J Neurol Res

Journal of Neurology Research, ISSN 1923-2845 print, 1923-2853 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Neurol Res and Elmer Press Inc

Journal website http://www.neurores.org

Original Article

Volume 6, Number 1, February 2016, pages 12-17

Mitochondrial Respiratory Chain Enzymatic Activities on Skin Fibroblasts in Patients With Mutant Glucocerebrosidase and PARK2 Genes

Figure 1. Scatter plots of values of enzymatic activities of complexes I - V in Parkinson’s disease (PD) patients and in controls. All respiratory chain enzyme activities are normalized to the activity of citrate synthase (CS). Horizontal bars represent the mean values.

**Table 1.** Details of PCR, Mismatched PCR and Restriction Fragment Length Polymorphisms
Mutation	Primer sequence	PCR product size (bp)	Restriction enzyme	Restriction position	Restriction product size (bp)
bp: base pairs; WT: wide-type; MT: mutant-type.
C1309delG	GBA-F9-11: AAC CAT GAT TCC CTA TCT TC GBA-R9-11: ACC ACC TAG AGG GGA AAG TG	1,290	SalI	WT 358 MT none	WT 358, 932 MT 1,290 WT/MT 358, 932, 1,290
IVS10-9_10 GT>AG	Hpy188III	WT 817, 889 MT 817, 889, 1005	WT 72, 401, 473 MT 72, 116, 285, 473 WT/MT 72, 116, 285, 401, 473
IVS2+1G>A	GBA-F2: CTT CTC TTC ATC TAA TGA CCC TGA GBA-R2: CCA CCG AGC TGT AGC C	1,065	HphI	WT 524, 665, 848, 880, 908, 977 MT 665, 848, 880, 908, 977	WT 28, 32, 69, 88, 141, 183, 524 MT 28, 32, 69, 88, 183, 665 WT/MT 28, 32, 69, 88, 141, 183, 524, 665
N386K	GBA-N386K-F: CTG GAC CGA CTG GAA CCT TGC CCT GTA (T is a mismatch nucleotide) GBA-N386K-R: 5’-GTG CCC GCC CTC CAC TCA CCT	160	RsaI	WT 116 MT none	WT 44, 116 MT 160 WT/MT 44, 116, 160
M1T	Parkin_1_E1_F: ACG TAA GAT TGC TGG GCC TGA AG Parkin_1_E1_R: GGC ACT TTG GCC CCG TCA TTG AC	392	NlaIII	WT 224, 267 MT 224	WT 43, 126, 223 MT 169, 223 WT/MT 43, 126, 169, 223

Table 1. Details of PCR, Mismatched PCR and Restriction Fragment Length Polymorphisms

Mutation

Primer sequence

PCR product size (bp)

Restriction enzyme

Restriction position

Restriction product size (bp)

bp: base pairs; WT: wide-type; MT: mutant-type.

C1309delG

GBA-F9-11:
AAC CAT GAT TCC CTA TCT TC
GBA-R9-11:
ACC ACC TAG AGG GGA AAG TG

1,290

SalI

WT 358
MT none

WT 358, 932
MT 1,290
WT/MT 358, 932, 1,290

IVS10-9_10 GT>AG

Hpy188III

WT 817, 889
MT 817, 889, 1005

WT 72, 401, 473
MT 72, 116, 285, 473
WT/MT 72, 116, 285, 401, 473

IVS2+1G>A

GBA-F2:
CTT CTC TTC ATC TAA TGA CCC TGA
GBA-R2:
CCA CCG AGC TGT AGC C

1,065

HphI

WT 524, 665, 848, 880, 908, 977
MT 665, 848, 880, 908, 977

WT 28, 32, 69, 88, 141, 183, 524
MT 28, 32, 69, 88, 183, 665
WT/MT 28, 32, 69, 88, 141, 183, 524, 665

N386K

GBA-N386K-F:
CTG GAC CGA CTG GAA CCT TGC CCT GTA (T is a mismatch nucleotide)
GBA-N386K-R:
5’-GTG CCC GCC CTC CAC TCA CCT

160

RsaI

WT 116
MT none

WT 44, 116
MT 160
WT/MT 44, 116, 160

M1T

Parkin_1_E1_F:
ACG TAA GAT TGC TGG GCC TGA AG
Parkin_1_E1_R:
GGC ACT TTG GCC CCG TCA TTG AC

392

NlaIII

WT 224, 267
MT 224

WT 43, 126, 223
MT 169, 223
WT/MT 43, 126, 169, 223

**Table 2.** Phenotypic and Genotypic Data of Parkinson’s Disease Patients and Controls
	Code	Gender	Age (years)	Causative gene	Mutation
SD: standard deviation.
Patients	1	Male	51	None	-
	2	Male	57	None	-
	3	Male	47	None	-
	4	Male	37	None	-
	5	Male	45	None	-
	6	Male	46	None	-
	7	Female	53	None	-
	8	Male	56	PARK2	exon 8 deletion
	9	Female	43	PARK2	p.Met1Thr
	10	Female	41	PARK2	Exon 8 deletion
	11	Male	56	GBA	IVS11-9,10 gt>ag
	12	Female	57	GBA	c.1309delG
	13	Female	41	GBA	p.Asp386Lys
	14	Female	49	GBA	IVS2+1g>A
Mean ± SD	48.5 ± 6.7
Controls	1	Male	53	None	-
	2	Female	45	None	-
	3	Female	59	None	-
	4	Female	46	None	-
	5	Female	45	None	-
Mean ± SD	49.6 ± 6.2

Table 2. Phenotypic and Genotypic Data of Parkinson’s Disease Patients and Controls

Code

Gender

Age (years)

Causative gene

Mutation

SD: standard deviation.

Patients

Male

None

Male

None

Male

None

Male

None

Male

None

Male

None

Female

None

Male

PARK2

exon 8 deletion

Female

PARK2

p.Met1Thr

Female

PARK2

Exon 8 deletion

Male

GBA

IVS11-9,10 gt>ag

Female

GBA

c.1309delG

Female

GBA

p.Asp386Lys

Female

GBA

IVS2+1g>A

Mean ± SD

48.5 ± 6.7

Controls

Male

None

Female

None

Female

None

Female

None

Female

None

Mean ± SD

49.6 ± 6.2

**Table 3.** Enzymatic Activities (Mean ± SEM) of Complexes I - V Corrected by Citrate Synthase Activity
	Control (N = 5)	Patients
SEM: standard error of measurement.
Complex I (/× 10³)	8.92 ± 0.55	10.78 ± 0.45	11.48 ± 0.50	10.09 ± 0.68	9.44 ± 0.71	10.57 ± 1.10
Complex II (/× 10³)	107.9 ± 11.57	117.4 ± 7.45	122.1 ± 13.77	112.6 ± 6.40	125.70 ± 6.25	102.70 ± 6.90
Complex III (/× 10²)	53.11 ± 3.69	51.69 ± 3.00	48.88 ± 2.60	54.50 ± 5.43	47.42 ± 11.66	59.81 ± 3.64
Complex IV (/× 10³)	27.15 ± 2.28	29.30 ± 1.31	31.61 ± 2.04	26.99 ± 1.21	27.61 ± 1.35	26.52 ± 2.01
Complex V (/× 10)	34.13 ± 2.39	32.95 ± 2.06	35.08 ± 3.96	30.83 ± 1.08	31.63 ± 2.41	30.23 ± 0.94
Citrate synthase (mOD/min)	13.32 ± 0.19	12.66 ± 0.13	12.46 ± 0.14	12.87 ± 0.21	12.97 ± 0.30	12.79 ± 0.31

Table 3. Enzymatic Activities (Mean ± SEM) of Complexes I - V Corrected by Citrate Synthase Activity

Control (N = 5)

Patients

All patients (N = 14)

No mutation (N = 7)

All mutations (N = 7)

PARK2 (N = 3)

GBA (N = 4)

SEM: standard error of measurement.

Complex I (/× 10³)

8.92 ± 0.55

10.78 ± 0.45

11.48 ± 0.50

10.09 ± 0.68

9.44 ± 0.71

10.57 ± 1.10

Complex II (/× 10³)

107.9 ± 11.57

117.4 ± 7.45

122.1 ± 13.77

112.6 ± 6.40

125.70 ± 6.25

102.70 ± 6.90

Complex III (/× 10²)

53.11 ± 3.69

51.69 ± 3.00

48.88 ± 2.60

54.50 ± 5.43

47.42 ± 11.66

59.81 ± 3.64

Complex IV (/× 10³)

27.15 ± 2.28

29.30 ± 1.31

31.61 ± 2.04

26.99 ± 1.21

27.61 ± 1.35

26.52 ± 2.01

Complex V (/× 10)

34.13 ± 2.39

32.95 ± 2.06

35.08 ± 3.96

30.83 ± 1.08

31.63 ± 2.41

30.23 ± 0.94

Citrate synthase (mOD/min)

13.32 ± 0.19

12.66 ± 0.13

12.46 ± 0.14

12.87 ± 0.21

12.97 ± 0.30

12.79 ± 0.31