3.3.1 Patients and controls
Patients with m.3243A>G mutation were selected from the MRC Centre Mitochondrial Disease Patient Cohort. Only six patients with this mutation had serial skeletal muscle biopsies available for use in this study (Table 3.1). Skeletal muscle biopsies collected from these patients for diagnostic purposes were approved for access by the Newcastle and North Tyneside 1 committee (REC 2005/202). Additional skeletal muscle biopsies required for this project were collected under REC reference 11/NE/0337 (Newcastle and North Tyneside 2), with informed signed consent from all participants, and stored at the NHS Highly Specialised Rare Mitochondrial Disease Laboratory in Newcastle (REC 08/H0906/28+5).
Three skeletal muscle biopsies were obtained from controls for use in this project (Table 3.2). Tissue from controls 1 and 2 was obtained following orthopaedic surgery with patient consent for the donation of tissue for use by Newcastle University for research purposes (REC 09/H1010/20 (NorthWest – Haydock. R&D number 5004)). Access to control 3 (disease control) was granted under REC reference 2005/202 as excess to diagnostic requirements.
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Table 3.1: Patients with m.3243A>G selected from the MRC Centre Mitochondrial Disease Patient Cohort. GI=Gastrointestinal; RQ=right
quadriceps; LQ=left quadriceps; LG=left gastrocnemius; NMDAS=Newcastle Mitochondrial Disease Adult Scale; *R = retrospective clinical data only. Scaled NMDAS scores are recorded within one year of the muscle biopsy date. Where patient biopsies were collected before 2005, no NMDAS data is available and so retrospective scoring is required (*R).
Patient Interval (years)
Biopsy one Biopsy two
Age Muscle Symptoms NMDAS Age Muscle Symptoms NMDAS
P1 (F) 4.0 54.6 LG Mild hearing loss, GI dysmotility
4.0 58.6 LQ Mild hearing loss, GI dysmotility, ataxia
4.2
P2 (F) 11.2 23.9 LQ Ataxia *R 35.1 LQ Ataxia, deafness, SLE, myopathy,
fatigue
8.5
P3 (M) 13.6 32.8 LQ Asymptomatic *R 46.4 RQ Asymptomatic 0.0
P4 (M) 13.9 40.9 LG Deafness, diabetes, fatigue *R 54.8 RQ Deafness, diabetes, fatigue, ataxia, myopathy, dysphonia/dysarthria
12.0
P5 (F) 14.2 29.3 LQ N/A *R 43.5 RQ Diabetes, deafness, GI dysmotility,
LVH
13.8
P6 (F) 14.3 59.1 RQ Deafness, LVH, ptosis, myopathy, fatigue,
migraine,
*R 73.4 LQ Deafness, ataxia, LVH, ptosis,
myopathy, fatigue
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Control (sex) Age at biopsy Classification
C1(M) 18 Orthopaedic surgery
C2(M) 46 Orthopaedic surgery
C3(F) 63 Disease control
Table 3.2: Control cases used for quantitative immunofluorescent analysis of complex I and complex IV in skeletal muscle biopsies. Skeletal muscle biopsies were obtained from
three controls, either during orthopaedic surgery or as excess to diagnostic requirements.
3.3.2 Post mortem tissue
Tissue was collected post mortem from five patients with m.3243A>G mutation (Table 3.3). Samples from multiple muscles from each case were collected and stored at the NHS Highly Specialised Rare Mitochondrial Disease Laboratory in Newcastle (REC 2005/202). DNA was extracted from muscle blocks as described in section 2.7.2 and stored at -80°C until use for molecular genetic analysis.
3.3.3 NMDAS
All recorded NMDAS data for these six patients was collated for analysis of clinical disease progression. Multiple NMDAS assessments were available for all but one patient (P6). Due to the subjective nature of part one of the NMDAS assessment, scores from this section were removed from the final analysis, leaving total NMDAS scores calculated from sections two and three only. Total scores were scaled to account for unassessed fields by dividing the total score by the number of assessed fields, and then multiplying by the total number of fields.
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Patient (sex) Age at death Muscles biopsied Clinical details
PM1 (F) 59 Biceps, diaphragm, gastrocnemius, psoas, quadriceps, tibialis,
triceps
MELAS, MIDD, pseudo-obstruction
PM2 (M) 45 Biceps, diaphragm, gastrocnemius, paraspinal, psoas,
quadriceps, tibialis, triceps
MELAS *1
PM3 (M) 35 Biceps, psoas, quadriceps Myopathy, cardiomyopathy, renal
tubule acidosis
PM4 (M) 30 Biceps, quadriceps Diabetes, ptosis, PEO, cardiomyopathy,
deafness
PM5 (M) 20 Diaphragm, psoas Mild deafness *2
Table 3.3 Post-mortem cases with m.3243A>G mutation. Multiple muscles were biopsied from six post-mortem cases with m.3243A>G. Cases
included had at least two different muscle types biopsied. MELAS = mitochondrial encephalopathy, lactic acidosis and stroke-like episodes; MIDD = maternally inherited diabetes and deafness; PEO = progressive external ophthalmoplegia. *1 previously published case = patient 3 in
Lax et al. (2016). *2 previously published case = case 1 in Ng et al. (2016).
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All statistical modelling was performed by Dr. John Grady. Box-Cox (Box and Cox, 1964) indicated the square root was the optimal transform for total NMDAS scores in order to satisfy normality assumptions. The transformed data were then used in a random effects mixed model where total NMDAS scores were the dependent variables and age at
assessment was the sole independent variable. The model included a random effect of the gradient with respect to age, which generated a gradient for each individual patient. This model was constrained to have no intercept, such that the NMDAS score at birth is fixed at zero. The rate of clinical progression for each patient is represented by the gradient
calculated by the model.
3.3.5 Immunofluorescent staining of cryosectioned skeletal muscle biopsies
10µm sections were cut from patient and control skeletal muscle biopsies and left to dry before storing at -80°C. Sections were allowed to dry at room temperature for up to one hour prior to beginning the protocol outlined in section 2.5.2. Fluorescent images were acquired at 20x magnification using an Axio Imager M1 microscope (Carl Zeiss) and
processed with AxioVison software (Carl Zeiss). Images were analysed using Imaris (version 7.6.5, Bitplane) and R script as described in sections 2.5.5 and 2.5.7.
3.3.6 Cloning (to make plasmid p.7D1. β2M)
A plasmid construct was engineered for use as a standard curve. A 275bp portion of the β2M gene (GenBank accession number NG_012920.1) was amplified by forward primer 5’-
TCCAAAGATTCAGGTTTACTCACG-3’ (nt.8941-nt.8964) and reverse primer 5’-
CCCACTTAACTATCTTGGGCTGT-3’ (nt.9215 -nt.9193). Plasmid p7D1 (Rygiel et al., 2015), containing a portion of the MT-ND1 gene, was cut with Sma1 restriction enzyme and β2M template inserted by blunt-ended ligation to produce plasmid p7D1.β2M (7469bp). Incorporation of the β2M sequence was confirmed by Sanger sequencing as outlined in section 2.10.9.
3.3.7 Pyrosequencing for determination of m.3243A>G heteroplasmy level
The PCR and pyrosequencing protocols are outlined in section 2.9. Patient DNA samples were split into two batches for pyrosequencing. P1, P2 and P3 were run on the same plate
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and P4, P5, and P6 were batched together on a separate plate. Each patient sample was diluted to approximately 10ng/µl and loaded in triplicate alongside a positive control (85% heteroplasmy), negative control (0% heteroplasmy) and a no-template control (no DNA). m.3243A>G heteroplasmy level was calculated using Pyromark Q24 software as described by White et al. (2012).
3.3.8 Real-time PCR assay for determination of mtDNA copy number
A six-point standard curve comprising a 1:10 serial dilution of p.7D1.β2M was prepared and loaded in triplicate onto each sample plate. Patient DNA samples were first diluted to 10ng/µl concentration for the β2M assay, and then diluted 1:100 in nuclease-free water for the MT-ND1 assay. Each patient sample was loaded in sextuplicate onto six replicate plates, as required for a 10% mean detectable difference in mtDNA copy number (Grady et al., 2014). As for the pyrosequencing protocol, patients were batched and serial biopsies were loaded onto the same plate. Further details of the MT-ND1 and β2M assays are provided in sections 2.8.3 and 2.8.4, respectively.