Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency

Transcription

1 Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency Tobias B. Haack 1,2,11, Katharina Danhauser 2,1,11, Birgit Haberberger 2,1, Jonathan Hoser 3, Valentina Strecker 4, Detlef Boehm 5, Graziella Uziel 6, Eleonora Lamantea 7, Federica Invernizzi 7, Joanna Poulton 8, Boris Rolinski 9, Arcangela Iuso 1, Saskia Biskup 5, Thorsten Schmidt 3, Hans-Werner Mewes 3,10, Ilka Wittig 4, Thomas Meitinger 1,2, Massimo Zeviani 7 *, Holger Prokisch 1,2 * 1 Institute of Human Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany 2 Institute of Human Genetics, Technische Universität München, Munich, Germany 3 Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Germany Research Center for Environmental Health, Neuherberg, Germany 4 Molecular Bioenergetics, Medical School, Goethe-Universität Frankfurt, Frankfurt am Main, Germany 5 CeGaT GmbH, Tübingen, Germany 6 Unit of Child Neurology, Neurological Institute Carlo Besta IRCCS Foundation, Milan, Italy 7 Unit of Molecular Neurogenetics, Neurological Institute Carlo Besta IRCCS Foundation, Milan, Italy 8 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, The Womens Centre, John Radcliffe Hospital, Oxford, UK 9 Städtisches Klinikum München GmbH, Department Klinische Chemie, Munich, Germany 10 Chair of Genome Oriented Bioinformatics, Center of Life and Food Science, Freising-Weihenstephan, Technische Universität München, Munich, Germany 11 These authors contributed equally to this work * Corresponding Authors: Holger Prokisch, Institute of Human Genetics, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str. 22, Munich, Germany, Phone: , Fax: Prokisch@helmholtz-muenchen.de Massimo Zeviani, Center for advanced diagnostics and research on Neurological Mitochondrial Disorders of Infancy and Childhood Bicocca Laboratories Department of Experimental Research and Diagnostics C. Besta National Institute of Neurology via Libero Temolo 4, Milano, Italy, Phone: , Fax: , Zeviani@istituto-besta.it

2 Supplementary Table 1 Distribution of the coverage of the targeted bases (mean coverage: 47.6-fold) Bases covered at least Bases Percent of target 10x 30,035, x 30,633, x 31,240, x 31,854, x 32,484, x 33,125, x 33,779, x 34,459, x 35,174, x 35,973, All bases 37,806,

3 Supplementary Table 2 Identification of candidate genes for complex I deficiency by exome resequencing Filter Number of SNVs / genes Total SNVs 14,167 Synonymous SNVs 3,921 NS/SSV 4,928 Novel NS/SSV 533 Homozygous NS/SSV 23 Heterozygous NS/SSV 510 Compound heterozygous NS/SSV 25 With predicted mitochondrial localization 1 Among the identified SNVs NS/SSV (nonsynonymous or splice acceptor and donor site variants) were subsequently filtered for novel variants and cellular localization under a recessive model.

4 Supplementary Table 3 Ratios of transitions (Ti) to transversions (Tv) and synonymous (SV) to non-synonymous variants (NSV) overall Transitions Transversions Ti:Tv SV NSV SV:NSV all 14,167 10,450 3, :1 3,921 4,500 1:1.15 known 12,537 9,559 2, :1 3,732 4,023 1:1.08 novel 1, : :2.52

5 Supplementary Table 4 Densitometric quantification of mitochondrial complexes in 2D BN/SDS-PAGE from human fibroblasts, expressed as percent of untreated control cells normalized to monomeric ATP synthase Complex Control cells (%) Patient cells (%) untreated transduced Supercomplex (s.d.) Independent experiments/ number of gels (10.2) 65.0 (29.1) (37.2) 5 / 8 3 / 6 2 / 4

6 Supplementary Note A: Case report Index Patient (I:A) and affected brother (I:B): A baby girl was born after an uneventful pregnancy and delivery on the 39 th + 6 d week of gestation, and showed after 24 h sudden and severe cardio-respiratory depression requiring assisted ventilation (intubation). Severe metabolic acidosis (ph 6.55, BE 28), normal blood glucose, and slightly elevated Ca 2+ in serum (6.3 mg/dl n.v. 3-6 mg/dl) were documented. After this episode that resolved within two days, anemia (Hb 10 gr/dl) required one transfusion on the 3 rd day after birth. After 20 days she suffered of a new episode of metabolic acidosis (ph 7.13, BE 17, Ca mg/dl, glucose 54 mg/dl). Echocardiography revealed left ventricle hypertrophy. No hyperparathyroidism was found, that could explain the increase of Ca 2+ in serum. Neurologically, she had severe reduction of spontaneous movements, muscular hypotonia and hyporeactivity. The EEG was characterized by severe reduction of recordable electric activity, but a brain echography was normal. After a brief period of improvement, the neurological conditions deteriorated, the body weight decreased, and she had persistent metabolic acidosis. The patient died at the age of 46 days. In skin fibroblasts, complex I was 39% of the normal mean, while the other complexes were normal, except for a slightly reduced ATPase (complex V) activity. In liver biopsy, complex I activity was undetectable, and that of complex V was 38.6% of the normal mean; in muscle, complex I activity was 9% of the normal control mean, and complex V was 52%. The patient s younger brother manifested at birth the same features as his sister, consisting of muscular hypotonia and heart hypertrophy with lactic acidosis. Analysis of urinary organic acids at one month of age revealed severe lactic aciduria but no dicarboxylic aciduria, which is the metabolic signature of fatty acid ß-oxidation disorders. The serum acyl carnitine and free fatty-acid profiles, analyzed recently, were also normal. Being aware of the rapid downhill course of her sister, we then undertook a vigorous, immediate correction of the acidosis and the administration of a multivitamin cocktail including carnitine 100 mg/kg/day, coenzyme Q 50 mg/day and riboflavin 50 mg/day. After one year the riboflavin treatment was increased to two times 50 mg/day. Patient I:B is now 5 years old, with no cognitive impairment and normal psychomotor development. The only problem reported by the parents is mild exercise intolerance; however, this patient, who is now 5 years old, is not objectively weak, with no sign of myopathy. A stable, functionally compensated, hypertrophy of

7 the left ventricle was recently documented by ultrasound examination. However, plasma lactate is persistently high (4 mm, n.v. < 1.8), but no metabolic decompensation has ever occurred after the first episode at birth. B: Free fatty acids and acylcarnitine analysis in patient I:B at the age of 5 years Medium and Long Chain Fatty Acid analysis from a blood sample by GC/MS (in µm): C8:0 - undetectable (n.v. 0-15) C10:1w=6 - undetectable (n.v. <1.0) C10:0-3 (n.v. 0-20) C12:1 - undetectable (n.v. <1.0) C12:0-16 (n.v. 0-50) C14:1w=9 - undetectable (n.v. <1.0) C14:1w=5-2 (n.v. <13) C14:0-80 (n.v ) C16:1w=9-12 (n.v. <45) C16:1w=7-127 (n.v ) C16: (n.v ) C18:0-477 (n.v ) Plasma acylcarnitine analysis by Electrospray tandem mass spectrometry (ESI- MS/MS; values given in µm): Free carnitine (n.v ) C2: (n.v. <7.00) C3: (n.v. <1.50) C4: (n.v. <0.60) C5: (n.v. <0.30) C6: (n.v. <0.15) C8: (n.v. <0.20) C10: (n.v. <0.20) C10: (n.v. <0.30) C12: (n.v. <0.15) C12: (n.v. <0.25) C14: (n.v. <0.12) C14: (n.v. <0.18) C14: (n.v. <0.22) C16: (n.v. <0.10) C16: (n.v. <0.22) C18: (n.v. <0.25) C18: (n.v. <0.20) C12-OH (n.v. <0.07) C14-OH (n.v. <0.07) C16-OH (n.v. <0.05) C18-OH (n.v. <0.05)