Homozygous missense mutation in BOLA3 causes ... - Springer Link

J Inherit Metab Dis (2013) 36:55–62 DOI 10.1007/s10545-012-9489-7

ORIGINAL ARTICLE

Homozygous missense mutation in BOLA3 causes multiple mitochondrial dysfunctions syndrome in two siblings Tobias B. Haack & Boris Rolinski & Birgit Haberberger & Franz Zimmermann & Jessica Schum & Valentina Strecker & Elisabeth Graf & Uwe Athing & Thomas Hoppen & Ilka Wittig & Wolfgang Sperl & Peter Freisinger & Johannes A. Mayr & Tim M. Strom & Thomas Meitinger & Holger Prokisch

Received: 7 February 2012 / Revised: 22 March 2012 / Accepted: 10 April 2012 / Published online: 5 May 2012 # SSIEM and Springer 2012

Abstract Defects of mitochondrial oxidative phosphorylation constitute a clinical and genetic heterogeneous group of disorders affecting multiple organ systems at varying age. Biochemical analysis of biopsy material demonstrates isolated or combined deficiency of mitochondrial respiratory chain enzyme complexes. Co-occurrence of impaired activity of the pyruvate dehydrogenase complex has been rarely

reported so far and is not yet fully understood. We investigated two siblings presenting with severe neonatal lactic acidosis, hypotonia, and intractable cardiomyopathy; both died within the first months of life. Muscle biopsy revealed a peculiar biochemical defect consisting of a combined deficiency of respiratory chain complexes I, II, and II+III accompanied by a defect of the pyruvate dehydrogenase

Communicated by: Shamima Rahman Tobias B. Haack, Boris Rolinski and Birgit Haberberger authors contributed equally. Electronic supplementary material The online version of this article (doi:10.1007/s10545-012-9489-7) contains supplementary material, which is available to authorized users. T. B. Haack : B. Haberberger : T. M. Strom : T. Meitinger : H. Prokisch (*) Institute of Human Genetics, Technische Universität München, Trogerstrasse 32, 81675 Munich, Germany e-mail: [email protected] T. B. Haack : B. Haberberger : J. Schum : E. Graf : T. M. Strom : T. Meitinger : H. Prokisch Institute of Human Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany

F. Zimmermann : W. Sperl : J. A. Mayr Department of Paediatrics, Paracelsus Medical University, Salzburg, Austria V. Strecker : I. Wittig Molekulare Bioenergetik, Zentrum der Biologischen Chemie, Goethe-Universität Frankfurt, Frankfurt am Main, Germany

B. Rolinski Elblandkliniken, Elblandkliniken Gmbh, Riesa, Germany

T. Hoppen Department of Paediatrics, Gemeinschaftsklinikum Koblenz-Mayen, Koblenz, Germany

B. Rolinski : U. Athing Department Klinische Chemie, Städtisches Klinikum München GmbH, Munich, Germany

P. Freisinger Department of Paediatrics, Städtisches Klinikum Reutlingen, Reutlingen, Germany

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J Inherit Metab Dis (2013) 36:55–62

complex. Joint exome analysis of both affected siblings uncovered a homozygous missense mutation in BOLA3. The causal role of the mutation was validated by lentiviralmediated expression of the mitochondrial isoform of wildtype BOLA3 in patient fibroblasts, which lead to an increase of both residual enzyme activities and lipoic acid levels. Our results suggest that BOLA3 plays a crucial role in the biogenesis of iron-sulfur clusters necessary for proper function of respiratory chain and 2-oxoacid dehydrogenase complexes. We conclude that broad sequencing approaches combined with appropriate prioritization filters and experimental validation enable efficient molecular diagnosis and have the potential to discover new disease loci.

caused by mutations in PDHB, PDHX, DLAT, DLD, PDP1, (Patel et al 2011) and recently identified mutations in TPK1 (Mayr et al 2011a, b) and LIAS (Mayr et al. 2011a, b). Only few individuals with a combination of these biochemical signatures, referred to as "multiple mitochondrial dysfunctions syndrome (MMDS1, MIM #605711, and MMDS2, MIM #614229)", have been reported to date (Seyda et al 2001). In these patients, microcell-mediated chromosome transfer located the genetic defect to chromosome 2p142p13 but the affected genes remained elusive for more than ten years and have been identified only very recently (Cameron et al 2011a, 2011b).

Clinical data Introduction Next-generation sequencing (NGS) is the method of choice for molecular diagnosis of disorders untraceable with conventional capillary sequencing due to the inability to prioritize few candidate genes. Reasons for this are genetic and clinical heterogeneity or the simple fact that a substantial portion of disease-causing genes is still unknown. An example for such a group of disorders are defects of the oxidative phosphorylation system (OXPHOS), affecting 1 in 5000 live births (Skladal et al 2003), being linked to more than 100 loci and associated with a wide spectrum of clinical manifestation at varying age (Munnich and Rustin 2001). Diagnosis is difficult and mainly relies on biochemical assessment of biopsy material showing isolated or combined deficiency of mitochondrial respiratory chain enzyme complexes. While mutations in both nuclear and mitochondrial DNA (mtDNA) encoded genes have been identified as the molecular genetic correlate of combined defects of the mitochondrial respiratory chain (Sasarman et al 2008), reduced activities of the pyruvate dehydrogenase complex (PDHc) are mainly due to mutations in the X-chromosomal PDHA1 (Lissens et al 2000), followed by a smaller number of cases Table 1 Biochemical analysis in individuals #49720 and #56712

CI-V, mitochondrial respiratory chain complex I-V; CS, citrate synthase; PDHc, pyruvat dehydrogenase complex; Enzyme activities are given relative to gram noncollagen protein. Italics and bold indicate abnormal value; N.A., not assessed.

Enzyme activities

CS CI CII+III CII CIII CIV CV PDHc

In this study, we used exome sequencing to investigate two siblings with MMDS associated with an early onset fatal course of the disease leading to death within the first year of life. The children (#49720, male and # 56712, female) were the first and the second child of healthy non-consanguineous parents. Family history did not indicate inherited neuromuscular or metabolic disorders. Both children showed a very similar clinical course. The boy was born at term after an uneventful pregnancy and delivery. Initial postnatal adaption was normal. In the first week of life feeding difficulties were noted. At the 17th day of life he had just regained his birth weight of 3350 g. An increased lactate level in plasma was noted (7.7 mmol/L, normal 0.5 – 2.2) when he was admitted to hospital due to his failure to thrive. He then developed progressive muscular hypotonia, respiratory insufficiency and severe lactic acidosis with lactate levels up to 32.6 mmol/L. Other laboratory abnormalities included increased plasma glucose (10.5 mmol/L, normal 3.9 – 6.1) as well as a slightly increased glycine concentration (533 μmol/L, normal 224–514) in amino acid analysis. Organic acids analysis in urine revealed elevated metabolites of the citric acid cycle (succinate, fumarate, malate, aconitic acid, citric acid) together with increased

In fibroblasts

In muscle

#49720

Reference range

#49720

#56712

Reference range

398 13 36 13 517 275 246 1.5

225-459 18-53 79-219 54-124 208-648 270-659 78-287 6.0-19.7

144 10.3 2.6 2.3 N.A. 114 N.A. 0.4

91 4.8 2.3 0.7 N.A. 44 N.A. 0.7

45-100 15.8-42.8 6.0-25.0 16-42.6 112-351 1.5-3.9


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excretion of lactate and pyruvate pointing to a mitochondrial disorder. The boy stabilized under parenteral nutrition and buffering with bicarbonate and application of water-soluble vitamins. Biochemical assessment of a skeletal muscle biopsy showed combined deficiency of respiratory chain complexes I, II, and II+III accompanied by a defect of the pyruvate dehydrogenase complex (Table 1). The same biochemical signature was also found in patient-derived fibroblasts. Moreover, quantification of fluorescent-labelled mitochondrial complexes by 2D BN/SDS-PAGE (Haack et al. 2010) showed a

clear decrease in complex I-containing supercomplexes (Fig. 1). Echocardiography performed at the age of 40 days indicated a hypertrophic cardiomyopathy of the left ventricle with and end-diastolic dimension (EDD) of the septum interventricularis of 11 mm (normal 2.3 – 5.1 mm), the left ventricular posterior wall of 13 mm (normal 2.5 – 4.7 mm) and a shortening fraction of 21 % (normal 28 – 44 %). Brain MRI performed at the age of 9 weeks demonstrated delayed myelination and T2-weighted bifrontal and biparietal hyperintense lesions supra- and periventricular as well as in the midbrain in

Fig. 1 Separation and quantification of fluorescent-labelled mitochondrial complexes by 2D BN/SDS-PAGE. 3D visualization of quantified mitochondrial membrane complexes from (a) control and (b) patient #49720. NHS-Fluorescein-labelled (Thermo) mitochondrial membrane fractions (isolated from 10 mg wet weight of fibroblasts) were resuspended in 20 μl solubilisation buffer (50 mM NaCl, 50 mM imidazole, 2 mM aminohexanoic acid, and 1 mM EDTA, pH 7), solubilized with 5 μl digitonin (20 %) and separated by 2D BN/SDS-PAGE. 2D gels were scanned with Typhoon 9400 scanner (GE Healthcare) and analyzed by DIA modul (Differential In-Gel Analysis) of the DeCyderTM 2D 7.0 software (GE Healthcare) for densitometric quantification of

fluorescence intensities of selected clearly visible protein spots. (c) Mitochondrial complexes were quantified by densitometry, normalized to porin complexes and expressed as percent of healthy control cells (two technical replicates). Silver stained 2D-gels from (d) control and (e) patient fibroblasts are shown for comparison. Rectangle shows a representative gel area of the 3D view in (a and b). Arrows indicate proteins used for quantification. Assignment of complexes: S, supercomplexes composed of respiratory chain complexes I, III, and IV; V, complex V or ATP synthase; III, complex III or cytochrome c reductase; IV, complex IV or cytochrome c oxidase; Phb1/2, prohibitin complex and P for porin complexes

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the formatio reticularis (Fig. 2). The clinical course showed a rapid neurological deterioration with seizures, severe muscular hypotonia, persistent lactic acidosis and finally death due to multi organ failure at the age of 3 months. The girl was born after uneventful pregnancy and delivery. Initial development was normal without feeding difficulties. At the 15th day of life, the girl was admitted to the hospital with respiratory insufficiency, gray skin, agitation and severe lactic acidosis (22.7 mmol/L, normal 0.5 – 2.2). Further analysis showed slightly elevated glycine (541 μmol/L, normal 224–514) in plasma and excessively elevated excretion of lactate and pyruvate in urine. Therapy was initiated with continuous intravenous application of bicarbonate and Trisbuffer, and parenteral nutrition with 4–5 g/kg body weight/day (20 %) fat. In addition, L-carnitine (3×100 mg/d, p.o.), biotin (20 mg/d, p.o.), thiamin (50 mg/d, i.v.), ubiquinone (60 mg/d, p.o.), and riboflavin (3×40 mg/d) was administred. The girl responded well to this therapy regimen and lactate levels decreased to 2.4 mmol/L but remained slightly elevated. Skeletal muscle biopsy revealed a combined respiratory chain defect of complex I, and II and a PDHc deficiency as observed in her brother (Table 1). In addition, complex IV was decreased. Hypertrophic cardiomyopathy was documented by echocardiography at the age of 75 days (EDD septum interventricularis 9 mm (normal 2.3 – 5.1 mm); left ventricular posterior wall 10 mm (normal 2.5 – 4.7 mm); shortening fraction 14 % (normal 28 – 44 %)). T2-weighted MRI showed hyperintense signals in the right capsula interna, bilateral in the tegmentum mesencephali, and diffuse white matter lesions. She developed progressive encephalopathy with extrapyramidal signs. The girl died at the age of 3 months.

Methods and results We used exome sequencing to identify the disease-causal variants in the two affected siblings. The exome of individual #49720 was sequenced as paired-end 54 bp reads using two


lanes of a flow cell on a Genome Analyzer IIx system (Illumina) following targeted enrichment of exonic sequences (SureSelect Human All Exon 38 Mb kit, Agilent). In individual #56712, the SureSelect Human All Exon 50 Mb Kit (Agilent) was used for in-solution capture of the exome followed by subsequent sequencing as 76 bp paired-end runs on a HisSeq2000 (Illumina). We achieved an average coverage of 107x and 128x in individuals #49720 and #56712, respectively; 87.7 % and 91.8 % of the exome were covered at least 20x (Supplementary Table 1) allowing a high-confidence variant call. Read alignment to the human reference assembly hg19 was done with BWA (version 0.5.8). SAMtools (version 0.1.7) was used for detection of single-nucleotide variants (SNVs) and small insertions and deletions (indels). As the biochemical signature of the disease is rare, we expected to find private variants and excluded variants present in 818 control samples from patients with unrelated phenotypes. Based on the autosomal recessive pattern of inheritance, we then focused on genes carrying compound heterozygous or homozygous variants. These sequential filtering steps left two genes shared by both individuals, MOGS and BOLA3, and only BOLA3 after filtering for genes encoding potential mitochondrial proteins (MitoP2 score >1 (Elstner et al 2009), Table 2). MOGS codes for glucosidase I (GCSI), which is localized in the endoplasmic reticulum and has been reported as the cause of a congenital disorder of glycosylation, type IIb (MIM 606056) (De Praeter et al 2000). The MOGS variant c.2336G>A (NM_006302) corresponds to a change of a moderately conserved amino acid residue, p.Arg779Gln, and its effect is judged to be "neutral" by PolyPhen2, accordingly. There was no experimental evidence for a mitochondrial localization of BOLA3. The prediction was based on its amino acid sequence (MITOPRED score 85.9, TargetP score for mitochondrial targeting peptide 0.764). The identified missense mutation in BOLA3, c.200T>A, p.Ile67Asn, affects an isoleucine fully conserved in 38 vertebrates down to Danio rerio, as well as in the nematode Caenorhabditis elegans Table 2 Variants identified in affected individuals by exome sequencing

Fig. 2 T2 weighted brain MRI performed at the age of 9 weeks. Arrows indicate hyperintense lesions (a) periventricular parietal and in the (b) midbrain in the formatio reticularis

Patient ID

#49720

#56712

Present in both

Synonymous NSV NSV not present in 818 controls >2 NSV / gene

8,761 8,058 201

11,015 10,824 294

6,985 6,264 72

8

16

Mitochondrial localization

1

1

2 (BOLA3, GCSI) 1 (BOLA3)

NSV 0 missense, nonsense, stop loss, splice site disruption, insertions, deletions; mitochondrial localization refers to proteins with a MitoP2score >1.0.


(Fig. 3). Concordant with a potential pathogenic role, both parents were heterozygous carriers. In order to confirm the deleterious impact of the BOLA3 variant, we performed complementation experiments in patient fibroblast cell lines to test for functional rescue of impaired enzyme activities. Human BOLA3 has two isoforms [NM_0013035505.1 and NM_212552.2 (Fig. 2)]. In contrast to isoform 1, isoform 2 lacks the third exon of BOLA3 resulting in two transcripts with alternative stop codons and more than 50 % of the sequence being different. The mutation identified in our patient is located in exon 3 and solely affects isoform 1. We therefore regarded isoform 1 to be associated with the observed phenotype and expressed the wildtype cDNA in patient fibroblast cells using a lentiviral vector following the manufacturer’s protocol (p.Lenti6.3/V5-TOPO Kit, version A10291, Invitrogen) (Danhauser et al 2011). Control and patient fibroblasts were seeded at 20,000 cells/well in 80 μl DMEM, followed by incubation at 37° C, 5 % CO2 for 24 h. Oxygen consumption rate (OCR) was measured using a XF96 extracellular flux analyzer (Seahorse bioscience) after adding oligomycin (1 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.4 μM) and rotenone (5 μM). Uncoupled complex I activity was calculated as rotenone-sensitive OCR showing a significant increase from 32 % to 82 % residual activity upon expression of BOLA3wt cDNA (Fig. 4). Immunohistochemical investigations of assembled complex II in fibroblasts by confocal microscopy demonstrated a clear reduction of SDH, which was restored after expression of BOLA3wt cDNA (Fig. 4). The BOLA3 orthologue in Saccharomyces cerevisiae has been postulated to play a role in the regulation of iron-sulfur (Fe-S) clusters (Li et al) which are present in all affected

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respiratory chain enzyme complexes. Impairment of Fe-S clusters biogenesis is able to explain the defect found in complex I, II, and II+III, while PDHc lacks Fe-S clusters. Lipoic acid functions as a coenzyme of the three mitochondrial dehydrogenases PDHc, α-KGDH and BCKDH. In PDHc it is covalently bound to the E2 and E3BP subunits. Lipoate synthetase, encoded by LIAS, contains two Fe-S clusters. We therefore tested the levels of lipoic acid in patient fibroblast by immunoblotting. This experiment showed a clear decrease in lipoic acid (Fig. 4), which was restored after expression of the BOLA3wt cDNA. Recently, mutations in LIAS (Mayr et al 2011a, b) have been reported to cause impaired PDHc function via reduced levels of lipoic acid providing an explanation for the observed PDHc defect.

Discussion Advances in NGS technology enable the efficient analysis of a number of candidate genes, the entire coding sequence or the entire genome. Sequencing of gene-panels like the "Mendelianome" (2993 known disease genes) (Bell et al 2011) or the MitoExome (1034 nuclear-encoded mitochondrial-associated genes and the mtDNA) (Tucker et al 2011), are designed to capture known disease variants or genes encoding proteins which are known or predicted to localize to mitochondria. In comparison to whole exome sequencing these restrictions can be more cost-efficient. However, novel disease genes or genes not predicted to cause mitochondrial disorders are missed. In our study, both, the disease-gene and the MitoCarta-based (Calvo et al 2012) approach would have missed the causal BOLA3 mutation. Unbiased sequencing of the whole exome or genome has a better chance to discover the genetic defect. However, the

Fig. 3 Gene structure of BOLA3 and position of the identified missense mutation

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Fig. 4

Complementation experiments in patient fibroblast cell lines confirm the pathogenic role of the BOLA3 variants. (a) Rescue of complex I (CI) defect by lentiviral-mediated expression of BOLA3wt cDNA in patient fibroblasts. Control and patient fibroblasts were seeded at 20,000 cells/well in 80 μl DMEM, and incubated at 37°C, 5 % CO2 for 24 h. Complex I activity was determined as rotenonesensitive oxygen consumption rates using the XF96 extracellular flux analyzer (Seahorse bioscience). Data shown are mean of >10 biological replicates ± s.d. *PA

p.Ile67Asn

#56712

f

15 days

3

c.200T>A

p.Ile67Asn

reported patient

m

4

11

c.123dupA

p.Glu42ArgfsX13

Clinical features

Feeding difficulties, recurrent metabolic crisis with lactic acidosis, elevated levels of serum glycine, hyperthropic cardiomyopathy, epileptic seizures, progressive encephalopahty Feeding difficulties, recurrent metabolic crisis with lactic acidosis, elevated levels of serum glycine hyperthropic, cardiomyopathy, progressive encephalopahty Epileptic seizures, developmental delay, dilated cardiomyopathy, elevated levels of serum and CSF glycine, lactic acidosis, progressive course, CI 30 %, CII 50 %, CIII 60 %, CIV normal, PDHc 5-10 %, OGDHc 30 %

f, female; m, male; CI-IV, mitochondrial respiratory chain complex I-IV; PDHc, pyruvat dehydrogenase complex; 2-OGDH, 2-oxo-glutaric acid dehydrogenase; AO, age at onset; CSF, cerebrospinal fluid.

impaired glycine metabolism contributes to the phenotype of mutant BOLA3. In summary, the peculiar constellation of biochemical defects subsumed as MMDS together with a fast progression of the disease warrants genetic testing of BOLA3. Elevated glycine levels might also be indicative yet not exclusive for BOLA3 mutations as they are also detected in primary forms of NKH or other genetic defects disrupting lipoic acid metabolism. BOLA3 is a poorly understood member of the mammalian BolA-like protein family that is conserved from prokaryotes to eukaryotes (Fig. 3). The BolA homologue Fra2 together with the monothiol glutaredoxins Grx3 and Grx4 have been shown to play a key role in iron regulation in Saccharomyces cerevisiae (Li et al 2010). In humans it can be speculated that BOLA3 is interacting with glutaredoxin 5, which is involved in biogenesis of 2Fe-2S and 4Fe-4S clusters (Cameron et al 2011a, b). Along this line, apart from preserved function of CIII, the pattern of the respiratory chain complex defects overlaps with their dependence on Fe-S clusters. The observed decrease in levels of lipoic acid suggests an impairment of the lipoic acid synthetase. Future studies have to elucidate the exact role of BOLA3 in the complex process of Fe-S cluster assembly and maintenance. Acknowledgements We are grateful to the patients and their family for their participation and especially to A. Huber, R. Hellinger and A. Löschner for their technical support. Web resources The URLs for data presented herein are as follows: MitoP2, http://www.mitop.de MITOPRED, http://bioapps.rit.albany.edu/MITOPRED/ TargetP, http://www.cbs.dtu.dk/services/TargetP/ Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/

Funding T.M. and H.P. were supported by the Impulse and Networking Fund of the Helmholtz Association in the framework of the

Helmholtz Alliance for Mental Health in an Ageing Society (HA215) and the German Federal Ministry of Education and Research (BMBF) funded German Center for Diabetes Research (DZD e.V.) and Systems Biology of Metabotypes grant (SysMBo #0315494A). H.P. was supported by the grant RF-INN-2007-634163 of the Italian Ministry of Health. T.M., P.F., and H.P. were supported by the BMBF funded German Network for Mitochondrial Disorders (mitoNET #01GM1113C). F.A.Z., W.S., and J.A.M. were supported by the GENOMIT project funded by the FWF (I 920‐B13) and the Vereinigung zur Förderung der pädiatrischen Forschung und Fortbildung Salzburg. I.W. was supported by the Bundesministerium für Bildung und Forschung (BMBF #01GM1113B; mitoNET – Deutsches Netzwerk für mitochondriale Erkrankungen) and by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 815, Project Z1 (Redox-Proteomics). T.B.H. was supported by the NBIA disorders association. The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors.

Conflict of interest None.

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