The landscape of gene loss and missense variation across the mammalian tree informs on gene essentiality

A comparative analysis of 462 placental mammals has produced two gene-level measures of evolutionary constraint that complement human sequencing metrics used to identify important and disease-associated genes. The Gene Identity Score of Mammalian Orthologs, or GISMO, quantifies the frequency of gene loss across the mammalian tree, while GISMO-mis measures missense variation relative to synonymous variation in codon alignments spanning up to 462 species. Both measures were associated with established human constraint scores, but their relationships with coding-sequence length were much weaker than that of LOEUF, a human loss-of-function constraint metric. The study found that the mammalian measures also identified short genes that the human-only approach did not classify as intolerant.

The researchers used human GENCODE version 38 annotations as the reference and applied TOGA, a tool for inferring orthologs from genome alignments, to compare genes across 462 placental mammals. TOGA identified disruptive mutations including frameshifts, premature stop codons, splice-site disruptions and larger deletions. A gene was classified as lost when its intact reading frame was absent in a species and all corresponding transcripts met the criteria for loss. Across the dataset, each gene had a median of 18 loss events, while each species had a median of 1,130 likely gene-loss events. Only six genes had no observed loss across the complete mammalian sample: NAP1L1, ZC3H14, MEIS2, STRIP1, CCT5 and ATP6V0D1.

GISMO was designed as a weighted estimate of the proportion of species in which a gene has been lost. Each loss is weighted according to evolutionary distance from humans, so losses in more distantly related species contribute more heavily. A lower score indicates that a gene is rarely lost and is therefore more constrained across the sampled mammals. The researchers also examined a phylogenetically adjusted version, which was strongly correlated with the regular score, but the nonphylogenetic version showed stronger empirical associations with independent constraint measures. They attributed some of this performance to TOGA’s requirement for multiple disruptions in an open reading frame before classifying a gene as lost, which can reduce the risk of counting a single questionable disruption as a loss event. The metric covered 97.7% of genes in GENCODE version 38, the highest proportion among the conservation scores benchmarked in the study.

The second measure, GISMO-mis, captures a different aspect of constraint. For each gene, the researchers generated a mammalian consensus sequence and counted missense and synonymous differences in each species relative to that consensus. They then calculated the mean missense-to-synonymous ratio across available species. A lower ratio indicates that amino-acid-changing substitutions are relatively depleted compared with synonymous changes, consistent with stronger functional constraint at the protein level. Codon alignments were included when a gene had a single ortholog in at least 75% of queried species; species with more than four predicted orthologs were excluded. The method extends the logic of the missense-to-synonymous substitution ratio by using a multi-species consensus and by handling ambiguous alignments conservatively.

Tests against independent gene sets showed that the most constrained deciles were enriched for genes whose disruption causes early lethality in mice or loss of viability in cell lines. For mouse heterozygous knockouts, the odds ratios were 3.43 for GISMO and 5.78 for GISMO-mis. The corresponding values for homozygous knockouts were 8.21 and 13.1, while essential cell-line genes had odds ratios of 4.34 and 10.70. By contrast, the least constrained deciles contained the greatest proportions of genes classified as nonessential or associated with olfaction. Frequently lost genes also tended to be expressed in fewer tissues in the GTEx dataset, with expression defined as more than 0.3 transcripts per million. The distributions of GISMO scores did not drift as gene-family size increased, indicating no observed association with that feature in the benchmarked data.

Both measures were correlated with human constraint estimates derived from large-scale sequencing data. GISMO correlated with the gnomAD LOEUF score at Spearman’s R of 0.38, while GISMO-mis reached R of 0.56, with both P values below 1 × 10−300. GISMO-mis also correlated with the missense constraint score MOEUF at R of 0.61 and with the predicted missense effect metric AlphaMissense at R of 0.84. Both measures were associated with haploinsufficiency and triplosensitivity scores, while GISMO-mis had stronger correlations than GISMO for each dosage measure. In a UK Biobank copy-number-variant dataset generated from exome sequencing in 197,306 people, the most constrained deciles of both metrics had the fewest deletions and duplications. GISMO showed an even stronger correlation with deletions than pHaplo, a dosage-sensitivity metric derived from lower-resolution microarray data across about one million human samples. These results establish associations with independent measures of constraint, rather than showing that the scores are interchangeable.

A major difference from LOEUF was the reduced sensitivity of the mammalian measures to coding-sequence length. LOEUF was strongly correlated with coding-sequence length at R of −0.50, whereas the correlations for GISMO and GISMO-mis were each −0.12. When the researchers defined intolerance as the most constrained 15% of genes, GISMO identified 2,897 genes with a median coding-sequence length of 1,502 base pairs, and GISMO-mis identified 2,502 with a median of 1,302 base pairs. LOEUF identified 2,968 intolerant genes with a longer median of 2,267 base pairs. Genes considered intolerant by GISMO or GISMO-mis but tolerant by LOEUF were enriched in mouse phenotypes involving sperm immotility, absence of the acrosome and abnormal actin cytoskeleton morphology. The analysis was comparative and computational, so these enrichments identify previously unclassified constrained genes for further study rather than demonstrating human clinical outcomes.

The study also examined whether the evolutionary measures captured disease-associated genetic signals. Using linkage disequilibrium score regression, the researchers partitioned common-variant heritability across 276 independent UK Biobank traits and several additional disease phenotypes. Heritability showed a strong linear enrichment across the constraint deciles of both GISMO and GISMO-mis and was lowest in the least constrained deciles, with consistent results at 10-, 50- and 100-kilobase flanking windows. Genes in the most constrained deciles also showed stronger associations with rare variants linked to reduced fecundity, including schizophrenia, autism and neurodevelopmental disorders. The findings indicate that deeply conserved genes contribute disproportionately to both common- and rare-variant signals in these phenotypes, but they do not establish that conservation itself causes the associations. The researchers also found that genes with high LOEUF constraint but low GISMO conservation were disproportionately X-linked, raising the possibility of differences related to hemizygosity, sex-biased selection or technical challenges in short-read variant detection; this discordant subset was not enriched for Mendelian disease genes or essential phenotypes when restricted to the X chromosome.

GISMO had a distinct association with recessive disease genes because gene loss across species can reflect selection against homozygous loss-of-function alleles. In a set of 1,183 autosomal recessive genes assembled from OMIM, the most constrained GISMO deciles were enriched with an odds ratio of 3.37 and a 95% confidence interval of 2.91 to 3.89. GISMO-mis did not show significant enrichment for this set, with an odds ratio of 0.91 and P = 0.41. Both measures were enriched for 709 dominant disorder genes: the odds ratio was 9.92 for GISMO and 4.38 for GISMO-mis. In the Deciphering Developmental Disorders study of about 14,000 trios, GISMO distributions for recessive developmental-disorder genes with confident or strong evidence were similar to those of Mendelian recessive genes, whereas genes with limited or moderate evidence were unclear. The authors described GISMO and GISMO-mis as complementary: GISMO emphasizes disruption of an entire gene across species, while GISMO-mis emphasizes amino-acid-level constraint. They recommend using the continuous scores where possible and calibrating categorical thresholds against relevant gene sets such as ClinGen annotations, mouse knockout phenotypes or disease-gene catalogs.

Several limitations constrain the interpretation and future refinement of the results. Mammalian conservation may not reflect recent human selective pressures or human-specific innovations, and a gene tolerated in other mammals may nevertheless be essential in humans. The available species may also introduce survivorship and accessibility biases, so broader taxonomic sampling could improve the metrics. GISMO combines signals associated with heterozygous and homozygous selection and therefore does not distinguish between them by itself; pairing it with LOEUF and GISMO-mis can help separate those relationships. Although the nonphylogenetic version of GISMO does not distinguish multiple independent losses from one ancestral loss, it performed better against independent measures of functional constraint than the phylogenetic alternative, and a phylogenetic version was also released. Recent human-specific duplications showed a slight correlation with the conservation score of the original paralog, but such cases were few. Overall, the analyses support gene loss and missense fixation across mammals as measurable signals of selection and show that these scores can rank gene importance, inform disease-gene prioritization and provide evolutionary evidence alongside variation in modern human populations.

Subject of Research: Evolutionary constraint and gene essentiality across 462 placental mammals

Article Title: The landscape of gene loss and missense variation across the mammalian tree informs on gene essentiality

Article References: Liao, C., Ye, R., Ivankovic, F., Fu, J. M., Walters, R., Lowther, C., Valkanas, E., Churchhouse, C., Samocha, K. E., Lindblad-Toh, K., Karlsson, E., Hiller, M., Talkowski, M. E., & Neale, B. M. (2026). The landscape of gene loss and missense variation across the mammalian tree informs on gene essentiality. Nature Genetics. https://doi.org/10.1038/s41588-026-02783-9

Image Credits: AI Generated

DOI: 10.1038/s41588-026-02783-9

Keywords: comparative genomics, gene essentiality, mammalian evolution, gene loss, missense variation, evolutionary constraint, disease-gene prioritization, rare variants, common-variant heritability, copy-number variation, landscape, gene