giovanni montini milano, italy · interplay of mitochondrial and nuclear genes in the biogenesis of...

Nefrologia pediatrica tra aggiornamento e linee guida

Sabato 14 novembre 2015

Università degli Studi di Milano-Bicocca, Monza

Citopatia mitocondriale con coinvolgimento renale:

nuove prospettive fisiopatologiche e terapeutiche

Giovanni Montini

Milano, [email protected]

CLINICA PEDIATRICA

Direttore : Prof Andrea Biondi

Characteristics of mitochondrial genetics

Mutant and wild type mtDNA molecules segregate randomly in

daughter cells during each cell division

Random drift

A mutation must affect a critical proportion (usually >70~%) of

the total mtDNA molecules within a cell or tissue to cause a

biochemical effect resulting in a clinical phenotype

Threshold effect

Wild type and mutant mtDNA molecules can coexist in different

proportions within cells of the same tissue or in different

tissues of the same individual

Heteroplasmy

Both genders might be affected by mtDNA mutations, but only

females transmit mutations to their children

Maternal

inheritance

F Emma, G Montini, SM Parikh, L Salviati

Nature Reviews in Nephrology in press

Interplay of mitochondrial and nuclear genes in

the biogenesis of the respiratory chain



Mitochondrial DNA

encodes for 13 structural

subunits of the RC, and

for the 2 rRNA and 22

tRNA required for

mitochondrial protein

synthesis.

Nuclear DNA encodes for

all other structural

subunits of the RC

complexes, cytochrome c,

and their assembly

factors and for the

enzymes required for

CoQ10 biosynthesis.



Citopatie mitocondriali e rene

• Malattie tubulari (più frequenti)

• Malattie glomerulari (secondarie a mutazioni della

via metabolica della biosintesi del coenzima Q10 o

mutazioni m.3243 A>G mutation in the

tRNALeu(UUR) gene)

• Il malfunzionamento mitocondriale concorre alla

fisiopatologia del danno tessutale

Podocytes

• Podocytes are highly differentiated cells with limited replicative capacity. Their functions are:

– they are major component of the glomerular filtration barrier,

– they support the other capillary components in counteracting endocapillary pressure

– they synthesize major cytoskeletal proteins and extracellular matrix components

– they have several immunological roles.

• Podocytes are particularly dependent on energy and are rich in mitochondria. Impairment of oxidative phosphorylation in podocytes results in

– excessive generation of ROS

– disruption of the glomerular filtration barrier

– proteinuria and ultimately the development of glomerular sclerotic lesions.

� Coenzyme Q10, Ubiquinone, ubidecarenone, Coenzyme Q, CoQ10, CoQ or Q10

� It is a 1,4-benzoquinone chemical compound, where Q refers to the redox active benzoquinone chemical group and 10 refers to the number of isoprenyl chemical subunits in its tail.

Coenzyme Q10 (1)

Coenzyme Q10 (2)

• Vitamin like substance, lipophilic

• First isolated in 1957 in beef heart

mitochondria (Crane FL et al., Isolation of a quinone

from beef heart mitochondria. Biochimica et Biophysica Acta,

1957)

• Highest concentrations in tissues

with high energy turnover (heart,

brain, liver, kidney)

• Humans and rodents mainly CoQ10

and CoQ9

PDSS1 and PDSS2 (COQ1)

Coenzyme Q10(3)

Co Q10 functionsElectron transporter in the mitochondrial respiratory chain

P. Mitchell

Nobel Prize for Chemistry 1978

Di Mauro S et al JCI 07

Coenzyme Q10(3)

Co Q10 functionsElectron transporter in the mitochondrial respiratory chain

Cofactor for dehydrogenases

Electron transporter outside the mitochondria

It is implicated in the inhibition of apoptosis

Regulates mitochondrial transmembranepermeability

It is required for pyrimidine nucleoside biosynthesis

Anti oxidant

P. Mitchell

Nobel Prize for Chemistry 1978

CoQ10 may be of benefit as an ingredient for topical cosmetic products.

CoQ10 has the beneficial effect of preventing photoaging. We were

able to demonstrate that CoQ10 reduce the level of oxidation.

Furthermore a reduction in wrinkle depth following CoQ10 application

was shown. CoQ10 was determined to be effective against UVA

mediated oxidative stress in human keratinocytes.

(100 single or combination-ingredient products. In 2002 200m $ sales in USA)

Accessed August 2014

Two sisters (14 and 12 yo) presented with encephalomyopathies.

Blood tests: Elevated lactic acid and creatine kinase

Muscle histology: ragged-red fibers and excessive lipid

Muscle biochemistry: Decreased

activities of mitochondrial respiratory chain complexes I+III and II+III.

Low CoQ10 (~5% of normal)

• 3 siblings with a complex clinical syndrome: progressive encephalopathy and SRNS.

• 2 developed ESRF and were transplanted at 8 and 9 years of age

• 1 died at 8 years of age after rapid neurologic deterioration

• Male, b 02.12.2000; Parents first cousins

• At 2 months nystagmus

• At 12 months NS

• Renal biopsy: FSGS

• Mutation analyses of nephrin and podocin negative.

• Treatment with steroids, diuretics, and cyclosporine ineffective

SL, male, b. 02.12.00

• Neurologic deterioration with progressive psychomotor

regression and loss of the ability to walk and stand.

Funduscopic examination showed optic atrophy.

• Brain MRI showed diffuse cerebral atrophy

• 18 mo ESRF PD Tx

• Low CoQ10 levels and complex II+III in the fibroblasts

• CoQ10 was initiated with neurologic improvement

Neurology 2005;65:606-608

NL, b. 10.10.02

� Female sibling with a preclinical diagnosis of CoQ10 deficiency at 12 months of age

� At 12 months of age Nephrotic Syndrome: urinary protein excretion: 55 g per square meter of bs per day, hypoalbuminemia (13.5 g/L of albumin), and severe generalized edema.

� Renal biopsy: mild focal segmental glomerulosclerosis; at EM : podocytes particularly rich in mitochondria.

2 podocytes with the cytoplasm completely occupied by mitochondria and hyperplasia of the podocyte villae. For the richness of mitochondria these cells appear similar to "oncocytes".

Detailed view of the mitochondria showing a reduced number of crests

After 8 yrs no neurological symptoms.

Normal renal function

Regression of NS

30 mg/Kg

� Visual acuity: 1/10

� Visual field restricted (rode cone dystrophy)

� Deafness; hearing aid

� Kidney transplantation

� Severe neurologic sequelae of encephalopathy, seizures, and hemiplegia

� Visual acuity: 8/10

� Normal visual field

� Normal hearing

� Normal kidney function; modest proteinuria

� Normal neurologic examination

SL CoQ10 late Tx NL CoQ10 early Tx

NL and SL long-term effects of CoQ10 Tx

ML 3 year old, normal boy: prental diagnosis, no mutation

c. 890 A>G Y297C

Homozygous missense mutation in COQ2

Am J Hum Genet 2006; 78: 345-349

CoQ10 Deficiency: Genetics

The A G transition at nucleotide 890 changes a highly conserved tyrosine tocysteine at amino acid 297 within a predicted transmembrane domain

Quinizii C et al

Yeast Model

Δ

COQ2

Transformation with WT or Mut

(human or yeast) Gene

Does not grow onnon-fermentable

media

(150)

(151)

(152)

(153)

(154)

Tubulopathy

Tubulopathy

Tubulopathy

Tubulopathy*

Tubulopathy*

RRM2BTWINKLEMPV17SUCLA2DGUOK

Defects in genes involved in mtDNA

maintenance

(147)

(148)

(149)

Tubulopathy

Tubulopathy

Tubulopathy

MRPS22YARS2SARS2

Defects in genes Involved in mtDNA Translation

(142)

(143)

(144)

(145)

(146)

Tubulopathy

Tubulopathy*

Tubulopathy

Tubulopathy

Tubulopathy*

COX10SURF1BCS1LUQCC2TMEM70

Defects of assembly factors

(90)

(89)

(88)

(85)

(86)

(63)

SRNS

SRNS

SRNS

SRNS

SRNS

Tubulopathy

PDSS1PDSS2COQ2COQ6ADCK4COQ9

Defects of electron carriers (including CoQ

biosynthesis)

ReferenceRenal phenotypeGeneCategory

Defects in nuclear genes that affect the

biogenesis of the respiratory chain and present

with a renal phenotype



Copyright ©2008 American Physiological Society

Saiki, R. et al. Am J Physiol Renal Physiol 295: F1535-F1544 2008;doi:10.1152/ajprenal.90445.2008

Fig. 8. Hematoxylin- and eosin-stained sections of kidneys from Q10-treated and untreated B6

Normal kidney in a Q10 treated162-day-old B6.Pdss2 mice. Urine albumin: 3.7 mg/24 h

Severe IN in an untreated

128-day-old B6.Pdss2 mice. Urine albumin: 57.34 mg/24 h

Note the severe interstitial

inflammation, tubular dilatation, and glomerular crescents

• Rare autosomal recessive trait (ca 60 patients have been reported), first described in 1989

• Clinically heterogeneous disorder of the respiratory chain

• Four major phenotypes

• Encephalomyopatic form

• Multisystemic infantile variant with renal disease

• Myopathic form

• Ataxic form

CoQ10 Deficiency: Clinical Presentations

Multisystemic Form

• Developmental delay, ataxia, pyramidal signs, mental retardation

• Nephrotic syndrome, renal failure, tubulopathy; FSGS, collapsing glomerulopathy, severe extracapillaryproliferation

• Cardiomyopathy

• Frequent visual and hearing involvement

• Early onset: from birth up to 2nd year of life

• Onset symptoms: nystagmus, severe myopia, deafness, delayed motor development, poor feeding, nephrotic syndrome

CoQ10 Deficiency: Clinical Presentations

• Blood lactate levels

• Decreased combined activity of complex II+III in muscle

biopsies toghether with normal both complex II and III

• Biochemical study of CoQ10 in both muscle and fibroblasts

seems advisable to demonstrate the deficiency in all patients

• CoQ10 concentration in peripheral blood mononuclear cells?

• Plasma concentration is inadequate as it is influenced by

diet.

• Screening using NGS and specific gene panels is becoming a

valuable diagnostic approach

CoQ10 Deficiency: Diagnosis

• Dietary contribution (3-5 mg per day) is minimal. The body relies on endogenous synthesis: supplementation is essential

in the treatment of CoQ10 deficiency

• Importance of CoQ formulation: soluble

formulations are better. Our patients both worsened their

symptoms when switched from vials to tablets (bioavailability of the

crystalline form is low and inconsistent, because of the poor solubility

and high molecular weight of this form)

• Now capsules containing oily formulation (absorption is enhanced in the presence of lipids)

• Remarkable safety profile (very few side effects).Safe at intakes of up 3000 mg/day

CoQ10 Deficiency: Treatment

� Coenzyme Q10 nephropathy should be considered when mitochondrial abnormalities in podocytes are present on EM, in familial cases of NS or in association with a multisystemic disease .

� Renal cortex, and cultured skin fibroblasts should be analysed for coenzyme Q10 content.

� If coenzyme Q10 deficiency is present, genetic studies should be performed.

� It is important that ubiquinone deficiency be recognized because this form of mitochondrion dysfunction respond well to oral coenzyme Q10

supplementation.

CoQ10 Deficiency: conclusions

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