Mitochondrial DNA point mutations and relative copy number in 1363 disease and control human brains

Coverage and quality of assembled mitochondrial genomes

Complete assembly of the mitochondrial genome was obtained in all cases, with a mean
read depth across the genome of 289 (SD?=?169.8) (range 66–1328) and a mean base quality
score of 36.8 (sd?=?0.25) (range 36.0–37.5) (Additional file 1: Figures S1 and S2). There was no difference in mean read depth or mean base quality
score for any group vs controls (Additional file 1: Figure S3).

Haplogroup associations

Haplogroups and phylogenetic relationships were determined for all 1363 samples (Fig. 1). There was no difference in major overall haplogroup frequency when compared to
2360 UK population controls (Additional file 1: Table S2), confirming the accuracy of haplogroup calling and that the cohort as
representative of the UK population. We saw no association between any disease cohort
and specific haplogroups in our study (Additional file 1: Table S3).

Fig. 1. Phylogenetic tree of the 1363 mtDNA sequenced derived from the MRC Brain Tissue Resource.
Major haplogroups are shown

Homoplasmic variants

One thousand, nine hundred twenty-three homoplasmic variants were detected within
the cohort, with a mean of 22.9 (sd?=?10.8) variants per sample. Four hundred sixty-seven
variants were defined as ‘common’ (minor population Allele Frequency, MpAF 0.05 within
their haplogroup), and included known haplogroup defining variants. One thousand,
four hundred fifty-six homoplasmic variants were defined as rare (MpAF 0.05 within
their haplogroup). Twenty-five of the rare variants were novel, not seen in the NCBI
database (n?=?30,506), MITOMAP (n?=?30,589) or the 1000 genomes database. Four of the novel variants were in non-coding
regions, two were in rRNA genes, and 19 were synonymous (Additional file 1: Table S4).

Association with disease – common homoplasmic variants

Here we considered all homoplasmic variants, and a subgroup analysis of all non-synonymous
homoplasmic variants. We saw no evidence of a disease association with any single
variant, the burden of homoplasmic variants in any gene, nor the burden of homoplasmic
variants in groups of genes forming a respiratory chain complex (Additional file 1: Figures S4–S8, Additional file 1: Table S5). However, when stratifying by age, there was a trend towards young onset
AD cases (age of death 60) having a greater number of total variants in MT-TR compared to controls (6/13 vs 9/139) (p?=?0.002) (Additional file 1: Figure S5).

Association with disease – rare homoplasmic variants

No single rare homoplasmic variant was present at greater frequency in any disease
compared to controls (Additional file 1: Figure S8). There was a trend towards a greater number of rare homoplasmic point
mutations in two genes in AD compared to controls; MT-RNR1 (AD; 30/282 (10.6%), Controls; 16/344 (4.7%)) (p?=?0.005)) and again MT-TR (AD; 6/282 (2.1%), Controls; 0/344) (p?=?0.008), although both failed to reach significance at the corrected threshold of
p?=?0.0014. (Additional file 1: Figure S8, Additional file 1: Table S5). When stratified by age, this suggested that the trend towards an excess
burden of rare homoplasmic variants in MT-RNR1 was likely driven by variants in young onset AD cases vs controls (AD: 9/53 (16.9%),
Controls (2/65), p?=?0.012 (3%) (Additional file 1: Figure S9). We also saw that young onset PD-DLB cases (death aged 70) had a significantly
greater number of rare homoplastic mutations in MT-CO2 (PD-DLB: 5/23 (21.7%), Controls: 5/213 (2.3%), p?=?0.0010 (Additional file 1: Figure S9). The majority of the variants in MT-CO2 in both cohorts were in non-coding D-loop, but when combined this did not reach the
corrected threshold for significance. There was no association between any rare non-synonymous
variant, nor the burden of rare non-synonymous variants in any gene or respiratory
chain complex in any disease group vs controls.

Heteroplasmic variants

Three hundred eleven heteroplasmic variants (10% MAF) were detected (mean HF?=?7%,
sd?=?1.0), in 440 cases, with 10 of these variants entirely novel. 55.7% of all heteroplasmic
variants occurred within the D-loop, 33.3% in coding regions, 4.8% in rRNA genes and
6.2% in tRNA genes (Fig. 2).

Fig. 2. Circos plot summarizing all of the genetic data from 1363 sequences derived form the
MRC Brain Tissue Resource. From outside the circle to inside: (1) mtDNA position,
(2) mtDNA genes, (3) mtDNA Complex, (4) frequency of variants in 40,440 mitochondrial
sequences in NCBI-GenBank, (5) mean read depth of 1363 samples per base, (6) Total
variants in all 1363 samples [circles], (7) Total Rare variants in 1363 samples [triangles],
(8) Total novel variants in 1363 samples [squares]. Colour code for circles (6) –
(8): Red – AD, green – ALS-FTD, blue – CJD, yellow – DLB-PD, grey – others; from inner
to outer, HF increasing. Key – AD –Alzheimer’s disease, CJD – Creutzfeldt Jacob Disease,
DLB-PD – Dementia with Lewy Bodies or Parkinson’s disease, FTD-ALS – Frontotemporal
Dementia or Amyotrophic Lateral Sclerosis

There was no association between any disease group and controls for any single heteroplasmic
variant, the total number of heteroplasmic variants, or the mean variant pathogenicity
score. There was also no association between the number of non-synonymous heterozygous
variants in any gene or complex and any disease group compared to controls (Additional
file 1: Tables S6–S8, Additional file 1: Figure S12).

Heteroplasmy and age

We subsequently used a Poison loglinear model to determine the relationship between
heteroplasmy and age within each group. There was no age correlation with the total
number of heteroplasmic variants, mean level of heteroplasmy (HF), nor the mean variant
pathogenicity score in any disease group (Fig. 3).

Fig. 3. The distribution and nature of major heteroplasmic mtDNA variation within 1363 cases
from the MRC Brain Bank Tissue Resource. a Top left – The mean number of heteroplasmic variants per sample in each cohort. b Top right – The distribution of heteroplasmic fraction (ratio of mutant to wild-type
allele) for heteroplasmic variants for each disease cohort. c Bottom left – The mean number of heteroplasmic point mutations by age for each disease
cohort. d Bottom right – The mean heteroplasmic fraction by age for each disease cohort. Key
– AD –Alzheimer’s disease, CJD – Creutzfeldt Jacob Disease, DLB-PD – Dementia with
Lewy Bodies or Parkinson’s disease, FTD-ALS – Frontotemporal Dementia or Amyotrophic
Lateral Sclerosis

mtDNA number

mtDNA copy number was significant lower in AD and CJD compared to controls (p?=?2.85?×?10
^?7
(AD), p?=?3.34?×?10
^?7
(CJD)), and we observed a strongly positive correlation between age and mtDNA copy
number in CJD (p?=?2.7?×?10
^?11
). No association with age was seen in other groups (Fig. 4). The frequency of cerebellar samples in the AD cohort was no different to controls
(p?=?0.64) or the FTD-ALS cohort (p?=?0.87). However, the CJD cohort did show a greater proportion of cases from the
cerebellum compared to all other cohorts (100%, p??0.001 vs all other groups).

Fig. 4. The relative mtDNA copy number in each disease cohort. a Top – Relative copy number of each cohort calculated as the ratio between the mean
mtDNA read depth and the mean exome read depth as previously described 5]. ** (P??0.01). b Bottom – The association between relative mtDNA copy number and age for all CJD cases
(n?=?182) with the Spearman Rank ? and p-value shown. Key – AD –Alzheimer’s disease,
CJD – Creutzfeldt Jacob Disease, DLB-PD – Dementia with Lewy Bodies or Parkinson’s
disease, FTD-ALS – Frontotemporal Dementia or Amyotrophic Lateral Sclerosis