Screening seven common mitochondrial mutations in 28 Egyptian patients with suspected mitochondrial...

Screening seven common mitochondrial mutations in 28 Egyptian

patients with suspected mitochondrial diseaseGhada M.M. Al-Ettribia, Laila K. Effata, Hala T. El-Bassyounib, Maha S. Zakib,Gamila Shanabc and Amr M. Karimc

Departments of aMedical Molecular Genetics, bClinicalGenetic Division of Human Genetics and GenomeResearch, National Research Center (NRC) andcDepartment of Biochemistry, Faculty of Science,Ain-Shams University, Cairo, Egypt

Correspondence to Laila K. Effat, MD, Departmentof Medical Molecular Genetics, National ResearchCenter (NRC), El-Bohouth St., Dokki, Cairo 12111,EgyptTel: + 20 233 387 953; fax: + 20 233 370 931/+ 20 100 233 9293; e-mail: [email protected]

Received 19 September 2012Accepted 26 September 2012

Middle East Journal of Medical Genetics

2013, 2:28–37

Aim

In this research, we aimed to study the clinical findings in a group of 28 Egyptian

patients with suspected mitochondrial disease and to test whether seven common

mitochondrial DNA (mtDNA) mutations causing mitochondrial disorders in different

populations are the same ones causing mutations in Egyptian patients.

Patients and methods

Twenty-eight Egyptian patients with suspected mtDNA disorders were subjected

to a thorough clinical examination, pedigree analysis, and biochemical investigations.

Neurophysiologic investigations were carried out including electroencephalogram,

electromyelogram, nerve conduction velocities, a complete eye evaluation including

electroretinogram and visual-evoked potential, a hearing test, brain MRI, and magnetic

resonance spectroscopy. Pathological examination on muscle biopsy stained by the

modified Gomori trichrome stain was also carried out. PCR-RFLP analyses were

carried out for the detection of seven (3243A4G, 3271T4C, 8334A4G,

8993T4G/C, 3256C4T, 4332G4A, and 12147G4A) mtDNA point mutations.

Results

All 28 patients showed variable multisystem affection. We divided the patients into

three groups according to the main findings and investigations. Group 1 included four

patients with suspected Leigh’s syndrome, group 2 included two patients with

suspected MELAS, and group 3 included 22 patients with suspected general

mitochondrial disorder. The molecular study indicated that the chosen point mutations

(3243A4G, 3271T4C, 8334A4G, 8993T4G/C, 3256C4T, 4332G4A, and

12147G4A) were not detected in this group of Egyptian patients.

Conclusion

The presence of other different mutations in either the nuclear or the mitochondrial

genomes might be the reason for the negative results obtained in this research.

Further confirmation by sequencing of mitochondrial and genomic genes is

recommended.

Keywords:

common mtDNA point mutations, mitochondrial respiratory chain disorders, molecular

diagnosis

Middle East J Med Genet 2:28–37& 2013 Middle East Journal of Medical Genetics2090-8571

IntroductionMitochondrial respiratory chain diseases (RCDs) are

a group of inherited disorders of energy metabolism.

Together, they form one of the most common groups of

inherited metabolic diseases, with a minimum birth

prevalence of 1 of 5000 (Calvo et al., 2006; Mancuso

et al., 2007; Neupert and Herrmann, 2007). The diagnosis

of mitochondrial RCDs remains a major challenge to the

clinician because of the varied clinical presentations,

frequent dependence on invasive testing procedures, the

huge genetic heterogeneity, and the absence of a reliable

screening or diagnostic biomarker that is both sensitive

and specific in all cases (Haas et al., 2007; Wong et al.,2010). Diagnostic criteria have been proposed that attempt

to take into account clinical manifestations, enzymatic

and physiologic analyses, tissue histochemical results,

levels of biochemical analytes, and DNA analysis for a

more reliable characterization of patients (Nissenkorn

et al., 1999; Bernier et al., 2002; Nonaka, 2002; Wolf and

Smeitink, 2002; Morava et al., 2006).

Many diseases involve multiple organ systems, in part

reflecting the dependence on the energy derived from

oxidative phosphorylation in a wide variety of tissues, and

this feature may be the initial indication of the correct

diagnosis. Any organ or tissue may be involved in this

group of diseases (Haas et al., 2007; Wong et al., 2010).

There are a number of ‘classic’ clinical syndromes with

stereotypical features such as MELAS (mitochondrial

myopathy, encephalopathy, lactic acidosis, and stroke-like

episodes), MERRF (myoclonic epilepsy and ragged red

fibers), Leigh’s syndrome (LS) (subacute necrotizing

encephalomyelopathy), and NARP (neurogenic muscle

weakness, ataxia, and retinitis pigmentosa). However, many

patients show only nonspecific features of developmental

28 Original article

2090-8571 & 2013 Middle East Journal of Medical Genetics DOI: 10.1097/01.MXE.0000422779.05483.d7

Copyright © Middle East Journal of Medical Genetics. Unauthorized reproduction of this article is prohibited.

mailto:[email protected]

delay or regression, further hindering accurate diagnosis

(McFarland and Turnbull, 2009; Wong et al., 2010).

Mitochondrial RCDs have a genetic etiology and this

genetic abnormality may be found in either mitochondrial

DNA (mtDNA) or nuclear DNA (nDNA) (Jacobs and

Turnbull, 2005; Greaves et al., 2006). Although molecular

testing is widely viewed as definitive, confirmation of the

diagnosis by molecular methods often remains a challenge

because of the large number of genes, the two-genome

complexity, and the varying proportions of pathogenic

mtDNA molecules in a patient, a concept termed hetero-

plasmy. Screening of common point mutations and large

deletions in mtDNA is typically the first step in the

molecular diagnosis (Wong et al., 2010). Although effective

treatments remain elusive, a definitive diagnosis is crucial

for appropriate symptom management, as well as accurate

prognostic and proper genetic counseling (Haas et al.,2007). Thus, identification of the most common mutation

in a population facilitates targeting and confirmation of the

diagnosis. In this respect, our aim was to choose seven

common mutations reported in different populations in

order to identify the existing panel of mutations in this

group of Egyptian patients with mitochondrial RCDs.

Participants and methodsParticipants and investigations

Twenty-eight Egyptian patients from 25 unrelated

families with suspected mtDNA disorders were included

in this study. Informed consent was obtained from all

parents according to the guidelines of the Ethical

Committee of the National Research Centre. All patients

were subjected to a thorough clinical examination,

pedigree analysis, and biochemical investigations [includ-

ing lactate, pyruvate, creatine phosphokinase (CPK),

ammonia, and other metabolic screening tests].

Neurophysiologic investigations were carried out includ-

ing electroencephalogram (EEG), electromyelogram

(EMG), nerve conduction velocities (NCV), complete

eye evaluation including electroretinogram and visual-

evoked potential, hearing test, brain MRI, and magne-

tic resonance spectroscopy (MRS), and pathological

examination on muscle biopsy stained by the modified

Gomori trichrome stain.

MethodsGenomic DNA was extracted from peripheral blood

leukocytes using a standard extraction method (Miller

et al., 1988). Patients were screened for seven of the most

common mutations in the mtDNA (3243A4G, 3271T4C,

8344A4G, 8993T4G/C, 3256C4T, 4332G4A, and

12147G4A) using PCR-RFLP analysis. The sequence of

primers and the restriction enzymes used are listed

in Table 1. Seven uniplex PCR reactions were carried out

in a 25ml total volume containing 10 ng genomic DNA,

0.2 mmol/l of each dNTP (Finnzyme, European Union), 1 U

Taq. Polymerase (Finnzyme, European Union), 1.2 pmol/ml of

the 3243 primers, 2.4 pmol/ml of the 3271 primers, 2.4 pmol/

ml of the 8344 primers, 2 pmol/ml of the 8993 primers, or

2.8 pmol/ml of the 3256, 4332, and 12147 primers. The

annealing temperatures used for each amplification were 53,

52, 55, 60, 50, 56, and 551C, respectively.

ResultsOf the 28 patients with suspected mitochondrial

disorders, 17 were females and 11 were males. Patients

were classified into three groups. Group 1 included four

patients with suspected LS (P1–4), group 2 included two

patients with suspected MELAS (P5 and 6), and group 3

included 22 patients with suspected general mitochon-

drial disorder (P7–28). Patients’ ages of onset ranged

from 4 months to 1 year in group 1, 7 months to 9 years in

group 2, and 2 months to 4 years in group 3 (mean age 1

year and 4.5 months).

P4 had 2 affected sibs who were normal until the age of

1.5 years; then, they developed fever, followed by sudden

loss of acquired milestones, and died at the age of 2.5

years. P8 and P10 each had one sib with the same

manifestations. P12 had two affected sibs: one with

microcephaly and moderate mental retardation (IQ: 58)

and the other with urethral stricture. P14 had one

affected sib with mental retardation. P18 had one sib

with floppiness since birth and died at 9 months of high

fever; also, her mother had an abortion in the first

trimester. The mother of P23 had an abortion in the

Table 1 Sequence of the primers and the names of the

restriction enzyme used in the study

Primer sequences

Restrictionenzymes

(Fermentas,European

union) References

3243A4G F: 50-CCT CCC TGTACG AAA GGA C-30

HaeIII King et al.(1992)

3243A4G R: 30-GTA ACA TGGGTA AGA TTA GCG-50

3271T4C F: 50- AGG ACA AGAGAA ATA AGG C-30

AflII Kim et al.2002

3271T4C R: 30- AAT TCC AGTCTC CAA GTT AAG GAG AAGAAT-50

8344A4G F: 50-CCC CCA TTA TTCCTA GAA CCA GGC G-30.

BanII Silvestri et al.(1993)

8344A4G R: 30-GGG TGT GGAGAA ATG TCA CTT TAC GGGG-50

8993T4G/C F: 50-CCG ACT AATCAC CAC CCA AC-30

HpaII Leshinsky-Silver et al.(2003)

8993T4G/C R: 30-TTC GGA GATGGA CGT GCT GT -50

3256C4T F: 50-GTT AAG ATGGCA GCG CCC GGT AAGCGC-30

HinPI Moraes et al.(1993)

3256C4T R: 30-AGT AAC ATGGGT AAG ATT AGC G-50

4332G4A F: 50-CCA TAC CCA TTACAA TCT CC-30

MaeI Sternberget al. (2001)

4332G4A R: 30-GAG CGT GACTAA AAA ATG GAC TC-50

12147G4A F: 50-CAC CTA TCCCCC ATT CTC C-30

Tsp509I Taylor et al.(2004)

12147G4A R: 30-GGA ATT AATGGC TCT TTC GAG TGT T-50

Study of mitochondrial disorders in Egyptians Al-Ettribi et al. 29


second trimester. P22a and b had a family history of a

matrilineal relative with epilepsy (Fig. 1a). P24 had a

family history of one matrilineal relative with hydro-

cephalus and neural tube defect and three other relatives

with mental retardation (Fig. 1b).

Patients of group 1 presented with either delayed motor

and mental milestones or loss of acquired milestones,

seizures, hypotonia with or without hyporeflexia, dysto-

nia, or muscle rigidity. P3 also had eye affection

manifested as ptosis, squint, and external ophthalmople-

gia, whereas P4 had dysmorphic features. EEG in all

patients showed either a bilateral focal epileptogenic

discharge or an epileptogenic dysfunction. The results of

EMG showed myopathy in two patients (P1 and P4),

with axonal demyelinating neuropathy in NCV of P4. The

results of brain MRI showed an abnormal signal of white

matter, basal ganglia, and midbrain with central and

cortical atrophic changes. All the patients had high blood

lactate levels and three of them (P1, P2, and P4) showed

a high lactate peak by the MRS (Table 2).

The two patients from group 2 had stroke and seizures.

One patient (P5) had hypertonia and hyperreflexia,

whereas the other (P6) had hypotonia and hyporeflexia.

P5 also had loss of acquired milestones and hepatomegaly.

The results of EEG in both patients indicated epilepto-

genic dysfunction. Brain MRI showed bilateral basal

ganglia degeneration, white matter affection, cortical

changes, and cerebellar atrophy. P5 had an abnormal

visual pathway in visual-evoked potential and high blood

lactate and CPK (Table 2).

Patients of group 3 (22 patients) had hypotonia with or

without hyporeflexia, muscle wasting, dystonia, brisk

reflexes, head nodding, drunken gait, hyperextensibility

of the fingers and wrist joint, or uncontrolled urination and

defecation. One patient (P11b) had hypertonia, hyper-

reflexia, Rocker bottom feet, and hyperextensibility of the

knee joint and fingers. Other manifestations found in this

group of patients were delayed mental and/or motor

milestones (19/22 patients), loss of acquired milestones

with or without failure of growth (3/22), mental retardation

(6/22), seizures (11), eye affection (nystagmus, squint,

cataract) (six), hearing loss (six), hepatomegaly with edema

(one), splenomegaly (one), heart disease (three), repeated

chest infection (one), dysmorphic features (three), micro-

cephaly (one), and dysphasia (one). The results of EMG

showed myopathy in 14 patients. NCV showed neuropathic

changes (demyelinating polyneuropathy or peripheral

polyneuropathy) (11/22). Ragged red fibers (RRF) were

found in muscle biopsy stained with Gomori trichome in

seven of 22 patients. EEG indicated focal epileptogenic

dysfunctions in 14 of 22 patients. For all 22 patients, the

results of brain MRI showed cortical changes with

myelination defect, cerebral and cerebellar atrophy, basal

ganglia degeneration, white matter signal with demyelina-

tion, hypogenesis of corpus callosum, thin corpus callosum,

a small retrocerebellar arachnoid cyst, or demyelination

around occipital horns (Fig. 2). Echo heart of two patients

(P18 and P21) showed a dilated hypertrophic left ventricle

and mild mitral regurge and cardiomyopathy. Patient 23

had an ECG signal of premature ventricular contraction

and right axis deviation, with Echo heart indicating dilated

cardiomyopathy. Biochemical investigations showed high

blood lactate (16/22), high blood pyruvate (9/22), high

blood CPK (10/22), high blood CPK-MB (3/22), and

high serum ammonia (1/22). Six of the patients in group 3

were brothers and sisters who came from three different

families (P11a and b, P19a and b, P22a and b). Each two

sibs had the same manifestations, except P11a and b; one

had delayed milestones and hypotonia and the other had

loss of acquired milestones, failure of growth, hypertonia,

and hyperreflexia (Table 2).

PCR-RFLP analyses for the detection of the seven

common mitochondrial mutations were negative in the

patients studied (Figs 3–9). In all cases, only a band

corresponding in size to that of the fragment containing

the normal nucleotide was observed: a 196 bp band for

the 2343A digested fragment (Fig. 3), a 172 bp band for

the 3271T fragment (Fig. 4), two bands (299 and 78 bp)

for the 8344A digested fragment (Fig. 5), a 554 bp band

for the 8993T fragment (Fig. 6), a 100 bp band for the

3256C fragment (Fig. 7), two bands (231 and 106 bp) for

the 4332G digested fragment (Fig. 8), and a 123 bp band

for the 12147G fragment (Fig. 9).

Figure 1

Two pedigrees (a) and (b) for patients 22 and 24, respectively, showingtheir family histories. In Fig. 1 (a), (a) under one index is for patient22a/F while (b) is for patient 22b/M.

30 Middle East Journal of Medical Genetics


Table 2 Clinical data of the 28 Egyptian patients

Suspectedsyndrome

Ageat onset Signs and symptom Clinical investigations Biochemical tests

1/M LS 4 m Delayed motor and mentalmilestones

Attack of convulsion and comaat 1 year

Myoclonic seizuresHypotoniaMuscle rigidityDystonia

EEG: bilateral focal epileptogenic dischargeEMG: myopathyBrain MRI: bilateral widely spread cerebral

white matter and basal ganglia areas ofabnormal signal intensity; central and corticalatrophic changes.

MRS: high lactate peakECG, Echo: normal

Normal blood and CSF lactateNormal metabolic screening

2/F LS 1 y Delayed developmentalmilestones

HypotoniaHyporeflexiaSeizures

EEG: epileptogenic dysfunctionBrain MRI: bilateral high signal of basal gangliaMRS: low NAA

(N-acetylaspartate)and high lactate

High blood lactate:4 (0.6–2.4 mmol/l)

3/F LS 1 y Delayed motor and mentalmilestones

HypotoniaHyporeflexiaDystoniaSeizuresPtosisSquintExternal ophthalmoplegia

EEG: epileptogenic dysfunctionBrain MRI: central atrophy; high signal

of the midbrain

High blood lactate/pyruvate

4/M LS 1 y Loss of acquired milestonesDysmorphic featuresHypotoniaSeizures

EEG: epileptogenic dysfunctionEMG: myopathyNCV: axonal demyelinating neuropathyBrain MRI: bilateral changes in the lenticular

nucleus and the red nucleus of the mid brainMRS: high lactate peak

High blood lactate

5/M MELAS 7 m Loss of acquired milestonesStrokeSeizuresHepatomegalyHypertoniaHyperreflexia

EEG: epileptogenic focusBrain MRI: bilateral basal ganglia degeneration

(lentiform); white matter affection; corticalchanges

VEP: abnormal visual pathway

High blood lactateHigh blood CPKNormal serum ammonia

6/F MELAS 9 y Attacks of loss of visionassociated with eitherdementia or hemiparesis,followed by dystonia

SeizuresHypotoniaHyporeflexia

EEG: active right occipitotemporalepileptogenic dysfunction

Brain MRI: mild cortical changes in bothoccipital and frontal lobes; cerebellar atrophy

Normal blood lactateNormal metabolic screening

7/F General MTdisorderwithoutopticatrophy

6 m Delayed developmentalmilestones

Dysmorphic featuresHypotoniaDysphasia

EEG: epileptogenic focusEMG: congenital myopathyNCV: demyelinating neuropathyBrain MRI: cortical changes with myelination

defect

High blood lactate:21 (4.5–19.8 mg/dl)

High blood pyruvate:0.8 (0.3–0.7 mg/dl)

High blood CPKNormal serum ammonia: 70

(68–136 mg/dl)8/F 8 m Delayed motor and mental

milestoneDysmorphic featuresIQ: 68HypotoniaHyporeflexiaSeizuresBilateral congenital cataract

EMG: myopathyBrain MRI: cerebral and cerebellar atrophy; mild

ventricular dilation

High blood lactateNormal blood CPK

9/F 1 y Delayed motor and mentalmilestones

HypotoniaSeizuresHearing lossNystagmusSquint

EEG: epileptogenic dysfunctionEMG: myopathyBrain MRI: cerebral changes; basal ganglia

degeneration

High blood lactateHigh blood pyruvate


HypotoniaSeizuresHearing lossSquint

EEG: bilateral focal epileptogenic dysfunctionMuscle biopsy: mitochondrial myopathy (RRF)Brain MRI: cerebral atrophyMRS: high lactate peak

High blood lactate:509 (4.5–19.8 mg/dl)

High blood CPK:261 (15–196 U/l)

11a/F 4 m Delayed motor milestonesWide base (drunken) gaitBilateral hyperextensibility of

the fingers and wrist jointHypotoniaNystagmusCongenital cataract

EEG: bilateral centrotemporal epileptogenicdysfunction

EMG and NCV: demyelinating polyneuropathyBrain MRI: increased deep white matter signal

with demyelination

High blood lactateHigh blood PyruvateNormal blood CPK

11b/M 1 y Loss of acquired skillsWide base (drunken) gait;

Rocker bottom feet

EEG: bilateral centrotemporal epileptogenicdysfunction

EMG and NCV: mild peripheral polyneuropathy

High blood lactateHigh blood CPK-MB:

35 (7–25 U/l)



Table 2 (continued)

Suspectedsyndrome


Hyperextensibility of knee jointand fingers

Failure of growthHypertoniaHyperreflexiaNystagmusCongenital cataract

Brain MRI: increased deep white matter signalwith demyelination

Normal blood CPK

12/F 1 y Loss of acquired skillsSeizuresHypotoniaHyporeflexiaIQ: 58Nystagmus

EEG: epileptogenic focusEMG and NCV: demyelinating polyneuropathyMuscle biopsy: mitochondrial myopathy (RRF)Brain MRI: periventricular white matter

demyelination; basal ganglia degeneration

High blood lactateHigh blood pyruvateHigh blood CPK


HypotoniaHyporeflexia

EMG and NCV: peripheral polyneuropathyMuscle biopsy: mitochondrial myopathy (RRF)Brain MRI: basal ganglia degeneration

14/M 2 y Delayed motor and mentalmilestones


EEG: epileptogenic dysfunctionEMG and NCV: demyelinating polyneuropathy,

myopathyBrain MRI: hypogenesis of corpus callosum

High blood lactateHigh blood CPK


Dysmorphic featuresRepeated chest infectionMental retardation (IQ: 70)


EMG and NCV: demyelinating polyneuropathyMuscle biopsy: mitochondrial myopathy (RRF)Brain MRI: periventricular demyelination

16/F 4 y Delayed motor milestonesMental retardationDystoniaHypotonia

Brain MRI: thin corpus callosum; smallretrocerebellar arachnoid cyst

High blood LactateHigh blood pyruvate


SNHLIQ: 67Hypotonia

EMG: myopathyBrain MRI: demyelination around occipital horns

High blood pyruvate: 103(39–82mmol/l)

High blood CPK: 4046(15–196 U/l)


Low birth weightIQ: 60Generalized tonic–clonic

seizuresHypotoniaHyperreflexiaMuscle wastingMild conductive hearing lossMild splenomegaly

EEG: epileptogenic dysfunctionEMG aand NCV: mild axonal neuropathic

changesBrain MRI: cerebral atrophy; white matter

demyelinationEcho heart: dilated hypertrophic left ventricle,

mild mitral regurge

High blood lactateHigh blood CPK: 1452

(15–196 U/l)High serum ammoniaNormal level of the following,

which rules out lysosomaldisorders: B-glucocerebrosidase,sphingomyelinase,chitotriosidase

19a/M 1 y Delayed motor and mentalmilestones

Hearing lossHeart defectHistory of bleeding

(ecchymosis)HypotoniaHyporeflexia

EMG: myopathyMuscle biopsy: mitochondrial myopathy (RRF)Brain MRI: cerebral atrophy; basal ganglia

degeneration

High blood CPK

19b/F 1 y Delayed motor and mentalmilestones

History of bleeding(ecchymosis)

Hearing lossHeart defectHypotoniaHyporeflexia

EMG: myopathyMuscle biopsy: mitochondrial myopathy (RRF)Brain MRI: cerebral atrophy; basal ganglia

degeneration

High blood CPK

20/M 5 m Delayed motor and mentalmilestones

HypotoniaBrisk reflexesSeizures

EEG: Eeileptogenic dysfunctionEMG: MyopathyBrain MRI: cerebral atrophy; basal ganglia

degeneration

Normal blood lactateHigh blood pyruvate: 17.7

(0.3–0.7 mg/dl)High serum ammonia103

(10–47 mmol/l)Normal amino acid and

metabolic screening21/F 3 y Delayed motor and mental

milestonesEdemaSeizuresHypotoniaHyporeflexiaMicrocephalyHepatomegaly

EEG: Epileptogenic dysfunctionEMG: myopathyNCV: neuropathic changesMuscle biopsy: mitochondrial myopathy (RRF)Brain MRI: cerebral atrophy; basal ganglia

degenerationEcho heart: csardiomyopathy



Table 2 (continued)

Suspectedsyndrome


22a/F 2 y Loss of acquired milestonesGeneralized convulsionsDevelopmental quotient: 21%

EEG: mild left frontotemporal epileptogenicdysfunction

EMG: myopathyNCV: average conduction and decreased

amplitudeBrain MRI: cerebral atrophy; basal ganglia

degeneration

High blood lactateHigh blood CPK-MB:

38 (0–25 U/l)High serum ammonia

22b/M 1.5–2 y Delayed motor and mentalmilestones

Head noddingGeneralized convulsions (tonic)HypotoniaBrisk reflexes

EEG: epileptogenic dysfunctionEMG: myopathyBrain MRI: cerebral atrophyMRS: high lactate signal

High blood lactateHigh blood pyruvateHigh blood CPK-MB

23/F 2 m Delayed motor and mentalmilestones

Congenital heart defectGeneralized convulsions (tonic)Hypotonia

EEG: epileptogenic dysfunctionEMG: MyopathyNCV: neuropathic changesBrain MRI: cerebral atrophyMRS: high lactate signalEcho heart: dilated cardiomyopathyECG: premature ventricular contraction,

right axis deviation


24/M 8 m Delayed motor and mentalmilestones

HypotoniaDystoniaBrisk reflexesCannot control urination and

defecation

EMG: myopathyNCV: neuropathic changesBrain MRI: cerebellar atrophy and basal ganglia

high signals


25/F 2 m Delayed developmentalmilestones

IQ: 80Myoclonic jerksBrisk reflexesGeneralized tonic–clonic

seizuresHypotonia

EEG: multifocal epileptogenic activityEMG: myopathyBrain MRI: periventricular deep white matter

changes; thin corpus callosum; corticalatrophic changes

High blood lactate:21 (5–12 mg/dl)

High blood pyruvate:0.9 (0.3–0.7 mg/dl)

Normal metabolic screeningNormal very long-chain fatty

acids

CPK, creatine phosphokinase; CSF, cerebrospinal fluid; EEG, electroencephalogram; EMG, electromyelogram; IQ, intelligent quotient; LS, Leigh’ssyndrome; m, months; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; MRS; magnetic resonancespectroscopy; MT, mitochondrial; NCV, nerve conduction velocities; RRF, ragged red fibers; VEP, visual-evoked potential; y, years.

Figure 2

Axial T2 for patient 19 showing a high signal of the lentiform nucleus; dilated lateral ventricles show central atrophic changes and basal gangliadegeneration, cortical atrophic changes, and cerebellar atrophy.



Figure 3

Eight percent polyacrylamide gel electrophoresis analysis of HaeIIIdigestion of the PCR product for the detection of mutation 3243A4G.Lanes 1 and 2: undigested PCR product (238 bp). Lanes 3–8:digested PCR products from six different patients showing thedigestion pattern of normal fragments only (196, 37, and 32 bp). Theenzyme cleaves mutant fragments, not present, into 97, 72, 37, and32 bp bands. M1: 50 bp DNA ladder. M2: ØX174 HaeIII molecularweight marker.

Figure 4

Eight percent polyacrylamide gel electrophoresis analysis of AflIIdigestion of the PCR product for the detection of mutation3271T4C. Lane 1: undigested PCR products (172 bp). Lanes 2–6:digested PCR products from five different patients showing thedigestion pattern of normal fragments only (172 bp). The enzymecleaves mutant fragments, not present, into 142 and 30 bp bands. M1:50 bp DNA ladder. M2: ØX174 HaeIII molecular weight marker.

Figure 7

Eight percent polyacrylamide gel electrophoresis analysis of HinpIdigestion of the PCR product for the detection of mutation 3256C4T.Lanes 1 and 2: undigested PCR products (124 bp). Lanes 3–9:digested PCR products from seven different patients showing thedigestion pattern of normal fragments only (100, 13, and 11 bp). Theenzyme cleaves mutant fragments, not present, into 111 and 13 bpbands. M1: 50 bp DNA ladder. M2: ØX174 HaeIII molecular weightmarker.

Figure 8

Eight percent polyacrylamide gel electrophoresis analysis of MaeIdigestion of the PCR product for the detection of mutation 4332G4A.Lanes 1–4: undigested PCR products (338 bp). Lanes 5–8: digestedPCR products from four different patients showing the digestionpattern of normal fragments only (231 and 106 bp). The enzyme doesnot cleave mutant fragments, not present. M1: 50 bp DNA ladder.M2: ØX174 HaeIII molecular weight marker.

Figure 5

Ten percent polyacrylamide gel electrophoresis analysis of BanIIdigestion of the PCR product for the detection of mutation8344A4G. Lane 1: undigested PCR product (418 bp). Lanes 2–7:digested PCR products from six different patients showing thedigestion pattern of normal fragments only (299, 78, and 41 bp). Theenzyme cleaves mutant fragments, not present, into 299, 52, 41, and26 bp bands. M1: 50 bp DNA ladder. M2: ØX174 HaeIII molecularweight marker.

Figure 6

Eight percent polyacrylamide gel electrophoresis analysis of HpaIIdigestion of the PCR product for the detection of mutation 8993T4C.Lane 1: undigested PCR products (554 bp). Lanes 2–5: digested PCRproducts from four different patients showing the digestion pattern ofnormal fragments only (554 bp). The enzyme cleaves mutant fragments,not present, into 347 and 207 bp bands. M1: 50 bp DNA ladder. M2:ØX174 HaeIII molecular weight marker.



DiscussionThe classification of mitochondrial disease is difficult.

A purely clinical classification can be helpful. However,

there is often considerable clinical variability and many

affected individuals do not fit neatly into one particular

disease category. The patients studied were divided into

three groups according to the clinical manifestations.

Four patients fulfilled the criteria of suspected LS. Their

symptoms began at the ages of 3 months and 1 year, and

the majority of them presented with abnormalities in the

central and peripheral nervous systems without the

involvement of other tissues, a feature that characterizes

LS (Yang et al., 2006; Finsterer, 2008), which has a typical

age of onset between 3 and 12 months (Thorburn and

Rahman, 2011). The neurological features in LS included

hypotonia, hyporeflexia, dystonia, muscle rigidity, and

seizures (myoclonic or generalized tonic–clonic) (Tsuji

et al. 2003; Thorburn and Rahman, 2011). The results of

neuroimaging showed focal and necrotizing lesions of the

basal ganglia or midbrain that was responsible for the

neurological abnormalities, reported in other studies

(Yang et al., 2006; Finsterer, 2008). One of the patients

also presented with squint, ptosis, and ophthalmoplegia

that were reported as brainstem, cerebral, and ophthal-

mologic symptoms associated with Leigh or Leigh-like

syndromes (Rahman et al., 1996; Debray et al., 2007;

Finsterer, 2008). Dysmorphic features were found in one

patient (P4). The lactate level was increased in blood

and/or cerebrospinal fluid in all four patients, a finding

present in most LS cases (Finsterer, 2008).

In the two patients with suspected MELAS, the

symptoms started at the age of 7 months and 9 years.

The onset of MELAS was initially described to occur in

the age range from younger than 2 years to older than 60

years, although almost 70% of patients presented with

initial symptoms between 2 and 20 years of age (Sproule

and Kaufmann, 2008). They had stroke attacks and

seizures, which are invariant criteria of the disease

(DiMauro and Hirano, 2010). P6 presented with stroke-

like episodes of transient cortical blindness associated

with dementia and hemiparesis preceded by dystonia.

This is in agreement with the findings of Sproule and

Kaufmann (2008). The third criterion for the diagnosis of

MELAS is lactic acidosis and/or RRF (DiMauro and

Hirano, 2010), and only one patient of this group (P5)

had a high blood lactate level. High blood and/or

cerebrospinal fluid lactic acid is an almost universal

finding, occurring in 94 of 101 (94%) patients in the study

of Hirano and Pavlakis (1994), seizures in 97 of 102

(96%), and stroke-like events in 106 of 107 (99%). The

typical results of brain MRI of MELAS were found in

terms of asymmetric lesions of the occipital and parietal

lobes that mimic ischemia that are often restricted to the

cortex with relative sparing of deep white matter

(Sproule and Kaufmann, 2008). However, the results of

the two patients indicated the presence of cortical

changes in P6 that occurred in both occipital and frontal

lobes, and were associated with cerebellar atrophy. In P5,

the cortical atrophy was associated with bilateral basal

ganglia degeneration and white matter affection.

The age of onset in group 3 ranged between 2 months

and 4 years; the mean age was 1.5 years. Mitochondrial

diseases may present at any age of onset (Haas et al.,2007). Seventy percent of MELAS cases have an onset at

less than 2 years to more than 20 years (Sproule and

Kaufmann, 2008), MERRF may occur in childhood

(DiMauro and Hirano, 2009), LS typically occurs

between 3 and 12 months of age and a later onset (41

year or in adulthood) occurs in up to 25% of cases

(Goldenberg et al., 2003), and NARP may occur in early

childhood (Thorburn and Rahman, 2011).

The patients in group 3 did not fulfill the clinical criteria

of any specific classical syndrome. Twelve patients

showed multisystem involvement (brain and muscles in

association with ear, eye, spleen, liver, heart, chest, or

blood), reflecting, in part, the dependence on energy

derived from oxidative phosphorylation in a wide variety

of tissues, a feature that is a hallmark of mitochondrial

diseases and may be the initial indication for the correct

diagnosis (McFarland and Turnbull, 2009; Wong et al.,2010). However, they manifested with the common

symptoms and signs of mitochondrial diseases that

include seizures (generalized convulsion and generalized

tonic–clonic), myoclonic jerks, unexplained hypotonia in

newborns, infants, or young children, dystonia, basal

ganglia diseases, and dilated cardiomyopathy with muscle

weakness, in conjunction with other nonspecific symp-

toms of mitochondrial disease such as hearing loss, axonal

neuropathy, and unexplained cerebral and cerebellar

atrophy (Haas et al., 2007; McFarland and Turnbull,

2009). It has been reported that when the nonspecific

symptoms present in combination, the likelihood of

a mitochondrial disorder increases particularly if the

nonspecific features involve different organ systems as in

our patients (Haas et al., 2007; Debray et al., 2008).

Sixteen of the patients in group 3 had high blood lactate

and/or high lactate peak on MRS and eight patients had high

Figure 9

Eight percent polyacrylamide gel electrophoresis analysis of Tsp509Idigestion of the PCR product for the detection of mutation12147G4A. Lane 1: undigested PCR products (144 bp). Lanes2–7: digested PCR products from six different patients showing thedigestion pattern of normal fragments only (123 and 21 bp).The enzyme cleaves mutant fragments, not present, into 74, 54, and21 bp bands. M1: 50 bp DNA ladder. M2: ØX174 HaeIII molecularweight marker.



blood pyruvate. Despite their lack of specificity, an elevated

plasma lactate or pyruvate level could be an important

marker of mitochondrial disease (Haas et al., 2007).

Seven of the 22 patients, with onsets at 1 or 3 years,

showed abnormal subsarcolemmal accumulations of

mitochondria on skeletal muscle biopsy stained with

Gomori trichrome, a unique feature of mitochondrial

disease described as the ‘RRF’. However, RRFs are rarely

seen in early childhood, when subsarcolemmal accumula-

tion of mitochondria may be mild or absent. In addition,

abnormal accumulation of mitochondria may be absent in

patients with proven mitochondrial disease such as LS

and NARP (McFarland and Turnbull, 2009; Wong et al.,2010; Thorburn and Rahman, 2011).

The nonspecific features of mitochondrial diseases makes

studies of family history difficult. The clinical variability

among siblings of P4, P12, P14, and P18 is a common

finding in mitochondrial disease caused by mtDNA

defects. This is believed to reflect the mitochondrial

‘genetic bottleneck’ (Chinnery and Schon, 2003), in

which a decrease in the number of mtDNA repopulating

the offspring occurs in the early stages of development

and causes a sampling effect and accounts for the rapid

changes in heteroplasmy levels between offspring (Turnbull

et al., 2010; Yu-Wai-Man and Chinnery, 2011). Also, the

variability in the symptoms between probands and their

maternal relatives, similar to that found in P22a and b

and P24, may be because of the presence of variable

heteroplasmic levels of the mtDNA mutations between

them. The presence of nonsymptomatic maternal relatives

of the other patients may be because of variations in the

expression of the disease or in the presence of low levels of

heteroplasmic mutations (Chinnery et al., 1999; Turnbull

et al., 2010).

Determining which one of dozens, if not hundreds, of

genes spanning two genomes responsible for mitochon-

drial dysfunction in a given patient is a challenge (Haas

et al., 2008). There are a number of recurrent mtDNA

point mutations, including the 3243A4G transition in

the mitochondrial transfer RNA Leucine1 gene (tRNA-

Leu (UUR); OMIM#590050), which is the most

common mtDNA point mutation. It accounts for more

than 85% of patients with MELAS and for a large number

of progressive external ophthalmoplegia patients who do

not have large-scale deletions. The less common

3271T4C mutation in the same gene was found in

about 10% of MELAS patients (DiMauro and Hirano,

2010; Krishnan et al., 2010). The point mutation

8344A4G in the tRNALys gene (OMIM#590060) has

been found in more than 85% MERRF patients (DiMauro

and Hirano, 2009). The 8993T4G/C mutations in the

mitochondrial ATP synthase subunit 6 gene (MT-ATPase 6;

OMIM#516060) are found in about 10–30% of individuals

with maternally inherited LS (Rahman et al., 1996; Makino

et al., 1998; Thorburn and Rahman, 2011), whereas the same

mutations account for more than 50% in NARP patients

with elevated blood lactate concentrations (Melone et al.,2004; Taylor et al., 2004; Rantamaki et al., 2005; Thorburn

and Rahman, 2011).

In their last update in 4 September 2012, the MITOMAP

website reported a ‘confirmed’ status for the 3256C4T

and 3271T4C mutations with MELAS, the 4332G4A

mutation with encephalopathy/MELAS overlapped

disease, and the 12147G4A mutation with MERRF-

MELAS/cerebral edema overlapped disease (http://mitomap.org/bin/view.pl/MITOMAP/ClinicalPhenotypesRNA) (MITO-

WEB, 2012). The confirmed status indicates that at least

two or more independent laboratories have published

reports on the pathogenicity of these mutations and it is

generally accepted by the mitochondrial research commu-

nity as being pathogenic (Bataillard et al., 2001; Sternberg

et al., 2001; Melone et al., 2004; Taylor et al., 2004; Nishigaki

et al., 2010). Considering the reported literature, we chose

these common mutations to verify whether these muta-

tions are also common in the Egyptian population.

The molecular analysis of the 28 patients did not indicate

any of the common mutations associated with mitochon-

drial respiratory chain disorders. A different genetic

pattern with different mutations responsible might be

found in Egyptians. The mutations could be very

different from that reported in the literature; thus,

further sequencing of the mitochondrial DNA is recom-

mended in order to unveil these mutations in Egyptian

patients with mitochondrial disorders. Also, the low-level

heteroplasmy of some mtDNA mutations in peripheral

blood samples could be responsible for a false-negative

result (Haas et al., 2008). The 3243A4G MELAS

mutation can be found in about 50% of the cases when

blood samples are used for PCR-RFLP. This is believed

to be the result of the rapidly replicating ability of the

blood cells, which will gradually eliminate the leukocytes

with a higher mutation load (Sue et al., 1998). However,

patients with the 8344A4G MERRF mutation have a

high percentage of mutant mtDNA (90%) in blood and

muscle and the degree of heteroplasmy is evenly

distributed between different organs (Lertrit et al.,1992; Tanno et al., 1993; Oldfors et al., 1995; Brinckmann

et al., 2010). Furthermore, white blood cells or any other

tissue type can be used to test for the 8993T4G/C

mutations (Thorburn and Rahman, 2011) as they do not

show any significant variation in the mutation load among

tissues (White et al., 1999).

ConclusionEstablishing a specific diagnosis in a patient with the

suspected mitochondrial disease is a complex endeavor that

requires the integration of clinical assessments, family

history, electrophysiological investigations, biochemical test-

ing, histopathological examination, and molecular testing.

Close collaboration between primary clinicians, geneticists,

pathologists, other clinical specialists, and diagnostic labora-

tories with expertise in mitochondrial biochemical and

molecular testing is critical to maximize the likelihood of

establishing a correct diagnosis. The negative molecular

results of the seven chosen common mutations in our study

could be a result of the presence of other mutations in either

of the two genomes or a result of the low heteroplasmic

levels of the mtDNA mutations in leukocytes.



http://mitomap.org/bin/view.pl/MITOMAP/ClinicalPhenotypesRNA

http://mitomap.org/bin/view.pl/MITOMAP/ClinicalPhenotypesRNA

AcknowledgementsConflicts of interestThere are no conflicts of interest.

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