IPNA-ESPN Master Junior Classes Mitochondrial Cytopathies

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1 IPNA-ESPN Master Junior Classes Mitochondrial Cytopathies Francesco Emma, MD Division of Nephrology and Dialysis Bambino Gesù Children s Hospital, IRCCS Rome, Italy

2 Mitochondrial cytopathies Heterogeneous group of diseases characterized by defects in oxidative phosphorylations. Involve several organs or tissues: - CNS - skeletal muscle - heart - liver - pancreas / endocrine system - hematopoietic system - kidneys (children >> adults). First symptoms: < 1 month of age in > 30% of cases < 2 years of age > 80% of cases Increasing number of tissues affected over time CNS almost consistently involved in late stages

3 Mitochondrial respiratory chain NADH-CoQ reductase succinate-coq reductase (SDH) red.coq-cyt c reductase cytochrome c oxidase (COX) ATP synthetase Di Mauro 2003

4 Mitochondrial genome Di Mauro 2003

5 mdna mutations/deletions Di Mauro 2003

mutations classic mendelian genetics all mitochondria are affected to the same extent structural

6 Type of mutations mtdna genes mutations de novo or maternal inheritance heteroplasmy threshold effect proliferative segregation mutations in trna genes mutations in other mtdna genes deletions Nuclear DNA gene mutations classic mendelian genetics all mitochondria are affected to the same extent structural mitochondrial genes functional mitochondrial genes genes involved in mitochondrial assembly genes involved in mitochondria dynamics

Activities of mitochondrial enzymatic complexes Polarographic studies measure oxygen consumption by isolated mitochondria or whole cells in the presence of different oxidative substrates such as

7 Activities of mitochondrial enzymatic complexes Polarographic studies measure oxygen consumption by isolated mitochondria or whole cells in the presence of different oxidative substrates such as pyruvate, glutamate, succinate, etc. Spectrophotometric studies enzyme activities: CI, CII, CIII, [CII+CIII], CIV, CV require normalization for the N of mitochondria: citrate synthetase When possible, studies should be performed on involved tissues

8 Organic aciduria in renal mitochondrial disease

9 Organic aciduria in renal mitochondrial disease NB: may be observed in the absence of hyperlactacidemia

10 Histo-enzymatic investigations Muscle Kidney SDH COX SDH COX Di Mauro 2003

11 Proximal tubular cells by electron microscopy Renal Fanconi syndrome RTA + glomerular proteinuria Renal Fanconi syndrome

Renal diseases in mitochondrial disorders generally Fanconi syndrome RTA isolated hyperaminoaciduria isolated hypomagnesemia Barrter-like Only a few case reports Egger 1981, Brum 1994, Lehenart 1995,

12 Renal diseases in mitochondrial disorders generally Fanconi syndrome RTA isolated hyperaminoaciduria isolated hypomagnesemia Barrter-like Only a few case reports Egger 1981, Brum 1994, Lehenart 1995, Donaldson 1985, Rötig 1995, Szabolcs 1994 Zupanc 1991, Birch-Machin 1989, Minchon 1983, Muller-Hocker 1983, Strausbie 1982, Niaudet et al 1997, Goto 1990, Zaffanello 2005, Eviatar 1990, Gruskin 1973, Matsutani 1992, de Lonley 2001 Tubular Tubularinterstitial Glomerular

in the proximal tubule The mutation introduces a new mitochondrial targeting motif

13 Autosomal dominant Fanconi syndrome Heterozygous missense mutation in the EHHADH gene EHHADH is an enzyme involved in peroxisomal oxidation of fatty acids EHHADH is expressed in the proximal tubule The mutation introduces a new mitochondrial targeting motif Impaired mitochondrial oxidative phosphorylation Dominant-negative effect Klootwijk et al, NEJM 2014

Mitochondrial glomerular diseases Mostly FSGS. Generally associated with other symptoms: - hearing loss - myopathy - developmental delay - ophtalmoplegia - pigmentary retinitis.

14 Mitochondrial glomerular diseases Mostly FSGS. Generally associated with other symptoms: - hearing loss - myopathy - developmental delay - ophtalmoplegia - pigmentary retinitis. Two clearly defined genetic defects: - CoenzymeQ10 synthesis defects - trnaleu mutations. Brun 1994, Rötig 1994, Lowik 2005, Scaglia 2004, Guery 2003, Moulonguet Doleris 2000, Hotta 2001, Diomedi-Camassei 2007, Quinzii 2006, personal observations

A3243G trna Leu mutation Generally FSGS, but also cases of TIN and cystic kidney disease Other symptoms: - hearing loss: 80-90% (often not severe) - diabetes mellitus: 20-30% - neuromuscular: 10-20%

15 A3243G trna Leu mutation Generally FSGS, but also cases of TIN and cystic kidney disease Other symptoms: - hearing loss: 80-90% (often not severe) - diabetes mellitus: 20-30% - neuromuscular: 10-20% - (cardiomyopathy, retinopathy) Age of diagnosis years Progression to CRF within 5 years from diagnosis in ±50% of cases NB: possible misdiagnosis with Alport syndrome Lowik 2005, Scaglia 2004, Guery 2003, Moulonguet Doleris 2000, Hotta 2001

Electron transporter in the mitochondrial respiratory

16 Coenzyme Q10 (ubiquinone) Available in food, but most originates from de novo synthesis. Electron transporter in the mitochondrial respiratory chain. Anti-oxidant. Other functions. P. Mitchell Nobel Prize for Chemistry 1978

17 Sources of ROS in cells Generation of superoxide during the oxidation of ubiquinol Conversion of superoxide to hydrogen peroxide and hydroxyl radical Formation of hydroxyl radical by inorganic chemical reactions

18 CoQ10 is an important anti-oxidant in the kidney Three-week-old heminephrectomized male Sprague Dawley rats

19 Coenzyme Q10 biosynthesis

Gen 2006 PDSS1: encephalopathy / no renal disease J Clin

20 First reports of a primary coenzyme Q10 deficiency Infantile encephalopathy with renal dysfunction Am J Hum Gen 2006 PDSS1: encephalopathy / no renal disease J Clin Invest, 2007 COQ2: encephalopathy / congenital nephrotic syndrome

21 Renal disease in COQ2 mutations Diomedi-Camassei JASN 2007

22 Renal disease in COQ2 mutations Collapsing GN Crescentic GN FSGS FSGS Diomedi-Camassei JASN 2007

23 Renal disease in COQ2 mutations Diomedi-Camassei JASN 2007

Renal disease in COQ2 mutations Respiratory chain activities Yeast complementation Strain CoQ6 ng/mg prot BY4741 1448 ± 20 BY4741

24 Renal disease in COQ2 mutations Respiratory chain activities Yeast complementation Strain CoQ6 ng/mg prot BY ± 20 BY4741 Dcoq2 <10 BY4741 Dcoq2 hcoq2 842 ± 12 BY4741 Dcoq2 hcoq2 (c.890 A>G) 92 ± 3 Lopez-Martin et al, Hum Mol Gen 2007 Diomedi-Camassei JASN 2007

Renal disease in COQ6 mutations 6 different mutations in

families. Early-onset SRNS with sensorineural deafness.

Knockdown of coq6 causes apoptosis that is partially

25 Renal disease in COQ6 mutations 6 different mutations in the monooxygenase 6 gene (COQ6) in 13 individuals from 7 families. Early-onset SRNS with sensorineural deafness. Lack of complementation in coq6-deficient yeast. Knockdown of coq6 causes apoptosis that is partially reversed by coenzyme Q10 treatment (podocyte & zebrafish embryos). coq6 is expressed in podocytes and in the stria vascularis of the inner ear in rats. Heeringa et al, J Clin Invest, 2011

26 Renal disease in PDSS2 mutations Leigh syndrome Nephrotic syndrome CoQ10 deficiency (muscle and fibroblasts) López et al., Am J Hum Genet, 2006

27 Prenyl-diphosphate synthase (Pdss2 kd/kd ) mice

28 Prenyl-diphosphate synthase (Pdss2 kd/kd ) conditional KO mice Albuminuria: 21 ± 5 Albuminuria: 0.2 ± 0.1 Pdss2 kd/kd Wild type Albuminuria: 33 ± 8 Albuminuria: 0.1 ± 0.0 KO podocytes KO tubular epithelium Peng et al, PLoS genetics, 2010

29 CoQ10 biosynthesis defects: treatment 30 mg/kg/day Montini et al. NEJM, 2008

30 CoQ10 biosynthesis defects: treatment PLoS One Jul 30;5(7):e11897 ATP returned to normal (2x) after 1 week, but NOT after 24 hours with ubiquinone; CoQ10 analogues did NOT normalizing the bioenergetics status of cells. In COQ2 mutant fibroblasts, ALL supplements normalized increased superoxide anion production and oxidative stress-induced cell death.

31 Known coenzyme Q10 biosynthesis defects in humans * Emma et al, Ped Nephrol 2010 * 2 additional patients born prematurely that died at the age of 5 and 6 months with encephalomyopathy, without evidence of renal involvement. Jakobs et al, J Neurol Sci 326, (2013)

COQ2 causes ultiple-system atrophy Adult-onset intractable neurodegenerative disease with autonomic failure and pyramidal dysfunction (Old designations: olivopontocerebellar ataxia, striatonigral

32 COQ2 causes ultiple-system atrophy Adult-onset intractable neurodegenerative disease with autonomic failure and pyramidal dysfunction (Old designations: olivopontocerebellar ataxia, striatonigral degeneration, Shy Drager syndrome) Predominant parkinsonism (sub-type P) or cerebellar ataxia (sub-type C) No renal disease Functionally impaired variants of COQ2 (homozygous or compound heterozygous) associated with an increased risk of multiple-system atrophy in multiplex families and patients with sporadic disease.

Compound heterozygous mutation in COQ2 in 1 patient with

33 Possible undiagnosed cases that could benefit from early treatment NGS in 36 patients with SRNS. Compound heterozygous mutation in COQ2 in 1 patient with isolated SRNS at 2 years of age, rapidly evolving into ESRD (c.683 A>G, c.701delt).

Conclusions Mitochondrial defects should be suspected in patients that present with proteinuria associated with multi-organ involvement.

34 Conclusions Mitochondrial defects should be suspected in patients that present with proteinuria associated with multi-organ involvement. Defects in CoQ10 biosynthesis should be suspected when EM studies show proliferation of abnormal mitochondria in podocytes. Ubiquinone deficiencies respond to oral CoQ10 supplementation. t-rna LEU mutations can cause FSGS. Several other glomerular and tubular disorders associated with mitochondrial cytopathies have been reported in the past decades.

35 Thank you

Mitochondrial Cytopathies Francesco Emma. Division of Nephrology and Dialysis Bambino Gesù Children s Hospital, IRCCS - Rome, Italy

Mitochondrial Cytopathies Francesco Emma Division of Nephrology and Dialysis Bambino Gesù Children s Hospital, IRCCS - Rome, Italy Prevalence of mitochondrial cytopathies Mitochondrial diseases can become