Prioritization of potential causative genes for schizophrenia in placenta

Our earlier work has shown that genomic risk for schizophrenia converges with early life complications in affecting risk for the disorder and sex-biased neurodevelopmental trajectories. Here, we identify specific genes and potential mechanisms that, in placenta, may mediate such outcomes. We performed TWAS in healthy term placentae (N = 147) to derive candidate placental causal genes that we confirmed with SMR; to search for placenta and schizophrenia-specific associations, we performed an analogous analysis in fetal brain (N = 166) and additional placenta TWAS for other disorders/traits. The analyses in the whole sample and stratifying by sex ultimately highlight 139 placenta and schizophrenia-specific risk genes, many being sex-biased; the candidate molecular mechanisms converge on the nutrient-sensing capabilities of placenta and trophoblast invasiveness. These genes also implicate the Coronavirus-pathogenesis pathway and showed increased expression in placentae from a small sample of SARS-CoV-2-positive pregnancies. Investigating placental risk genes for schizophrenia and candidate mechanisms may lead to opportunities for prevention that would not be suggested by study of the brain alone.


Supplementary Data 22. Overlap of TWAS-significant associations with schizophrenia in female and male placentae. A) TWAS-significant genes in both females and males, with
Bonferroni corrected p <0.05: Columns A-I report the placental genes associated with schizophrenia with p<0.05 after Bonferroni correction. Column I shows the consistency of the sign of the TWAS-associations in female and male placentae. B) TWAS-significant genes in both females and males -FDR corrected p <0.05: Columns O-Z report the placental genes associated with schizophrenia with p<0.05 after FDR correction. Column Z indicates the consistency of the sign of the TWAS-associations in female and male placentae.
Supplementary Data 23. Coexpression of gene transcripts in female placentae. The table reports the output of the WGCNA analysis in female placentae, at transcript level. Column P (in red) indicates the module membership of each transcript. Columns K-U report measures of connectivity, that is, total connectivity (kTotal: connectivity of each gene based on its r-values to all other genes in the whole network), local connectivity (kWithin: connectivity of each gene within a single module based on its rvalues to all other genes within the same module), and their differences (KOut, Kdiff).

Supplementary Data 24. Coexpression of gene transcripts in male placentae.
The table reports the output of the WGCNA analysis in male placentae, at transcript level. Column P (in red) indicates the module membership of each transcript. Columns K-U report measures of connectivity, that is, total connectivity (kTotal: connectivity of each gene based on its r-values to all other genes in the whole network), local connectivity (kWithin: connectivity of each gene within a single module based on its rvalues to all other genes within the same module), and their differences (KOut, Kdiff). Supplementary Data 27. Transcript count overlap between WGCNA coexpression modules in male and female placentae and placental TWAS schizophrenia genes. Shown are the number of placental TWAS schizophrenia transcripts detected in the whole sample (column D), in the female (E), and in the male sample (F), and also in each of the female-specific, male-specific, and overlapping modules (category in column A). Supplementary Data 32. Summary Statistics of prenatal cerebral cortex TWAS for schizophrenia, in the whole sample, of the genes and transcripts associated with schizophrenia with p<0.05, after Bonferroni-correction. A) Prenatal cortical brain TWAS genes and transcripts associated with schizophrenia in the whole sample: Columns A-Q report all the genes and transcripts with TWASsignificant association with schizophrenia. Column Q specifies whether the association is at gene or transcript level. B) Unique genes: Columns V-AD report the unique prenatal cerebral cortex genes associated with schizophrenia at gene and/or transcript level (for each gene the feature with the most significant association is selected). Column AD indicate the presence of transcripts associate with schizophrenia with opposite sign. Supplementary Data 38. Unique genes with TWAS-significant association with schizophrenia, at gene and/or transcript level, in prenatal cerebral cortex, in the male sample: Columns A-I report the unique prenatal cortical brain genes associated with schizophrenia at gene and/or transcript level (for each gene the feature with the most significant association is selected). Column I indicate the presence of transcripts associate with schizophrenia with opposite sign.

Supplementary Data 39. Genes with TWAS-significant association with schizophrenia, at gene and/or transcript level, in prenatal cerebral cortex, in the whole sample, in female, and in male. A)
The table reports all the TWAS significant genes, with p<0.05 after Bonferroni-correction. Column I indicates the presence of transcripts associated with schizophrenia with opposite sign. B) The table reports the TWAS significant genes in male and female prenatal cortical brains, with p<0.05 after Bonferroni-correction. C) The table reports the unique TWAS genes associated with schizophrenia in the whole sample, in male and females prenatal cortical brains, at gene and/or transcript level, with p<0.05 after Bonferroni-correction.
Supplementary Data 40. Larger set of genes with TWAS association with schizophrenia, at gene and/or transcript level, in prenatal brain, in the whole sample, in female, and in male. A) The table reports all the TWAS significant genes, with uncorrected p<0.05. B) The table reports the TWAS significant genes in male and female prenatal cortical brains, with p<0.05 after FDR-correction.

Supplementary Data 41. Placental-specific TWAS genes associated with schizophrenia. A)
The table reports all the TWAS genes associated with schizophrenia in placenta with p<0.05 after Bonferronicorrection, which do not show association with schizophrenia in prenatal brain (FDR>0.01). Column F reports whether the gene has been associated with schizophrenia by SMR analysis in fetal and adult brain and in blood (PMID: 35396580). B) Pathway enrichment results of placental-specific TWAS genes: Reported are the results of the Ingenuity Pathway Analysis for the placental-specific TWAS genes associated with schizophrenia. Statistics from right-tailed Fisher's Exact Test. C) Upstream Regulators enrichment results of placental-specific TWAS genes: Reported are the results of the Ingenuity Global Upstream Regulator Analysis for the placental-specific TWAS genes associated with schizophrenia. Statistics from right-tailed Fisher's Exact Test.
Supplementary Data 42. Prenatal cortex-specific TWAS genes associated with schizophrenia. The table reports all the TWAS genes associated with schizophrenia in midgestational cortical brain with p<0.05 after Bonferroni-correction, which do not show association with schizophrenia in placenta.

Supplementary Data 43. Placenta&brain-pleiotropic TWAS genes associated with schizophrenia.
A) The table reports all the TWAS genes associated with schizophrenia in placenta and in prenatal cerebral cortex with p<0.05 after Bonferroni-correction. Columns H indicates whether the association has opposite sign in brain and placenta. B) Pathway enrichment results of placenta&brain-pleiotropic TWAS genes: Reported are the results of the Ingenuity Pathway Analysis for the placenta&brainpleiotropic TWAS genes associated with schizophrenia, with predicted z-scores in placenta (P) and in fetal brain (Q). Statistics from right-tailed Fisher's Exact Test. C) Upstream Regulators enrichment results of placenta&brain-pleiotropic TWAS genes: Reported are the results of the Ingenuity Global Upstream Regulator Analysis for the placenta&brain-pleiotropic TWAS genes associated with schizophrenia, with predicted activation state and Z-scores in placenta (AB, AD) and fetal brain (AC, AE). Statistics from right-tailed Fisher's Exact Test.
Supplementary Data 45. Placental TWAS-significant associations with ADHD (Bonferronicorrected p<0.05). A) Placental TWAS genes and transcripts associated with ADHD in the whole sample: Columns A-O report all the genes and transcripts. B) Unique genes: Columns S-X report the unique placental genes associated with ADHD at gene and/or transcript level (for each gene the feature with the most significant association is selected). Column X indicates the presence of transcripts associated with ADHD with opposite sign. Columns Y-AB report gene variant-level colocalization probability (GRCP) and gene locus-level colocalization probability (GLCP), from the colocalization analyses at gene-level (Y, Z) and transcript level (AA, AB). (Bonferroni-corrected p<0.05). A) Placental TWAS genes and transcripts associated with depression in the whole sample: Columns A-O report all the genes and transcripts. B) Unique genes: Columns S-X report the unique placental genes associated with depression at gene and/or transcript level (for each gene the feature with the most significant association is selected). Column X indicates the presence of transcripts associated with depression with opposite sign. Columns Y-AB report gene variantlevel colocalization probability (GRCP) and gene locus-level colocalization probability (GLCP), from the colocalization analyses at gene-level (Y, Z) and transcript level (AA, AB).

Supplementary Data 47. Placental TWAS-significant associations with bipolar disorder (Bonferroni-corrected p<0.05). A) Placental TWAS genes and transcripts associated with bipolar disorder in the whole sample: Columns A-O report all the genes and transcripts. B) Unique genes:
Columns S-X report the unique placental genes associated with bipolar disorder at gene and/or transcript level (for each gene the feature with the most significant association is selected). Column X indicates the presence of transcripts associated with bipolar disorder with opposite sign. Columns Y-AB report gene variant-level colocalization probability (GRCP) and gene locus-level colocalization probability (GLCP), from the colocalization analyses at gene-level (Y, Z) and transcript level (AA, AB).

Supplementary Data 48. Placental TWAS-significant associations with birthweight (Bonferronicorrected p<0.05). A) Placental TWAS genes and transcripts associated with birthweight in the whole sample:
Columns A-O report all the genes and transcripts. B) Unique genes: Columns S-X report the unique placental genes associated with birthweight at gene and/or transcript level (for each gene the feature with the most significant association is selected). Column X indicates the presence of transcripts associated with birthweight with opposite sign. Columns Y-AB report gene variant-level colocalization probability (GRCP) and gene locus-level colocalization probability (GLCP), from the colocalization analyses at gene-level (Y, Z) and transcript level (AA, AB).

Supplementary Data 49. Placental TWAS-significant associations with height (Bonferronicorrected p<0.05). A) Placental TWAS genes and transcripts associated with height in the whole sample: Columns A-O report all the genes and transcripts. B) Unique genes:
Columns S-X report the unique placental genes associated with height at gene and/or transcript level (for each gene the feature with the most significant association is selected). Column X indicates the presence of transcripts associated with height with opposite sign. Columns Y-AB report gene variant-level colocalization probability (GRCP) and gene locus-level colocalization probability (GLCP), from the colocalization analyses at gene-level (Y, Z) and transcript level (AA, AB).

Supplementary Data 50. Placental TWAS-significant associations with BMI (Bonferroni-corrected p<0.05). A) Placental TWAS genes and transcripts associated with BMI in the whole sample:
Columns A-O report all the genes and transcripts. B) Unique genes: Columns S-X report the unique placental genes associated with BMI at gene and/or transcript level (for each gene the feature with the most significant association is selected). Column X indicates the presence of transcripts associated with BMI with opposite sign. Columns Y-AB report gene variant-level colocalization probability (GRCP) and gene locus-level colocalization probability (GLCP), from the colocalization analyses at gene-level (Y, Z) and transcript level (AA, AB).

Supplementary Data 51. Placental TWAS-significant associations with type 2 diabetes (Bonferronicorrected p<0.05). A) Placental TWAS genes and transcripts associated with diabetes in the whole sample:
Columns A-O report all the genes and transcripts. B) Unique genes: Columns S-X report the unique placental genes associated with diabetes at gene and/or transcript level (for each gene the feature with the most significant association is selected). Column X indicates the presence of transcripts associated with diabetes with opposite sign. Columns Y-AB report gene variant-level colocalization probability (GRCP) and gene locus-level colocalization probability (GLCP), from the colocalization analyses at gene-level (Y, Z) and transcript level (AA, AB).

Supplementary Data 53. Placental TWAS-significant associations with IQ (Bonferroni-corrected p<0.05). A) Placental TWAS genes and transcripts associated with IQ in the whole sample: Columns
A-O report all the genes and transcripts. B) Unique genes: Columns S-X report the unique placental genes associated with IQ at gene and/or transcript level (for each gene the feature with the most significant association is selected). Column X indicates the presence of transcripts associated with IQ with opposite sign. Columns Y-AB report gene variant-level colocalization probability (GRCP) and gene locus-level colocalization probability (GLCP), from the colocalization analyses at gene-level (Y, Z) and transcript level (AA, AB).
Supplementary Data 54. Placenta&Schizophrenia prioritized risk genes. List of 502 genes whose expression in placenta at gene and/or transcript level is predicted to be associated with schizophrenia, in the whole sample, and/or in male, and/or in female placentae (sample with the most significant association in column F with statistics in columns D,E; statistics in all samples in columns AA-AL). Column L reports whether the gene has TWAS-association with schizophrenia in fetal brain with FDR<0.01. Columns M-O reports whether the gene has been prioritized by the most recent schizophrenia GWAS (PMID: 35396580) based on fine mapping (M), SMR in adult and fetal brain and blood (N), or being annotated as a rare-variant priority gene (O). Columns P-Y indicate placental TWAS associations with other disorders and traits. Columns AM-BC summarize the results of the colocalization (COLOC) analysis: GRCP, that is, gene variant-level colocalization probability, in the analysis at gene level in the whole sample (AR), in the female (AV) and in the male (AZ) samples; GLCP, that is, gene locus-level colocalization probability, in the analysis at gene level in the whole sample (AS), in the female (AW) and in the male (BA) samples; GRCP in the analysis at transcript level in the whole sample (AT), in the female (AX) and in the male (BB) samples; GLCP in the analysis at transcript level in the whole sample (AU), in the female (AY) and in the male (BC) samples; maximum value of GRCP is reported in column AO, maximum value of GLCP is reported in column AP, maximum value of GRCP or GLCP is reported in column AQ; columns AM and AN report whether a gene has GRCP or GLCP > 0.5 (AM) and 0.1 (AN). Column J highlights whether the placental schizophrenia genes are prioritized based on absence of association in brain and absence of relevant association with other disorders and traits in placenta. Validation of association with schizophrenia with SMR is indicated in column K. The 139 prioritized genes validated with SMR are indicated in column I. In column I and J, we also report whether a prioritized gene has GRCP or GLCP higher than 0.5 or 0.1 (top-prioritized genes in column I). Columns Z indicates whether the placental TWAS gene has been detected in maternal blood as a cell-free RNA biomarker of pregnancy outcomes (PMID: 35140405). Recent changes of gene annotations are indicated in column C with Entrez Gene Name and other gene information. Gene synonyms, location and types are reported in columns BD, BE, and BF, respectively. See also Supplementary Fig. 1 for workflow on the prioritization of placenta&schizophrenia-risk genes, Supplementary Data 56-61 for detailed results from colocalization analysis, and Supplementary Data 55 and Supplementary Note 1 for biological information about selected genes.

Supplementary Data 55. Master regulators, diseases and functions, associated with the topprioritized placenta-schizophrenia-specific risk genes. A) Causal Network Analysis.
Reported in columns A-J are the results of the causal network analysis, in IPA, to identify master regulators of the 33 top-prioritized placenta-schizophrenia-specific risk, which show TWAS-significant association with schizophrenia in placenta, and not in fetal brain, are validated by SMR and colocalization analyses, and did not show any significant relevant association with any of the other disorders and traits analyzed. Pvalue of overlap is obtained by right-tailed Fisher's Exact Test; Network bias-corrected p-value is calculated by explicit permutation sampling to account for the presence of network hub genes in the list of top-prioritized placenta-schizophrenia-specific risk genes (PMID: 24336805). B) Isoprofiler analysis. Reported in columns P-AC are the results of the Isoprofiler analysis in IPA to identify functions and diseases terms associated with the top-prioritized placenta-schizophrenia-specific risk (please note that not all the 33 top-prioritized genes have known annotations). See also Supplementary Note 1 for biological information about selected genes.