Seminar paper 1: mtDNA

Question 1

What was the purpose of this paper?

Answer 1

Determine the underlying genetic cause of the 28-year-old male patient’s extreme exercise intolerance

Question 2

Why did the authors think it was a mitochondrial disease?

Answer 2

The symptoms, with the lactic acidosis after very little activity

Question 3

How did the authors look at variation in the patient’s mtDNA?

Answer 3

Sequenced it and compared it to his parents

Question 4

What was the main finding of this paper?

Answer 4

The mitochondria in the patient’s skeletal muscle were inherited from his father, while he had the maternal haplotype in every other tissue

Question 5

How did the authors know the patient’s skeletal muscle mitochondria were from his father?

Answer 5

The sequence background. It was way too different from his mother’s to just be simple variation, but it looked a lot like his father’s

Question 6

What were the two mutations considered promising to be the cause of the patient’s disease?

Answer 6

G1303A in the 12s rRNA gene

5132delAA in the ND2 gene (2 bp deletion)

Question 7

Why did the authors rule out G1303A as being the cause of the patient’s disease?

Answer 7

His father had it too, and he was unaffected. Position is not well conserved across mammals. It’s just sequence variation and not a pathological mutation

Question 8

What is homoplasmy?

Answer 8

Every molecule of mtDNA in every mitochondria in that tissue is the same. Either all mutant or all WT

Question 9

What is heteroplasmy?

Answer 9

There is a mix of mutant and WT mtDNA in that tissue

Question 10

Was the patient homoplasmic or heteroplasmic in his skeletal muscle?

Answer 10

First experiment showed homoplasmic for the mutant mtDNA, but another technique showed there to be about 10% maternal mtDNA. So the patient was heteroplasmic

Question 11

Why was it a surprise that the patient’s muscle had paternal mtDNA?

Answer 11

Traditionally thought that only the maternal mtDNA was ever inherited and all the paternal mtDNA was destroyed

Question 12

What did they have to rule out to say that the mitochondria in the patient’s skeletal muscle were actually inherited from his father?

Answer 12

1. Make sure the muscle and blood samples didn’t get mixed up by resampling
2. Make sure all the samples came from the patient by nuclear genotyping
3. Make sure the family was actually blood related by looking at microsatellites

Question 13

Why did the authors decide the 2 bp deletion was the cause of the patient’s disease?

Answer 13

It was absent in healthy relatives, and caused a frameshift mutation. Those don’t typically create much of a functional protein

Question 14

Where did the authors believe the 2 bp deletion in the patient came from?

Answer 14

Spontaneous mutation in a paternal mtDNA molecule, then this mutant mitochondria failed to be eliminated and it proliferated in the patient’s skeletal muscle

Question 15

At what points could the 2 bp deletion in the patient’s arose?

Answer 15

1. Early on in development
2. During gametogenesis in the father
3. In the sperm post-gametogenesis and pre-fertilization

Question 16

How did the authors determine that the deletion allele was predominant in the patient’s muscle?

Answer 16

PCR fragment analysis, using primers that annealed on either side of the suspected deletion. Then used fluorescent labels and capillary electrophoresis. They found the deletion to be predominant in the patient’s muscle, with a small amount of the full length fragment. The full-length fragment was the only one present in other tissues

Question 17

Why did the authors examine microsatellites in the family’s nuclear genome?

Answer 17

Inheritance of paternal mtDNA challenged what everyone thought. They had to prove that the individuals in the family were in fact related

Question 18

How do we read microsatellite nomenclature? Ex. D19S219

Answer 18

D: DNA
19: chromosome 19
S219: microsatellite number 219