLCARNITINE CAN BE ELEVATED IN NEWBORNS WITHCARNITINE ACYLCARNITINE TRANSPORTER DEFICIENCY
Mari Mori, MD, MS, Ashley S. Naughton, MD, Briana Scott, MD, Queenie Tan, MD, PhD,
Surekha Pendyal, MSc, MEd, RD, Sarah P. Young, PhD, Loren Pena, MD, PhD Duke University Medical Center, North Carolina, United States
Corresponding Author contact information: Mari Mori; phone 919-613- 8313; Email:email@example.com
Carnitine acylcarnitine transporter (CACT) deficiency is a rare autosomal recessive fatty acid oxidation disorder. To date, 61 cases have been reported worldwide, mostly before the advent of expanded newborn screening. We report a term infant, firstborn to parents of Asian origin, with non-ketotic hypoglycemia, bradycardia, poor cardiac contractility, hyperammonemia, coagulopathy, and rhabdomyolysis at day of life 3. Ketone bodies, propionylglycine, and methylmalonic aciduria were not present in urine organic acid profile; however, there were medium chain dicarboxylic acids suggestive of fatty acid oxidation defect. Plasma amino acid profile was not consistent with a urea cycle defect. Newborn screening and plasma acylcarnitine profile had elevations of long-chain saturated, unsaturated, and hydroxylated species including C16-OH. Based on these biochemical results, the patient was provisionally diagnosed with either long-chain 3-hydroxyacyl- CoA dehydrogenase or mitochondrial trifunctional protein deficiency (Tables 1 and 2).
The patient’s cardiac issues and biochemical abnormalities responded to aggressive glucose infusion, carnitine supplement, and she was discharged on predominantly medium chain triglycerides-containing formula mixed with canola oil to supplement essential fatty acid, and carnitine supplement. No mutations were detected on the HADHA or HADHB genes. Molecular testing confirmed CACT deficiency based on in heterozygous pathogenic mutations
c.199-2A>C and c.199-10T>G in trans in the SLC25A20 encoding CACT. The infant, now
5 month-old, has had episodes of hyperammonemia during intercurrent illnesses. Gastric tube placement is planned due to poor feeding and weight gain.
Travel Grant Awardees 2016
* Bianca Seminotti, PhD
University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC
Rangos Research Center
* Marta Frigeni, MD
Post-Doctoral Fellow Medical Genetics/Pediatrics, University of Utah
* Alexandre Umpierrez Amaral, MSc PhD
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade
Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
* Dr. Manuela Zlamy, PhD
Universitätsklinik für Pädiatrie I Medizinische Universität Innsbruck
* A.J. Bakermans, PhD
Department of Radiology (Z0-180) Academic Medical Center - University of Amsterdam
* Signe Mosegaard, BSc
Research Unit for Molecular Medicine, Aarhus University and Aarhus University Hospital,
* Guilhian Leipnitz, Ph.D.
Division Medical Genetics, Department Pediatrics, University of Pittsburgh, Pittsburgh, PA,
USA Department of Biochemistry, Universidade Federal do Rio Grande do Sul,
Porto Alegre, RS, Brazil
* Garen Gaston, MS
Department of Molecular & Medical Genetics at Oregon Health & Science University,
Children’s Hospital of Pittsburgh/UPMC - Medical Genetics - One Children's Hospital Drive
Faculty Pavilion 1st Floor - 4401 Penn Avenue - Pittsburgh, PA 15224
Keith McIntire - Coordinator, INFORM - firstname.lastname@example.org
SHORT TERM OUTCOME OF LCHAD DEFICIENCY IDENTIFIED
BY NEWBORN SCREENING
Alvaro Serrano Russi MD 1
1 Stead Department of Pediatrics; Division of Medical Genetics; University of Iowa, Iowa City, IA, USA
E-mail: Alvaroemail@example.com Phone: 319-356- 2674
Patient was born at 38 weeks gestation with a birth weight of 2175 grams. APGAR scores
of 8 and 9. She was small for gestational age and was found to have persistently decreased body temperatures associated with blood glucose values of 34 mg/dL to 64 mg/dL but dropping to 50 mg/dL with minimal fasting interval. Newborn screening collected
at 24 hours showed elevations in C14-OH, C16-OH, C16:1-OH and C18-OH.
Liver function testing and abdominal right upper quadrant ultrasound were normal. Echocardiogram showed normal function. She was managed locally with frequent feeding and was started on a mixture of breast milk and formula containing 55% MCT oil. Confirmatory testing in the first week of life showed elevations of C16 and C18
3- hydroxyacylcarnitine with an initial free carnitine of 20 nmol/mL. Urine organic acids showed hypoketotic C6-C10 dicarboxylic aciduria and a 3-hydroxydicaboxylic aciduria. Molecular genetic testing showed the homozygous c.1528G>C mutation in HADHA.
No mutation was found in HADHB.
At 5 months with acute illness and decreased oral intake, she had significantly elevated transaminases and CPK (AST: 514 U/L; ALT: 213 U/L; CPK: 8503 U/L) decreasing over five days of illness while following emergency protocol.
Baseline ocular exam at 6 months did not show evidence of retinopathy. Most recent clinic visit at 8 months showed she had recovered and was developmentally normal. Solid food was added on at 5-6 months of age monitoring levels of hydroxylated acyl carnitines
and essential fatty acids. Follow up of hydroxylated long chain acyl carnitines over time
* MITOCHONDRIAL ENERGY METABOLISM AND REACTIVE OXYGEN SPECIESLEVEL DISRUPTION IN ACAD9- DEFICIENT FIBROBLASTS
Guilhian Leipnitz, PhD 1,2*, Al-Walid Mohsen, PhD 1 , Anuradha Karunanidhi, MS 1,
Bianca Seminotti, PhD 1, Vera Y. Roginskaya, MS 2 , Bennett Van Houten, PhD 2 ,
Jerry Vockley, MD, PhD 1 .
1 Division Medical Genetics, Department Pediatrics, University of Pittsburgh,
Pittsburgh, PA, USA
2 Department of Biochemistry, Universidade Federal do Rio Grande do Sul,
Porto Alegre, RS, Brazil
3 Department Pharmacology and Chemical Biology, University of Pittsburgh,
Pittsburgh, PA, USA
firstname.lastname@example.org; phone: +1 412 692-91XX
Background: Acyl-CoA dehydrogenase 9 (ACAD9) is a homodimeric flavoprotein reported
to carry out a dual role catalyzing the first step in long-chain fatty acid b-oxidation and acting as an assembly factor for mitochondrial respiratory chain complex I. Individuals with complex I deficiency having mutations in ACAD9 gene present with progressive encephalomyopathy, recurrent Reye syndrome, and cardiomyopathy that can be fatal.
Objective: Evaluating the effect of ACAD9 deficiency on mitochondrial bioenergetics and reactive oxygen species production in fibroblasts of an ACAD9-deficient patient.Methods: Fibroblasts were cultured in medium with or without glucose to assess the ability of ACAD9 deficient cells to accommodate the shift of energy source from glucose and the effect of such shift on oxidative phosphorylation and ATP synthesis. We measured ACAD9 protein, oxygen consumption, mitochondrial membrane potential and mass, ATP production, and superoxide generation using western blotting, Seahorse flux analyzer, immunostaining, fluorimetry,
Results: Western blots demonstrated no significant alterations on ACAD9 protein content
in patient cells cultured in the absence or presence of glucose. Seahorse analyzer showed decreased basal respiration and reserve capacity in both media. Fluorescence staining
and luminometry demonstrated increased superoxide production, decreased mitochondrial membrane potential and ATP production in the presence or absence of glucose. However, mitochondrial mass was increased only in medium without glucose.
Conclusion: Our findings show that fibroblasts with ACAD9 deficiency have mitochondrial dysfunction characterized by bioenergetics disruption and increased superoxide generation, which may contribute to the pathophysiology underlying the symptoms observed
in ACAD9-related complex I deficiency.
The following Abstracts were selected for INFORM 2016
and were displayed during the Poster Session
MAGNETIC RESONANCE IMAGING AND SPECTROSCOPY OF THE LONG-CHAIN FATTY ACID β-OXIDATION DEFICIENT HEART
Adrianus J. Bakermans PhD 1, Jeannette C. Bleeker MD 2,3, Paul de Heer MSc 4 ,
Aart J. Nederveen PhD 1 , Ronald J.A. Wanders PhD 3 , and Gepke Visser MD PhD 2 .
1 Radiology, Academic Medical Center, University of Amsterdam, Amsterdam,
2 Paediatric Gastroenterology and Metabolic Diseases, Wilhelmina Children’s Hospital, UMC Utrecht, Utrecht, The Netherlands;
3 Laboratory Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
4 C.J. Gorter Center for High Field MR, Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands.
email@example.com - T: 0031 20 566 47 91
Cardiomyopathy can be a serious clinical complication in inborn errors of long-chain fatty acid β-oxidation (lcFAO). Underlying disease mechanisms are poorly understood and quantitative assessments are lacking, hampering evidence-based treatment. Potential contributors
to cardiomyopathy development include the toxic deposition of lipid intermediates and/or
a disturbed energy homeostasis. Using in vivo magnetic resonance imaging (MRI) and spectroscopy (MRS), we previously quantified left-ventricular dysfunction and hypertrophy, myocardial triglyceride accumulation and energy shortage in fasted long-chain acyl-CoA dehydrogenase knock- out mice.1,2
These MR methods are non-invasive and non-ionising, and - although technically challenging - can be exploited for investigations of the human heart. Our aim is to translate our preclinical animal studies into applications for investigations of human lcFAO disorders.
Proton MRS (1H-MRS) and MRI data (figure) acquired in two lcFAO-deficient patients (very long-chain acyl-CoA dehydrogenase deficiency, VLCADD) reveal elevated triglyceride levels and
a higher left-ventricular mass, which is indicative of mild hypertrophy. Ejection fraction was normal, but may become compromised during metabolic stress situations such as exercise
Ongoing work focusses on acquiring quantitative readouts of myocardial energy homeostasis (phosphocreatine, ATP) with phosphorus-31 MRS, and more detailed assessments of left-ventricular function (torsion, strain) through tagging MRI that may reveal subtle alterations prior to clinical symptoms cardiomyopathy.
* EXPERIMENTAL EVIDENCE THAT MONOCARBOXYLIC LONG-CHAIN HYDROXY FATTY ACIDS ACCUMULATING IN MTP AND LCHAD DEFICIENCIES MARKEDLY DISRUPT MITOCHONDRIAL BIOENERGETICSIN RAT SKELETAL MUSCLE
Alexandre Umpierrez Amaral 1 *, Cristiane Cecatto 1 , Kálita dos Santos Godoy 1
Ana Paula de Abreu Lopes 1 , Janaína Camacho da Silva 1 , Moacir Wajner 1,2
1 Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde,
Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
2 Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
*email: firstname.lastname@example.org Tel: +55 51 98378489
We studied the effects of the major monocarboxylic 3-hydroxylated fatty acids (LCHFA) accumulating in these disorders, namely 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, on important mitochondrial functions in rat skeletal muscle since muscular symptoms and recurrent rhabdomyolysis affect patients with mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxy- acyl-CoA dehydrogenase (LCHAD) deficiencies. These LCHFA markedly increased resting (state 4) respiration, decreased ADP-stimulated (state 3), uncoupled respiration and the respiratory control ratio (RCR). Similar effects were provoked
by 3HPA in permeabilized skeletal muscle fibers, an integrated cellular system, validating
the results obtained in isolated mitochondria. 3HTA and 3HPA also markedly decreased mitochondrial membrane potential, NAD(P)H content and Ca2+ retention capacity in Ca2+- loaded skeletal muscle mitochondria. These effects were totally prevented by the classical mitochondrial permeability transition (mPT) inhibitors cyclosporin A and ADP, as well as by ruthenium red, a mitochondrial Ca2+ uptake blocker, indicating the involvement of mPT and the participation of Ca2+ in 3HPA-induced deleterious effects. Mitochondrial membrane fluidity was also increased by 3HPA probably contributing to the nonselective permeabilization provoked by the LCHFA. In contrast and of interest, the dicarboxylic analogue of 3HTA, 3HTDA, was not able to alter any of the tested parameters, suggesting a selective action for the monocarboxylic LCHFA. These data indicate that the major fatty acids accumulating in MTP
and LCHAD deficiencies behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and mPT inducers in skeletal muscle. It is proposed that disturbance
of mitochondrial homeostasis may be involved in the muscular symptoms and episodes
of rhabdomyolysis characteristic of patients affected by these diseases
Financial support: We thank PROPESQ/UFRGS, FAPERGS and CNPq.
International Network for Fatty Acid Oxidation Research and Management