Table 2.

Limitations of MR

Limitation	Role in MR studies	Approaches to evaluating or avoiding the limitation
Low statistical power	MR studies are often of low power and effect estimates are imprecise because of this	Increase sample size and or combine genetic variants so they explain more of the variance of the intermediate phenotype
Reverse causation	A genetic variant may be causing the disease outcome which in turn causes the biomarker, or the causal direction could be in the opposite direction. 2SLS will not distinguish between these cases	Bi-directional MR can be used to distinguish between the two causal models
Population stratification	Spurious associations used as instruments can lead to faulty causal inference	Restrict analyses to ethnically homogeneous groups, and apply correction methods using ancestrally informative markers or principal components from genome-wide data. Perform analysis within a family study context, e.g. between siblings.
Reintroduced confounding though pleiotropy	A genetic variant may directly influence more than one post-transcriptional process. Known to be the case for some genetic variants	When possible utilize cis-variants with respect to the intermediate phenotype under study, as these may be less likely to have pleiotropic effects. Apply multiple instrument approaches with more than one independent genetic variant it is unlikely that pleiotropy will generate the same associations for different instruments
LD induced confounding	LD is crucial in genetic association studies as it allows marker SNPs to proxy for un-genotyped causal SNPs. However, this can reintroduce confounding if LD leads to the association of SNPs related to more than one post-transcriptional process. This case will be similar to the pleiotropy situation	Studies can be carried out in populations with different LD structures. Approaches to avoiding distortion by pleiotropy will also counter problems owing to LD
Canalization/developmental compensation	During development, compensatory processes may be generated that counter the phenotypic perturbation consequent on the genetic variant utilized as an instrument	No general approach developed, although context-specific biological knowledge can be applied. The period of the life course when influence of genetic variation on intermediate phenotypes emerge can indicate whether canalization could, in principle, be an issue
Lack of genetic variants to proxy for modifiable exposure of interest	No reliable genetic variant associations for many intermediate phenotypes of interest, although an increasing number of these now identified	Continued genome-wide and sequencing-based studies
Complexity of associations	Without adequate biological knowledge, misleading inferences regarding intermediate phenotypes and disease may be drawn	Increased biological understanding of genotype–phenotype links