COQ2 Nephropathy: a Newly Described Inherited Mitochondriopathy with Primary Renal Involvement Francesca Diomedi-Camassei,* Silvia Di Giandomenico,† Filippo M

JASN Express. Published on September 12, 2007 as doi: 10.1681/ASN.2006080833

CLINICAL RESEARCH www.jasn.org

COQ2 Nephropathy: A Newly Described Inherited Mitochondriopathy with Primary Renal Involvement Francesca Diomedi-Camassei,* Silvia Di Giandomenico,† Filippo M. Santorelli,† Gianluca Caridi,§ Fiorella Piemonte,‡ Giovanni Montini,¶ Gian Marco Ghiggeri,§ ࿣ Luisa Murer,¶ Laura Barisoni, Anna Pastore,† Andrea Onetti Muda,†† Maria Luisa Valente,** Enrico Bertini,† and Francesco Emma‡‡ Divisions of Pathology*, Molecular Medicine†, and Biochemistry‡, Department of Laboratory Medicine, and ‡‡Division of Nephrology and Dialysis, Department of Nephrology and Urology, Bambino Gesu` Children’s Hospital and Research Institute, and ††Department of Pathology, La Sapienza University, Rome, §Laboratory on Pathophysiology of Uremia, G. Gaslini Institute, Genoa, and ¶Department of Pediatrics, Division of Nephrology, Dialysis and Transplantation, and **Institute of Pathology, Azienda Ospedaliera-University of Padua, Italy; and ࿣ Department of Pathology, New York University, New York, New York

ABSTRACT

Primary coenzyme Q10 (CoQ10) deficiency includes a group of rare autosomal recessive disorders primarily characterized by neurological and muscular symptoms. Rarely, glomerular involvement has been reported. The COQ2 gene encodes the para-hydroxybenzoate-polyprenyl-transferase enzyme of

the CoQ10 synthesis pathway. We identified two patients with early-onset glomerular lesions that harbored mutations in the COQ2 gene. The first patient presented with steroid-resistant nephrotic syndrome at the age of 18 months as a result of collapsing glomerulopathy, with no extrarenal symptoms. The second patient presented at five days of life with oliguria, had severe extracapillary proliferation on renal biopsy, rapidly developed end-stage renal disease, and died at the age of 6 months after a course complicated by progressive epileptic encephalopathy. Ultrastructural examination of renal specimens from these cases, as well as from two previously reported patients, showed an increased number of dysmorphic mitochondria in glomerular cells. Biochemical analyses demonstrated decreased ϩ activities of respiratory chain complexes [II III] and decreased CoQ10 concentrations in skeletal muscle and renal cortex. In conclusion, we suggest that inherited COQ2 mutations cause a primary glomerular disease with renal lesions that vary in severity and are not necessarily associated with neurological signs. COQ2 nephropathy should be suspected when electron microscopy shows an increased number of abnormal mitochondria in podocytes and other glomerular cells.

J Am Soc Nephrol 18: 2773–2780, 2007. doi: 10.1681/ASN.2006080833

Coenzyme Q10 (CoQ10; ubiquinone) is an essential complex and has not yet been completely eluci- component of the mitochondrial electron-trans- dated. It involves at least 10 different enzymes that port chain and a lipid-soluble antioxidant mole- cule.1–4 In the mitochondrial respiratory chain, Received August 7, 2006. Accepted May 22, 2007. CoQ10 accepts reducing equivalents from NADH in complex I and acts as an electron shuttle between Published online ahead of print. Publication date available at complex II and complex III.5,6 In addition, its re- www.jasn.org. duced form, also termed ubiquinol, protects mem- Correspondence: Dr. Francesca Diomedi-Camassei or Dr. brane phospholipids and serum lipoprotein from Francesco Emma, Division of Pathology and Division of Nephrol- ogy, Bambino Gesu` Children’s Hospital and Research Institute, lipid peroxidation and prevents mitochondrial Piazza S. Onofrio 4-00165 Rome, Italy. Phone: ϩ39-06-6859- membrane protein from oxidative damage induced 2740; Fax: ϩ39-06-6859-2602; E-mail: [email protected] and by reactive oxygen species.4,7 [email protected]

J Am Soc Nephrol 18: 2773–2780, 2007 ISSN : 1046-6673/1810-2773 2773 CLINICAL RESEARCH www.jasn.org have been isolated from various genomes and are termed Thereafter, his encephalopathy continued to worsen, lead- COQ1 through COQ10.8,9 ing to a state of unresponsiveness, hypotonia, respiratory

Primary CoQ10 deficiency has been reported in a group of failure, and death at the age of 6 mo. Brain MRI showed rare autosomal recessive disorders that are generally character- cortical and subcortical stroke-like lesions in the frontal, ized by different combinations of central nervous system, skel- insular, and temporal regions and signs of diffuse cerebral etal muscle, and peripheral nerve clinical symptoms.9–19 Early atrophy. MRI spectroscopy revealed a significant peak of diagnosis is crucial, because oral supplementation with CoQ10 lactate in multiple single voxels, corresponding to the fron- has been shown to improve clinical symptoms.20–22 tal cortex and basal ganglia. Increased lactate levels were

COQ2 is part of the CoQ10 pathway and encodes the para- also documented in the cerebrospinal fluid (106 mg/dl; nor- hydroxybenzoate-polyprenyltransferase (EC 2.5.1.39), which mal value 10 to 22). In the urine, organic acid analysis catalyzes the prenylation of parahydroxybenzoate with a poly- showed a marked increase in lactate and pyruvate excretion. prenyl group.2 Mutations in the COQ2 gene first were identi- Serum lactate levels were normal. Serum ammonium levels 8 ␮ fied in two siblings with primary CoQ10 deficiency, and an- were moderately increased (86 mol/L; normal value 22 to other child was recently reported.23 One sibling of the former 55). Serology for hepatitis B, hepatitis C, Epstein-Barr virus, report presented with progressive encephalomyopathy and HIV, rubella, cytomegalovirus, and toxoplasma gondii was steroid-resistant nephrotic syndrome (NS), whereas his negative. The family history is remarkable for an older sister younger sister presented with isolated NS.8,22 Herein, we de- who had died of acute respiratory distress and metabolic scribed the renal pathology of these cases and two additional, acidosis at 18 h of life. Of note, her serum ammonium levels unrelated patients who bore new inherited COQ2 mutations had been elevated (520 ␮mol/L) shortly after birth. The and presented with steroid-resistant NS or neonatal renal eldest sister, now aged 6 yr, is healthy. failure. Patients 3 and 4 have been previously reported.8,9,22 Briefly, they were born from consanguineous parents who originated from north Africa. Patient 3 presented in the first RESULTS year of life with nystagmus secondary to bilateral optic nerve atrophy, seizures, and developmental delay. At 12 mo Clinical History of age, he developed severe steroid-resistant NS secondary Patient 1 is a 22-mo-old boy who was born after an uneventful to FSGS, which progressed to end-stage renal failure during pregnancy to unrelated healthy parents who originated from a period of 6 mo. He received a successful transplant at the Eastern Europe. Family history is unremarkable. A 6-yr-old age of 3 yr. His younger sister (patient 4) developed NS brother is healthy. At 18 mo of age, the patient rapidly devel- secondary to FSGS at 12 mo of life without any clinical signs oped severe steroid-resistant NS. Serology for hepatitis B, hep- of neurologic involvement.8 atitis C, Epstein-Barr virus, HIV, and parvovirus B19 was negative. Serum lactate was normal. The renal pathology revealed Genetic Analyses a collapsing glomerulopathy for which angiotensin-converting No mutations in the NPHS1, NPHS2, and PDSS2 genes were enzyme inhibitor and indomethacin treatment was started. detected in patients 1 and 2. Patient 1 showed a combined The NS, however, continued to worsen, leading to severe ana- heterozygous COQ2 mutation. He inherited a c.590G3A sarca that was unresponsive to infusions of human albumin. (p.Arg197His) mutation from his mother and a c.683A3G Six weeks after the initial symptoms, he underwent a unilateral (p.Asn228Ser) mutation from his father. His healthy brother nephrectomy and began peritoneal dialysis. Remarkably, he carries the paternal mutation. Patient 2 harbored a homozy- had no signs of neuromuscular involvement. Electroencepha- gous c.437G3A (p.Ser146Asn) COQ2 mutation. Both parents logram and brain MRI were also normal. At 21 mo of age, a are heterozygous carriers. These new mutations were not de- muscle biopsy was obtained. Since that age, the patient is re- tected in 500 control chromosomes. The c.890 A3G mutation ceiving oral supplementation of CoQ10 (30 mg/kg per d). His (p.Tyr297Cys) in patients 3 and 4 has been reported else- neurologic examination remains completely normal after where.8,22 8-mo of follow-up. The newly identified mutations, as well as the variant de- Patient 2 was a 6-mo-old boy born from distantly related tected before affect amino acid residues that are highly pre- healthy parents who originated from a small village in served in mammal, fly, worm, and yeast (Figure 1).8,9 On the southern Italy. Pregnancy was complicated by oligohy- basis of the predicted structure of the protein,2 the mutation in dramnios at the end of gestation. At5doflife, the patient patient 2 is located 26 amino acids upstream of the putative was found to be severely oliguric and hypertensive. At 10 d substrate-binding site (UbiA), the Arg197His mutation of pa- of life, a surgical renal biopsy demonstrated severe crescen- tient 1 is located three amino acids downstream of the same tic glomerulonephritis. Aggressive prednisone treatment domain, and the Asn228Ser mutation is located in the first failed to improve renal function, and peritoneal dialysis was putative transmembrane domain. The mutation detected in started at 3 wk of age. At 3 mo of age, he developed drug- patients 3 and 4 is predicted to be located in the third putative resistant seizures that evolved into a status epilepticus. transmembrane domain.8

2774 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2773–2780, 2007 www.jasn.org CLINICAL RESEARCH

Figure 1. Sequence homologies between COQ2 homologue genes of close and distantly related species. Patients’ mutations are indicated by arrows. Conservative mutations are indicated with gray characters; nonconservative mutations are indicated with nonbold letters.

Microscopic Features Patient 1. Examination of the renal biopsy revealed marked podocyte hypertrophy and hyperplasia forming pseudocrescents and wrinkling of the glomerular basement membranes (GBM) consistent with collapsing glomerulopathy. No immune- complex deposition was detected. The tubulointerstitial com- partment revealed extensive microcyst formation, focal tubular atrophy, and interstitial fibrosis. On ultrastructural examination, podocytes were characterized by extensive foot process effacement, partial loss of primary processes, and marked hypertrophy. Podocyte cell bodies were filled with numerous dysmorphic mitochondria, lacking cristae, or with abnormally enlarged ones and an electron-lucent central core. Dysmorphic mitochondria were also present in other glomerular cells, in- Figure 2. Renal histology and ultrastructural findings. Numbers cluding parietal, endothelial, and mesangial cells, and in myo- correspond to the patient numbers, as used in the text. Left cytes of small arteries, interstitial fibroblasts, and tubular epi- panels show light microscopy. Right panels show a representative thelial cells. No electron-dense deposits or tubuloreticular visceral epithelial cell by electron microscopy (EM). In all cases, inclusions were identified. A fragment of skeletal muscle was intense mitochondrial proliferation was observed. P1, Collapsed also examined and revealed accumulation of periodic acid- capillary tuft, surrounded by hyperplastic podocytes (silver stain). Schiff–positive granules at the periphery of the myocytes, un- EM image shows wrinkling and folding of the GBM and enlarged derneath the sarcolemma (ragged red fibers). Occasionally, podocytes containing numerous dysmorphic mitochondria. P2, myocytes lost their typical striate appearance or appeared pale Extracapillary proliferation with collapse of the glomerular tuft. and small, indicating atrophy (Figure 2). Tubular structures show mild degenerative changes of epithelial cells and contain abundant cellular debris (periodic acid-Schiff Patient 2. [PAS] stain). EM image shows numerous dysmorphic mitochondria in the cytoplasm of podocytes. P3, FSGS lesion with partial Analysis of the renal biopsy revealed severe extracapillary pro- sclerosis of the capillary tuft and mild tubular dilation (PAS stain). liferation, occasionally positive for fibrinogen, with collapse of EM image shows folded and collapsed GBM and swollen podo- the GBM. There was no evidence of immune complex deposi- cyte cell containing numerous dysmorphic mitochondria. Diffuse tion by immunofluorescence or electron microscopy. On ul- pedicle fusion and microvillous transformation of cellular profile trastructural analysis, podocytes and parietal epithelial cells are evident. P4, Mild FSGS lesions and tubular dilation (hematox- were filled with numerous dysmorphic mitochondria, which ylin and eosin stain). EM image shows numerous mitochondria in were variable in shape and size, often enlarged, had an elec- the cytoplasm of visceral epithelial cells that have a oncocyte-like tron-lucent core, and contained few short cristae. feature and shows diffuse pedicle fusion and microvillous transformation of cellular membrane. Magnifications: ϫ40 in P1; ϫ20 Patients 3 and 4. in P2, P3, and P4. Patients 3 and 4 had classic FSGS lesions on renal biopsy, with segmental solidification of the tuft, adhesion of the tuft to the his sister (patient 4). Ultrastructural studies showed diffuse efface- Bowman’s capsule, and occasional accumulation of hyaline ma- ment of foot processes, osmiophilic depositions in the subendo- terial. Sclerosing lesions in patient 3 were more extensive than in thelial space, swelling of endothelial cells, and increase in mesan-

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21 gial matrix. In both patients, numerous and abnormal ture. Although primary CoQ10 deficiencies have been recog- mitochondria were present and were characterized by an onco- nized since the late 1980s, no mutation had been described in cytic-like aspect. Podocyte mitochondrial proliferation extended the ubiquinone synthesis genes until recently.8,19 also to fibroblasts and endothelial, mesangial, and tubular cells in We identified two novel COQ2 mutations and analyzed patient 3; it was limited to glomerular cells in patient 4. the renal pathology of all currently reported patients. The pathogenicity of these mutations is supported by their ab- Histochemistry sence in 500 control DNA samples, the ultrastructural find- Renal cortex of patients 1 and 2 showed marked decrease in ing of abnormal mitochondria proliferation, and the dem- tubular cytochrome C oxidase (COX) and succinate dehydro- onstration of low CoQ10 levels in examined tissues (Table genase (SDH) activity, when compared with control speci- 2). In addition, Lopez-Martin et al.9 recently gave func- mens. SDH and COX histochemistry in skeletal muscle of the tional support to our clinical and pathologic observation same patients demonstrated a reaction homogeneity between showing that COQ2 cDNA harboring the Tyr297Cys muta- type 1 and type 2 fibers and mildly increased staining, indicat- tion of patients 3 and 4 is unable to complement function- ing mitochondrial proliferation. Lipid content was not in- ally COQ2-deficient yeast strains, as opposed to the wild- creased (Figure 3). type cDNA. These authors also showed that when the same mutation is introduced in the wild-type COQ2 yeast gene,

Mitochondrial Respiratory Chain Complexes and CoQ6 concentrations decrease and colony growth in respi- Measurements of CoQ10 Levels ratory chain–dependent medium is markedly reduced. Results of biochemical analyses in renal cortex and skeletal muscle Taken together, these data allow identification of a new of patients 1 and 2 are reported in Figure 4 and Table 1. As shown, entity within the category of mitochondrial cytopathies, complexes II and III activities were in the low level of the reference characterized by inherited COQ2 mutations, proliferation range or moderately decreased. The combined complex [IIϩIII] of dysmorphic mitochondria, and primary glomerular activity was more severely decreased, suggesting ubiquinone de- damage. We propose to refer to this entity as “COQ2 ne- pletion. This was confirmed by direct measurement of CoQ10 lev- phropathy,” because the kidney seems to be a primary tar- els, which were markedly reduced. Low levels of complexes I, II, get, as illustrated by the fact that two of the reported patients and IV activities were also observed. presented with isolated renal symptoms (four additional

patients with CoQ10 deficiency but without renal involvement were screened and no COQ2 and PDSS2 mutations DISCUSSION were found). This finding was somewhat surprising, be-

cause CoQ10 is ubiquitously expressed but may be related to 3 Renal dysfunction associated with mitochondriopathies is high CoQ10 content in human kidney. On the contrary, generally a rare event. Three cases of ubiquinone deficiency residual coenzyme activity may have prevented cell damage that presented with central and peripheral nervous system in- in other organs, as suggested by CoQ10 muscular levels in volvement and was associated with NS secondary to FSGS in patient 1, which were marginally reduced. Dietary uptake the first decade of life were previously reported in the litera- may have also provided enough ubiquinone to overcome 3 impaired CoQ10 synthesis in some tissues. However, the prevalence of renal symptoms in COQ2 defects may be related to differential expression of proteins involved in ubiquinone metabolism.3 In support of this hypothesis, we did not detect COQ2 mutations in four different patients

with congenital ataxia, severe CoQ10 deficiency, and no glomerular involvement, as stated before (data not shown). A prominent aspect of “COQ2 nephropathy” is the heterogeneous pattern of glomerular lesions (Table 2, Figure 2). Ul- timately, all reported lesions could be related to visceral epithelial cell damage, as illustrated by the unifying finding of abnormal mitochondria proliferation in these cells. FSGS lesions similar to those found in patients 3 and 4 have already been associated with mutations in the mitochondrial Figure 3. Kidney and muscle histochemistry. P, patient; C, con- genome [3243A3G in the tRNALeu(UUR) gene], which may trol tissues (from age-matched children); M, skeletal muscle; K, 6,24–26 kidney. Skeletal muscle SDH and COX histochemistry shows loss cause isolated glomerular disease. Podocyte damage sec- of type 1/type 2 fiber heterogeneity and mildly increased stain- ondary to inherited mitochondrial dysfunction may cause vis- ing, which suggests mitochondrial proliferation. Renal SDH and ceral cell depletion, accumulation of extracellular matrix, and COX histochemistry shows markedly decreased tubular activity. ultimately sclerosis of the glomerular tuft.27 In other cases, the Magnifications: ϫ40 in muscle; ϫ20 in kidney. same mitochondrial disease seems to trigger epithelial cell pro-

2776 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2773–2780, 2007 www.jasn.org CLINICAL RESEARCH

Figure 4. Biochemical data. Biochemical measurements in renal cortex (top) and muscle (bottom) homogenates. Complex I through IV values are expressed as unit per the activity of citrate synthase (CS). CS is expressed as nmol/min per mg protein; CoQ10 is expressed as ␮mol/g tissue. Gray areas indicate control values. Reference values for skeletal muscle were obtained from 52 control samples and for renal cortex were obtained from 13 control samples (both indicated as third, 50th, and 97th percentiles). F, patient 1; Œ, patient 2.

Table 1. Biochemical data (absolute values)a Parameter CS I II III II ؉ III IV CoQ10 Kidney cortex Patients 1 161 14 23 34 3 227 4.5 2 290 N/A 46 24 1.6 224 3.7 Controls P3 88 30.6 34.0 14.0 3.4 163 26 P50 131 44.0 64.0 42.0 11.5 338 94 P97 201 67.6 78.3 74.7 17.0 465 180 P (Mann-Whitney) NS N/A NS NS Ͻ0.03 NS Ͻ0.04 Skeletal muscle patients 1 350 14.0 39.0 57.0 6.5 603 12 2 356 7.1 46.0 54.0 4.7 276 0.8 Controls P3 274 29.0 60.0 101.1 20.9 902 49.2 P50 195 28.0 33.0 46.0 18.0 556 28.2 P97 140 17.1 25.3 29.8 7.8 363 17.6 P (Mann-Whitney) Ͻ0.03 NS NS NS Ͻ0.05 NS Ͻ0.02 aBiochemical measurements in renal cortex and skeletal muscle homogenates. Complexes I through IV and citrate synthase (CS) values are expressed in nmol/ ␮ min per mg protein. CoQ10 values are expressed in mol/g tissue. N/A, not available).

liferation (in particular podocyte proliferation), associated cases the pathway taken by injured podocytes leads to prolif- with GBM collapse. Whereas increased apoptosis of podocyte erative lesions.29 cells may explain the mechanisms underlying FSGS formation The collapsing lesions described in patient 1 are compa- in mitochondrial cytopathies,28 it remains unclear why in some rable to those described in a murine model of collapsing

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Table 2. Clinical, pathological, biochemical, and genetic features of reported patientsa Patient Parameter 1234 Reference This report This report Refs. 8,9,22, this report Refs. 8,9,22, this report Family history Consanguinity No Yes Yes Yes Other siblings 1 healthy brother 1 healthy sister, 1 dead sister (at2dof Patient 4 Patient 3 life) Gender Male Male Male Female Renal involvement renal symptoms SRNS ARF SRNS NS Age of onset 18 mo Birth 11 mo 12 mo Renal pathology Collapsing GN Crescentic GN FSGS FSGS Mitochondria abnormalities by Yes Yes Yes Yes EM Histochemistry (SDH/COX) Decreased Decreased N/A N/A Outcome ESRF (20 mo) Death (6 mo) ESRF (18 mo) Normal renal function Extrarenal involvement None Epileptic encephalopathy, hypotonia Epileptic encephalopathy, optic None nerve atrophy Biochemistry data ϩ ϩ ϩ CoQ10 levels Decreased (kidney muscle) Decreased (kidney muscle) Decreased (muscle Decreased (fibroblasts) fibroblasts) Mitochondrial complex activity Decreased ͓IIϩIII͔ (kidney ϩ muscle) Decreased ͓IIϩIII͔ (kidney ϩ muscle) Decreased ͓IIϩIII͔ (fibroblasts) Decreased ͓IIϩIII͔ ͓ ϩ ͔

mScNephrol Soc Am J decreased I III (muscle) (fibroblasts) COQ2 mutations p.Arg197His p.Asn228Ser p.Ser146Asn p.Tyr297Cys p.Tyr297Cys aARF, acute renal failure; ESRF, end-stage renal failure; GN, glomerulonephritis; NS, nephrotic syndrome; SRNS, steroid-resistant nephrotic syndrome. 18: 7328,2007 2773–2780, www.jasn.org CLINICAL RESEARCH glomerulopathy (kd/kd), in which animals spontaneously CONCISE METHODS develop proteinuria and renal disease associated with the presence of numerous dysmorphic mitochondria in all cell Histology, Immunofluorescence, and Histochemistry types, including podocytes.30 In this model, mitochondrial Formalin-fixed renal specimens were embedded in paraffin and dysfunction is due to a mutation in the gene encoding for a stained with hematoxylin and eosin, periodic acid-Schiff, trichrome, prenyltransferase-like mitochondrial protein that has ex- and periodic acid-methenamine silver stains using standard tech- tensive homologies with the human transprenyltransferase niques. Frozen renal tissues were immunostained with antibodies di-

(PDSS2) gene, which is involved in the CoQ10 synthesis rected against IgA, IgG, IgM, C3, C1q, C4d, fibrinogen, and albumin. pathway.31 Mutations in PDSS2 were not detected in pa- Electron microscopy was performed on glutaraldehyde-fixed tissues tients 1 and 2 but were recently reported in one patient who and processed for ultrastructural analysis according to standard lab- presented with severe Leigh syndrome and NS associated oratory protocols. In patients 1 and 2, 5-␮m frozen sections from 19 with CoQ10 deficiency. muscle and kidney were incubated with cytochrome C and diamino- Conversely, patient 2 presented with acute renal failure sec- benzidine or with succinate and nitroblue-tetrazolium to assess the ondary to extracapillary proliferation, indicating that cells in- activities of COX and SDH, respectively, following established proto- volved in this process included parietal epithelial cells in addition cols.35 to podocytes. Renal lesions were remarkable by their advanced stage at 10 d of life, indicating strongly that they had developed in Biochemical Analysis utero. This is also suggested by the obstetric report indicating oli- Spectrophotometric measurements of mitochondrial respiratory gohydramnios during the last weeks of gestation. chain enzyme activities and citrate synthase activity were performed 36 Biochemically, these lesions are characterized by a mod- on renal cortex and muscle tissues as previously reported. CoQ10 erate reduction of complex II and complex III activities in concentrations were assayed by reverse-phase HPLC according to es- renal and muscle tissues and by a remarkable reduction of tablished protocols.37 Normal values for skeletal muscles have been their combined, ubiquinone-dependent [IIϩIII] activity established in our laboratory.37 No reference data are available for the (Figure 4, Table 1). Direct CoQ10 measurements confirm renal cortex; therefore, values from patients were compared with 13 these functional data. The histochemical analyses, however, pediatric control specimens that were obtained after surgical nephrec- show discordant results between kidney and muscle. In skel- tomy for nephroblastoma. Parenchymal integrity was checked by etal muscles, high SDH activity is an indicator of increased standard light microscopy techniques. Particular attention was paid mitochondria number. Conversely, renal SDH activity was to analyze samples containing only renal cortex. decreased, despite equally intense mitochondrial proliferation. This seeming discrepancy may indicate more severe Molecular Genetics mitochondrial injury in the kidney. In this tissue, ubiquinol Genomic DNA was purified from peripheral blood. Screening for depletion may have increased reactive oxygen species activ- mutations in the nephrin and podocin genes (NPHS1 and NPHS2) ity,4 causing damage of inner membrane enzymes, such as was performed as previously reported.38 Direct sequencing after PCR SDH.4 SDH has been shown to be particularly sensitive to amplification was used to screen for mutation in the COQ2 (GenBank oxidative stresses in comparison with other enzymes of the no. NM_015697) and PDSS2 genes (GenBank no. NM_020381). All Krebs cycle and electron transport chain complexes.32 The analyses were performed using standard techniques with BigDye 3.1 decreased activities of other respiratory chain complexes chemistry, following the manufacturer’s protocols (Applied Biosys- observed in our patients may be related to similar mecha- tems, Foster City, CA). Intronic oligonucleotide primers sequences nisms of mitochondrial damage. are available upon request. Mitochondrial dysfunction and altered mitochondrial gene expression have also been documented in patients with NS 33,34 secondary to nephrin mutations, suggesting that, regard- DISCLOSURES less of the initial insult, mitochondria play an important role in None. podocyte metabolism and may be actively involved in the pathophysiology of various forms of NS. In conclusion, COQ2 mutations cause a renal disease that REFERENCES is characterized by variable renal lesions and widespread proliferation of dysmorphic mitochondria in glomerular 1. Turunen M, Olsson J, Dallner G: Metabolism and function of coenzyme Q. Biochim Biophys Acta 1660: 171–199, 2004 cells. The clinical picture can be heterogeneous, and neuro- 2. Forsgren M, Attersand A, Lake S, Grunler J, Swiezewska E, Dallner G, muscular symptoms may complicate the course of the dis- Climent I: Isolation and functional expression of human COQ2, a gene ease. 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