Bi-allelic Mutations in the Mitochondrial Ribosomal Protein MRPS2 Cause Sensorineural Hearing Loss, Hypoglycemia, and Multiple OXPHOS Complex Deficiencies
2018; Elsevier BV; Volume: 102; Issue: 4 Linguagem: Inglês
10.1016/j.ajhg.2018.02.012
ISSN1537-6605
AutoresThatjana Gardeitchik, Miski Mohamed, Benedetta Ruzzenente, Daniela Karall, Sergio Guerrero‐Castillo, Daisy Dalloyaux, Mariël van den Brand, Sanne van Kraaij, Ellyze van Asbeck, Zahra Assouline, Marlène Rio, Pascale de Lonlay, Sabine Scholl‐Buergi, David F.G.J. Wolthuis, Alexander Hoischen, Richard J. Rodenburg, Wolfgang Sperl, Zsolt Urbán, Ulrich Brandt, Johannes A. Mayr, Sunnie Wong, Arjan P.M. de Brouwer, Leo Nijtmans, Arnold Münnich, Agnès Rötig, Ron A. Wevers, Metodi D. Metodiev, Éva Morava,
Tópico(s)RNA and protein synthesis mechanisms
ResumoBiogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation. Biogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation. Mitochondria are essential organelles that harbor the oxidative phosphorylation system (OXPHOS), an ATP-producing system encompassing five multi-subunit enzymatic complexes whose biogenesis is strictly dependent on the coordinated expression of genes encoded by nuclear and mitochondrial DNA (mtDNA). mtDNA encodes 13 subunits that are essential structural components of OXPHOS complexes I, III, IV, and V, in addition to encoding two rRNAs (16S and 12S) and 22 tRNAs required for the translation of the subunits.1Pearce S. Nezich C.L. Spinazzola A. Mitochondrial diseases: translation matters.Mol. Cell. Neurosci. 2013; 55: 1-12Crossref PubMed Scopus (51) Google Scholar Mitochondrial translation is executed by a dedicated translation machinery that includes many regulatory factors and a mitochondrial-specific ribosome composed of two subunits: the 28S (mt-SSU) and 39S (mt-LSU) ribosomal subunits. These subunits are large ribonucleoprotein complexes containing around 80 nuclear-encoded structural mitoribosomal proteins (MRPs) and mt-DNA-encoded RNAs: 12S rRNA in the mt-SSU and 16S rRNA, in addition to either mt-tRNAVal or mt-tRNAPhe, in the mt-LSU subunit.2Amunts A. Brown A. Toots J. Scheres S.H.W. Ramakrishnan V. Ribosome. The structure of the human mitochondrial ribosome.Science. 2015; 348: 95-98Crossref PubMed Scopus (326) Google Scholar, 3Greber B.J. Boehringer D. Leibundgut M. Bieri P. Leitner A. Schmitz N. Aebersold R. Ban N. The complete structure of the large subunit of the mammalian mitochondrial ribosome.Nature. 2014; 515: 283-286Crossref PubMed Scopus (179) Google Scholar, 4Greber B.J. Boehringer D. Leitner A. Bieri P. Voigts-Hoffmann F. Erzberger J.P. Leibundgut M. Aebersold R. Ban N. Architecture of the large subunit of the mammalian mitochondrial ribosome.Nature. 2014; 505: 515-519Crossref PubMed Scopus (167) Google Scholar, 5Rorbach J. Gao F. Powell C.A. D'Souza A. Lightowlers R.N. Minczuk M. Chrzanowska-Lightowlers Z.M. Human mitochondrial ribosomes can switch their structural RNA composition.Proc. Natl. Acad. Sci. USA. 2016; 113: 12198-12201Crossref PubMed Scopus (45) Google Scholar The three-dimensional structures of the mammalian3Greber B.J. Boehringer D. Leibundgut M. Bieri P. Leitner A. Schmitz N. Aebersold R. Ban N. The complete structure of the large subunit of the mammalian mitochondrial ribosome.Nature. 2014; 515: 283-286Crossref PubMed Scopus (179) Google Scholar, 6Kaushal P.S. Sharma M.R. Booth T.M. Haque E.M. Tung C.S. Sanbonmatsu K.Y. Spremulli L.L. Agrawal R.K. Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome.Proc. Natl. Acad. Sci. USA. 2014; 111: 7284-7289Crossref PubMed Scopus (42) Google Scholar, 7Kaushal P.S. Sharma M.R. Booth T.M. Haque E.M. Tung C.S. Sanbonmatsu K.Y. Spremulli L.L. Agrawal R.K. Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome.Proc. Natl. Acad. Sci. USA. 2014; 111: 7284-7289Crossref PubMed Scopus (57) Google Scholar and human2Amunts A. Brown A. Toots J. Scheres S.H.W. Ramakrishnan V. Ribosome. The structure of the human mitochondrial ribosome.Science. 2015; 348: 95-98Crossref PubMed Scopus (326) Google Scholar mitoribosomes have only been elucidated recently. Considering the many factors involved in this process, it is not surprising that an increasing number of gene mutations cause defective mitochondrial translation and are linked to mitochondrial disease.1Pearce S. Nezich C.L. Spinazzola A. Mitochondrial diseases: translation matters.Mol. Cell. Neurosci. 2013; 55: 1-12Crossref PubMed Scopus (51) Google Scholar, 3Greber B.J. Boehringer D. Leibundgut M. Bieri P. Leitner A. Schmitz N. Aebersold R. Ban N. The complete structure of the large subunit of the mammalian mitochondrial ribosome.Nature. 2014; 515: 283-286Crossref PubMed Scopus (179) Google Scholar Mitochondrial translation defects usually result in a combined OXPHOS complex deficiency, leading to disorders with severe multisystem involvement and often an early lethal outcome. To date, mutations in eight mitoribosomal protein-encoding genes—MRPS7 (MIM: 611974), MRPS16 (MIM: 609204), MRPS22 (MIM: 605810), MRPS23 (MIM: 611985), and MRPS34 (MIM: 611994) from the mt-SSU and MRPL3 (MIM: 607118), MRPL12 (MIM: 602375), and MRPL44 (MIM: 611849) from the mt-LSU—have been linked to mitochondrial disease in a total of 22 subjects (summarized in Table 1).Table 1Overview of the Clinical Features of Subjects Carrying Mutations in Mitoribosomal SubunitsReferenceMRPS2MRPS2MRPS7MRPS16MRPS22MRPS22MRPS22MRPS23MPRS34MRPL3MRPL12MRPL44MRPL44this article (S1)this article (S2)Menezes et al.16Menezes M.J. Guo Y. Zhang J. Riley L.G. Cooper S.T. Thorburn D.R. Li J. Dong D. Li Z. Glessner J. et al.Mutation in mitochondrial ribosomal protein S7 (MRPS7) causes congenital sensorineural deafness, progressive hepatic and renal failure and lactic acidemia.Hum. Mol. Genet. 2015; 24: 2297-2307Crossref PubMed Scopus (53) Google ScholarMiller et al.11Miller C. Saada A. Shaul N. Shabtai N. Ben-Shalom E. Shaag A. Hershkovitz E. Elpeleg O. Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation.Ann. Neurol. 2004; 56: 734-738Crossref PubMed Scopus (186) Google ScholarSaada et al.13Saada A. Shaag A. Arnon S. Dolfin T. Miller C. Fuchs-Telem D. Lombes A. Elpeleg O. Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation.J. Med. Genet. 2007; 44: 784-786Crossref PubMed Scopus (128) Google ScholarSmits et al.9Smits P. Saada A. Wortmann S.B. Heister A.J. Brink M. Pfundt R. Miller C. Haas D. Hantschmann R. Rodenburg R.J. et al.Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy.Eur. J. Hum. Genet. 2011; 19: 394-399Crossref PubMed Scopus (80) Google ScholarBaertling et al.19Baertling F. Haack T.B. Rodenburg R.J. Schaper J. Seibt A. Strom T.M. Meitinger T. Mayatepek E. Hadzik B. Selcan G. et al.MRPS22 mutation causes fatal neonatal lactic acidosis with brain and heart abnormalities.Neurogenetics. 2015; 16: 237-240Crossref PubMed Scopus (27) Google ScholarKohda et al.14Kohda M. Tokuzawa Y. Kishita Y. Nyuzuki H. Moriyama Y. Mizuno Y. Hirata T. Yatsuka Y. Yamashita-Sugahara Y. Nakachi Y. et al.A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies.PLoS Genet. 2016; 12: e1005679Crossref PubMed Scopus (185) Google ScholarLake et al.12Lake N.J. Webb B.D. Stroud D.A. Richman T.R. Ruzzenente B. Compton A.G. Mountford H.S. Pulman J. Zangarelli C. Rio M. et al.Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.Am. J. Hum. Genet. 2017; 101: 239-254Abstract Full Text Full Text PDF PubMed Scopus (60) Google ScholarGalmiche et al.8Galmiche L. Serre V. Beinat M. Assouline Z. Lebre A.S. Chretien D. Nietschke P. Benes V. Boddaert N. Sidi D. et al.Exome sequencing identifies MRPL3 mutation in mitochondrial cardiomyopathy.Hum. Mutat. 2011; 32: 1225-1231Crossref PubMed Scopus (107) Google ScholarSerre et al.33Serre V. Rozanska A. Beinat M. Chretien D. Boddaert N. Munnich A. Rötig A. Chrzanowska-Lightowlers Z.M. Mutations in mitochondrial ribosomal protein MRPL12 leads to growth retardation, neurological deterioration and mitochondrial translation deficiency.Biochim. Biophys. Acta. 2013; 1832: 1304-1312Crossref PubMed Scopus (65) Google ScholarDistelmaier et al.10Distelmaier F. Haack T.B. Catarino C.B. Gallenmüller C. Rodenburg R.J. Strom T.M. Baertling F. Meitinger T. Mayatepek E. Prokisch H. Klopstock T. MRPL44 mutations cause a slowly progressive multisystem disease with childhood-onset hypertrophic cardiomyopathy.Neurogenetics. 2015; 16: 319-323Crossref PubMed Scopus (38) Google ScholarDistelmaier et al.10Distelmaier F. Haack T.B. Catarino C.B. Gallenmüller C. Rodenburg R.J. Strom T.M. Baertling F. Meitinger T. Mayatepek E. Prokisch H. Klopstock T. MRPL44 mutations cause a slowly progressive multisystem disease with childhood-onset hypertrophic cardiomyopathy.Neurogenetics. 2015; 16: 319-323Crossref PubMed Scopus (38) Google ScholarNumber of subjects112 siblings13 siblings11164 siblings111Dysmorphic features+−−+NR+NRNR+−+−−Cardiac involvement−−−++++NR−+NR++Hypotonia++NR+++NRNR+NR++NRSkin involvement+−−redundant skin of the neck−redundant skin of the neck−NR−−NR−−Structural brain abnormalities−−NR+NR++NR+++NRNRHearing impairment++++NRNR−NRNR−NRNRNRNRDevelopmental delay+/−+−NRNR++NRNR++++−+/−Growth delayFTT−FTTSGANRNR+NR+FTTSGA, FTTNRNRIncreased lactate levels++++++++++NR+++++Combined OXPHOS deficiency+++++++++++++Hypoglycemia++NRNRNRNRNR+NRNRNRNRNRAge of presentationinfantileinfantileinfantileneonatalneonatalneonatalneonatalinfantileneonatal or infantileinfantileneonatalinfantileneonatalAge of death (or age at last follow up)alive at 11 yearsalive at 11 years14 years; alive at 17 years3 days2–22 daysalive at 5 years3 daysalive at 1 year and 6 monthsrange: dead at 8.5 months to alive at 17 yearstwo died at 15 and 17 months; two are alive at 3 years2 yearsalive at 8 yearsalive at 26 yearsAbbreviations are as follows: −, not present; +/−, mildly affected; +, present; ++, severely affected; NR, not reported; FTT, failure to thrive; SGA, small for gestational age. Open table in a new tab Abbreviations are as follows: −, not present; +/−, mildly affected; +, present; ++, severely affected; NR, not reported; FTT, failure to thrive; SGA, small for gestational age. Most subjects presented in the neonatal period, and about one-third of the subjects died before the age of 1 year. However, besides the common feature of severe lactic acidosis, these disorders show a large variety in clinical presentation. Some have specific clinical features such as early-onset cardiomyopathy,8Galmiche L. Serre V. Beinat M. Assouline Z. Lebre A.S. Chretien D. Nietschke P. Benes V. Boddaert N. Sidi D. et al.Exome sequencing identifies MRPL3 mutation in mitochondrial cardiomyopathy.Hum. Mutat. 2011; 32: 1225-1231Crossref PubMed Scopus (107) Google Scholar, 9Smits P. Saada A. Wortmann S.B. Heister A.J. Brink M. Pfundt R. Miller C. Haas D. Hantschmann R. Rodenburg R.J. et al.Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy.Eur. J. Hum. Genet. 2011; 19: 394-399Crossref PubMed Scopus (80) Google Scholar, 10Distelmaier F. Haack T.B. Catarino C.B. Gallenmüller C. Rodenburg R.J. Strom T.M. Baertling F. Meitinger T. Mayatepek E. Prokisch H. Klopstock T. MRPL44 mutations cause a slowly progressive multisystem disease with childhood-onset hypertrophic cardiomyopathy.Neurogenetics. 2015; 16: 319-323Crossref PubMed Scopus (38) Google Scholar developmental abnormalities,10Distelmaier F. Haack T.B. Catarino C.B. Gallenmüller C. Rodenburg R.J. Strom T.M. Baertling F. Meitinger T. Mayatepek E. Prokisch H. Klopstock T. MRPL44 mutations cause a slowly progressive multisystem disease with childhood-onset hypertrophic cardiomyopathy.Neurogenetics. 2015; 16: 319-323Crossref PubMed Scopus (38) Google Scholar, 11Miller C. Saada A. Shaul N. Shabtai N. Ben-Shalom E. Shaag A. Hershkovitz E. Elpeleg O. Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation.Ann. Neurol. 2004; 56: 734-738Crossref PubMed Scopus (186) Google Scholar, 12Lake N.J. Webb B.D. Stroud D.A. Richman T.R. Ruzzenente B. Compton A.G. Mountford H.S. Pulman J. Zangarelli C. Rio M. et al.Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.Am. J. Hum. Genet. 2017; 101: 239-254Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar corpus callosum agenesis,13Saada A. Shaag A. Arnon S. Dolfin T. Miller C. Fuchs-Telem D. Lombes A. Elpeleg O. Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation.J. Med. Genet. 2007; 44: 784-786Crossref PubMed Scopus (128) Google Scholar hypoglycemia,14Kohda M. Tokuzawa Y. Kishita Y. Nyuzuki H. Moriyama Y. Mizuno Y. Hirata T. Yatsuka Y. Yamashita-Sugahara Y. Nakachi Y. et al.A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies.PLoS Genet. 2016; 12: e1005679Crossref PubMed Scopus (185) Google Scholar Leigh syndrome,12Lake N.J. Webb B.D. Stroud D.A. Richman T.R. Ruzzenente B. Compton A.G. Mountford H.S. Pulman J. Zangarelli C. Rio M. et al.Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.Am. J. Hum. Genet. 2017; 101: 239-254Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar or short stature and dysmorphic features.9Smits P. Saada A. Wortmann S.B. Heister A.J. Brink M. Pfundt R. Miller C. Haas D. Hantschmann R. Rodenburg R.J. et al.Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy.Eur. J. Hum. Genet. 2011; 19: 394-399Crossref PubMed Scopus (80) Google Scholar, 12Lake N.J. Webb B.D. Stroud D.A. Richman T.R. Ruzzenente B. Compton A.G. Mountford H.S. Pulman J. Zangarelli C. Rio M. et al.Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.Am. J. Hum. Genet. 2017; 101: 239-254Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 13Saada A. Shaag A. Arnon S. Dolfin T. Miller C. Fuchs-Telem D. Lombes A. Elpeleg O. Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation.J. Med. Genet. 2007; 44: 784-786Crossref PubMed Scopus (128) Google Scholar Wrinkled or redundant skin has been observed in mitochondrial translation defects caused by mutations in MRPS229Smits P. Saada A. Wortmann S.B. Heister A.J. Brink M. Pfundt R. Miller C. Haas D. Hantschmann R. Rodenburg R.J. et al.Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy.Eur. J. Hum. Genet. 2011; 19: 394-399Crossref PubMed Scopus (80) Google Scholar, 13Saada A. Shaag A. Arnon S. Dolfin T. Miller C. Fuchs-Telem D. Lombes A. Elpeleg O. Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation.J. Med. Genet. 2007; 44: 784-786Crossref PubMed Scopus (128) Google Scholar and MRPS16.11Miller C. Saada A. Shaul N. Shabtai N. Ben-Shalom E. Shaag A. Hershkovitz E. Elpeleg O. Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation.Ann. Neurol. 2004; 56: 734-738Crossref PubMed Scopus (186) Google Scholar Here, we report two unrelated subjects presenting with sensorineural hearing impairment, developmental delay, hypoglycemia, lactic acidemia, and a combined OXPHOS deficiency. Using exome sequencing, we identified bi-allelic mutations in MRPS2 (MIM: 611971), which encodes the mitochondrial ribosomal protein S2 and has not been implicated in disease until now. Our study adhered to the Declaration of Helsinki and was approved by the institutional review boards at each research site. Written informed consent was obtained from the parents of the subjects. Subject 1, a girl, was born at term after an uneventful pregnancy as the third child of non-consanguineous healthy parents of Austrian origin (Figure 1). Birth parameters were within normal limits. She had minor dysmorphic features, including low-set ears and slightly up-slanting palpebral fissures. Skin wrinkling, most pronounced on the abdomen and hands, was apparent from birth. During the first year of life she developed a failure to thrive. She had a psychomotor developmental delay in which the motor delay was most pronounced. She had an intermittent divergent strabismus of the left eye. At the age of 3 years, she had developed progressive sensorineural hearing loss, which required the use of a hearing aid and was corrected with bilateral cochlea implants. After this correction, both her speech and development improved markedly. Her last formal developmental assessment at the age of 5 years and 10 months showed an average developmental state of 2 years and 6 months of age. A cerebral MRI at the age of 3 years did not reveal any structural anomalies. She was prone to developing hypoglycemia. Biochemical evaluation revealed elevated liver enzymes (3- to 4-fold elevation in aspartate-amino transferase and alanine-amino transferase, repetitive elevated lactate levels (>8 mmol/L; reference values < 2 mmol/L), elevated serum alanine (up to 850 μmol/L; reference values = 99–350 μmol/L), and increased excretion of Krebs cycle intermediates (2-oxoglutaric acid between 50–220 μmol/mmol creatinine; reference values = 0–50 μmol/mmol creatinine and trace elevation of succinic acid). Glycosylation screening (transferrin and apolipoprotein-CIII isoelectric focusing) was unremarkable. Normal serum creatine kinase (CK) concentrations were found. Measurements of OXPHOS complex activities in liver, muscle, and fibroblasts showed a decrease in multiple enzyme complexes (Table 2).Table 2OXPHOS Complex Enzyme Activities in Muscle, Liver, and FibroblastsComplex Activity in MuscleComplex Activity in LiverComplex Activity in FibroblastsSubject 1Subject 2Subject 1Subject 2Subject 1Subject 2Complex I0. 14 (0.14–0.35)0.20 (0.13–0.29)0.02 ↓ (0.24; 0.59)0.02 ↓ (0.35–0.50)0.04 (0.04–0.12)0.16 (0.13–0.26)Complex II0.22 ↓ (0.23–0.41)0.19 ↑(0.09–0.15)1.43 (0.85; 1.80)0.9 ↓ (1.70–2.50)0.23 (0.18–0.43)0.36 ↑(0.25–0.34)Complex III0.81 ↓ (1.45–3.76)NP0.34 ↓ (2.18; 3.18)0.23 ↓ (1.00–1.40)0.96 (0.72–2.23)2.16 ↓ (2.23–2.98)Complex IV0.51 ↓ (0.82–2.04)0.42 ↓ (0.45–0.75)0.09 ↓ (1.44; 1.66)0.12 ↓ (1.00–1.40)0.24 ↓ (0.90–1.79)0.99 ↓ (1.14–1.54)Complex V0.67 (0.42–1.26)NP0.43 (0.31; 1.21)NP0.39 (0.39–0.79)0.48 (0.23–0.33)Laboratory reference values were available for muscle, fibroblasts, and liver and are shown in parentheses. Complex activities in liver for subject 1 are compared with those in two control samples that were used in the same experiment. These values are reported in parentheses. The following abbreviation is used: NP, not performed. Open table in a new tab Laboratory reference values were available for muscle, fibroblasts, and liver and are shown in parentheses. Complex activities in liver for subject 1 are compared with those in two control samples that were used in the same experiment. These values are reported in parentheses. The following abbreviation is used: NP, not performed. Subject 2, a boy, presented with fasting hypoglycemia at the age of 6 years. He was the first child of healthy unrelated parents of Tunisian origin and was born at term after an uneventful pregnancy. Birth parameters were within normal limits. Medical history was marked by several acute episodes of hypoglycemia after an overnight or a 12-hr fasting since the age of 18 months, especially when these episodes coincided with illness and poor oral intake, which associated with lactic acidosis. He could walk at 22 months. Speech development was delayed as a result of severe sensorineural deafness at the age of 2 years, necessitating the use of a hearing aid. Speech development improved after the hearing loss was corrected. At the age of 11 years he had a normal physical appearance, with normal growth parameters, but a moderate intellectual disability, frequent headache episodes, and muscular weakness of the lower limbs. He suffered from exercise intolerance marked by myalgia after walking. His brain MRI was normal. Metabolic investigations showed repetitive mildly increased lactate levels in plasma (2.2–3.8 mmol/L; reference values < 2 mmol/L) and urine (292 μmol/mmol creatinine; reference values = 25–100 μmol/mmol creatinine). A clinical fasting test showed hypoglycemia with hyperlactatemia (5 mmol/L) and slightly increased excretion of 2-oxoglutarate (37 μmol/mmol creatinine; control range < 27 μmol/mmol creatinine) in urine. Measurements of OXPHOS complex activities showed a decrease in multiple complexes in liver and fibroblasts and a complex IV deficiency in muscle. This subject had two unaffected siblings. A third sibling died during pregnancy (Figure 2B). The cause of her death is unknown, so it is unclear whether it is related to mitochondrial disease. Exome sequencing was performed to identify pathogenic variants underlying the disease in both subjects (for methods, see Supplemental Data). For subject 1, a series of filter steps, comparable to those described by Gilissen et al.,15Gilissen C. Arts H.H. Hoischen A. Spruijt L. Mans D.A. Arts P. van Lier B. Steehouwer M. van Reeuwijk J. Kant S.G. et al.Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome.Am. J. Hum. Genet. 2010; 87: 418-423Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar was applied to the variant data for the creation of a candidate gene list. Non-genic, intronic (except for splice sites), synonymous changes and common variants were excluded by comparison with dbSNPv132 and our in-house variant database (cutoff > 1%). Variants were further prioritized on the basis of PhyloP scores (cutoff > 3) as well as molecular pathways and segregation analysis. This resulted in the identification of a single candidate gene, mitochondrial ribosomal protein S2 (MRPS2; GenBank: NM_016034.3), carrying two heterozygous sequence variants, c.328C>T (p.Arg110Cys) and c.340G>A (p.Asp114Asn) (Figure 2A). Segregation analysis confirmed that both parents are heterozygous for one of these variants. One healthy sibling was homozygous for both wild-type alleles, whereas the second was heterozygous for the c.328C>T variant (data not shown). For subject 2, the pathogenic variant was selected according to similar criteria: exclusion of known SNPs reported with a frequency > 0.1% in dbSNP, 1000 Genomes, Exome Variant Server, or our in-house database; exclusion of non-coding variants; and selection of variants that were predicted to be possibly damaging by PolyPhen and SIFT. This filtering identified a homozygous variation (c.413G>A [p.Arg138His]) in only one gene: MRPS2, which encodes a known mitochondrial protein. Segregation analysis confirmed that both parents are heterozygous for this variant (Figure 2B). All identified variants affect highly conserved amino acids (Figure 2C) and were predicted to be pathogenic by at least two of the three in silico prediction programs that were used. These variants are reported in the GnomAD database only as heterozygous variants with very low minor allele frequencies (Table S1). Given that MRPS2 is a mitochondrial ribosomal protein, we proceeded to characterize the effects of the identified variants on mitochondrial physiology by using fibroblasts obtained from skin biopsies of both subjects (S1 and S2). A previously characterized fibroblast cell line (designated S3) carrying disease-causing mutations in MRPS22 was used as a positive control for mitoribosomal dysfunction.9Smits P. Saada A. Wortmann S.B. Heister A.J. Brink M. Pfundt R. Miller C. Haas D. Hantschmann R. Rodenburg R.J. et al.Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy.Eur. J. Hum. Genet. 2011; 19: 394-399Crossref PubMed Scopus (80) Google Scholar SDS-PAGE analysis of mitochondrial extracts (for methods, see Supplemental Data) from these fibroblasts showed decreased steady-state amounts of the protein MRPS2 in both S1 and S2 fibroblasts (Figure 3A). Similarly, the mt-SSU proteins MRPS5, MRPS18B, and MRPS28 were less abundant in subject fibroblasts. Amounts of the mt-LSU proteins MRPL37 and MRPL44 remained unchanged. Stability of newly imported mitoribosomal proteins and nascent 12S rRNA depends on their coordinated assembly into ribonucleoprotein complexes. Loss of individual MRPS proteins has been previously shown to result in decreased 12S rRNA steady-state abundance.9Smits P. Saada A. Wortmann S.B. Heister A.J. Brink M. Pfundt R. Miller C. Haas D. Hantschmann R. Rodenburg R.J. et al.Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy.Eur. J. Hum. Genet. 2011; 19: 394-399Crossref PubMed Scopus (80) Google Scholar, 11Miller C. Saada A. Shaul N. Shabtai N. Ben-Shalom E. Shaag A. Hershkovitz E. Elpeleg O. Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation.Ann. Neurol. 2004; 56: 734-738Crossref PubMed Scopus (186) Google Scholar, 13Saada A. Shaag A. Arnon S. Dolfin T. Miller C. Fuchs-Telem D. Lombes A. Elpeleg O. Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation.J. Med. Genet. 2007; 44: 784-786Crossref PubMed Scopus (128) Google Scholar, 16Menezes M.J. Guo Y. Zhang J. Riley L.G. Cooper S.T. Thorburn D.R. Li J. Dong D. Li Z. Glessner J. et al.Mutation in mitochondrial ribosomal protein S7 (MRPS7) causes congenital sensorineural deafness, progressive hepatic and renal failure and lactic acidemia.Hum. Mol. Genet. 2015; 24: 2297-2307Crossref PubMed Scopus (53) Google Scholar Consistent with this, steady-state abundance of 12S rRNA, but not that of 16S rRNA, was specifically decreased in S1 and S2 fibroblasts; a similar decrease was seen in S3 fibroblasts (Figure 3B) on northern blot analysis (for methods, see Supplemental Data). To further characterize the detrimental effects of MRPS2 mutations on mitoribosomal biogenesis, we analyzed the assembly and abundance of mt-SSU and mt-LSU particles with complexome profiling of mitochondrial extracts from control, S1, and S3 fibroblasts (for methods, see Supplemental Data). Complexome profiling enables the investigation of the abundance and composition of macromolecular protein complexes.17Heide H. Bleier L. Steger M. Ackermann J. Dröse S. Schwamb B. Zörnig M. Reichert A.S. Koch I. Wittig I. Brandt U. Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex.Cell Metab. 2012; 16: 538-549Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar Fully assembled mt-SSU particles were
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