The genetic architecture of human brainstem structures and their involvement in common brain disorders

Brainstem regions support vital bodily functions, yet their genetic architectures and involvement in common brain disorders remain understudied. Here, using imaging-genetics data from a discovery sample of 27,034 individuals, we identify 45 brainstem-associated genetic loci, including the first linked to midbrain, pons, and medulla oblongata volumes, and map them to 305 genes. In a replication sample of 7432 participants most of the loci show the same effect direction and are significant at a nominal threshold. We detect genetic overlap between brainstem volumes and eight psychiatric and neurological disorders. In additional clinical data from 5062 individuals with common brain disorders and 11,257 healthy controls, we observe differential volume alterations in schizophrenia, bipolar disorder, multiple sclerosis, mild cognitive impairment, dementia, and Parkinson’s disease, supporting the relevance of brainstem regions and their genetic architectures in common brain disorders.

Data sets were excluded from the study if one of the following requirements was not met: 1. the FOV included the whole brainstem, 2. the superior boundary of the midbrain approximated an axial plane through the mammillary body and the superior edge of the quadrigeminal plate, 3. the boundary between mibrain and pons approximated an axial plane through the superior pontine notch and the inferior edge of the quadrigeminal plate, 4. the boundary between between pons and medulla oblongata approximated an axial plane at the level of the inferior potine notch, 5. the inferior boundary of the medulla oblongata approximated an axial plane at the level of the posterior rim of the foramen magnum, 6. there were no substantial segmentation errors for the anterior and posterior boundaries of midbrain, pons, and medulla oblongata, and 7. the superior boundary of the SCP approximated the inferior boundary of the midbrain tectum, the inferior boundary of the SCP was defined by the merging with the cerebellum, and the anterior boundary of the SCP was defined by the posterior boundary of the pons. This visual quality control procedure excluded 11.4% (n = 6,513) of the data sets, mainly due to insufficient FOV, image quality, and segmentation errors in the clinical sample. SCP; superior cerebellar peduncle. adjusting for analyses of five volumes. Twenty-seven genes were associated with whole brainstem volume and 12, 33, 7, and 17 genes were associated with volumes of the midbrain, pons, superior cerebellar peduncle, and the medulla oblongata, respectively.

Supplementary Fig. 14 |
Volumes of brainstem structures in individuals with common brain disorders compared to healthy controls. Linear models were run covarying for sex, age, age², intracranial volume, and scanner site. The analyses for volumes of midbrain, pons, SCP, and medulla oblongata were run both with and without covarying for whole brainstem volume. The figure depicts the resulting case-control differences in mm 3 , whereas group differences in Cohen's d are shown in Fig. 5

Supplementary clinical analyses
Additional clinical analyses were run for individuals with multiple sclerosis (MS) or Parkinson's disease (PD) using linear models. All brain magnetic resonance imaging data from the individuals with MS were examined by two neuroradiologists and then divided into two groups according to presence of infratentorial lesions. There was no significant difference in brainstem volumes between patients with (n = 153) and without (n = 91) infratentorial lesions (all P > 0.05).
Patients without lesions had reduced volumes relative to the controls of the whole brainstem  where multiple disorders were studied on the same scanner, some of the same controls were used for different disorders. *All genetic analyses of the present study were run for the GWAS discovery and the GWAS replication sample.**Only a small minority in the clinical sample had genotype data and no genetic analyses were run for these individuals.

Supplementary Table 4. SNP-based heritability estimates for brainstem volumes
We estimated SNP-based heritability for the brainstem volumes, with and without accounting for whole brainstem volume using genome-wide complex trait analysis (GCTA) v1.92. For completeness, we also report heritabilities estimated with LD score regression v1.0.0.
Estimates from GCTA Estimates from LD-score regression Narrow-sense SNPbased heritability (SE) We conducted genome-wide associaton studies (GWAS) of the brainstem volumes in an additional 7,432 participants from the UK Biobank. We found that all of the lead single nucleotide polymorphisms (SNPs) of the discovery sample had the same directions of effect for most of the volumes (all but midbrain and medulla oblongata volumes). We also found that the majority of the lead SNPs had uncorrected P < 0.05 in the GWAS replication sample. Two of the lead SNPs had P < 5e-8 in the GWAS replication sample. The discovery and replication GWAS for all volumes were significantly correlated.

Replication of lead SNPs significant in discovery sample
Genetic correlation between discovery and replication GWAS Positional, expression quantitative trait loci (eQTL), and chromatin interaction mapping of the 125 independent significant single nucleotide polymorphisms identified 280 unique genes. The genome-wide gene-based association studies identified an additional 25 genes, thus resulting in a total of 305 brainstem-linked genes. Seventeen of these genes were identified by all four approaches and are shown below.  Table 7. Gene sets implicated by the significant genes.

Symbol
We conducted a gene-set analysis for curated gene sets and GO terms obtained from MsigDB (based on independent SNPs significant at the P < 5e-8 threshold) using hypergeometric tests in Functional Mapping and Annotation of GWAS (FUMA). The tests are described in detail on https://fuma.ctglab.nl/tutorial and all parameters used for the present study are available at https://fuma.ctglab.nl/browse/; ID 97-105. This identified 7 Gene Ontology sets significantly associated with whole brainstem volume, and 2, 8, 1, and 15 gene sets associated with volumes of the midbrain, pons, SCP, and medulla oblongata, respectively, after Bonferroni correction. GO_mf; Gene Ontology molecular function.

Supplementary Table 8. Pathway analyses for mapped genes (based on independent SNPs significant at the P < 5e-8 threshold).
We used the ConsensusPathDB to identify over-represented pathways for the mapped genes (based on independent SNPs significant at the P < 5e-8 threshold). ConsensusPathDB is a database system that integrates functional interactions, including binary and complex protein-protein, genetic, metabolic, signaling, gene regulatory and drug-target interactions, as well as biochemical pathways. The details of the statistical analyses employed by the platform can be found on http://cpdb.molgen.mpg.de/. There were 13 significant pathways for whole brainstem volume, and 1, 25, and 58 signficant pathways for pons, SCP, and medulla oblongata volume after FDR-correction. There was no significant pathway for genes associated with midbrain volume.

Supplementary Table 10. Pathway analyses for mapped genes (based on independent SNPs significant at the P < 1e-8 threshold).
We used the ConsensusPathDB to identify over-represented pathways for the mapped genes (based on independent SNPs significant at the P < 1e-8 threshold). ConsensusPathDB is a database system that integrates functional interactions, including binary and complex protein-protein, genetic, metabolic, signaling, gene regulatory and drug-target interactions, as well as biochemical pathways. The details of the statistical analyses employed by the platform can be found on http://cpdb.molgen.mpg.de/. There were 53, 19, 1, and 4 significant pathways for volumes of the whole brainstem, midbrain, pons, and medulla oblongata, respectively, after FDR-correction.