Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium

Abstract

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.

Figure 1. IMPC Mutant Models of Human Disease and Gene Function

Human disease models were identified by measuring the degree of phenotype similarity between IMPC null mutant mouse strains and their orthologous human disease genetic loci. Models of Mendelian Disease- of 889 potential disease models, 360 mutant strains had both phenotype overlap and an orthologous null allele to diseases with known mutations as described in OMIM and Orphanet; Novel Mendelian Disease Candidates- 135 strains had phenotype overlap and null alleles syntenic to linkage or cytogenetic regions associated with human diseases with unknown molecular mechanisms; New Functional Knowledge- of 2564 genes with a non-lethal IMPC phenotype, IMPC data provide the first functional experimental evidence for 1092 of these genes based on Gene Ontology Annotation.

Figure 2. New Mouse Models for Mendelian Human Disease

Gp9- Bernard-Soulier syndromes are bleeding disorders that result from mutations in the glycoprotein Ib platelet membrane receptor complex. Gp9^{tm1.1(KOMP)Vlcg} homozygotes have abnormal platelet development represented by an increased platelet volume (A; box plots representations throughout represent first and 3rd quartiles with the line indicating the median and whiskers representing the min and max values. Female control=479, female homozygous=8, male control=428, male homozygous=8; linear mixed-effects model without Weight; p=0) and decreased platelet numbers (B; Female control=439, female homozygous=8, male control=428, male homozygous=8; linear mixed-effects model without Weight; p= 2.31E-06). Bbs5- BBS5 is associated with Bardet-Biedl syndrome (BBS), a ciliopathy with multisystem involvement with severe and early-onset of symptoms. Bbs5^{tm1b(EUCOMM)Wtsi} homozygotes display profoundly increased body fat percentage (C; Female control=1276, female homozygous=8, male control=1296, male homozygous=8; linear mixed-effects model without Weight; p=1.99E-11) and impaired glucose tolerance as shown by the time series box plot (D; blood glucose levels at time points after 16 hours fasting followed by intra-peritoneal (IP) glucose injection. Female control=491, female homozygous=8, male control=509, male homozygous=8; linear mixed-effects model without Weight; p= 2.85 E-07). Whole body X-ray visualization of Bbs5 homozygous and control, showing increased body fat in mutant animals (E). Rnf216 - Gordon Holmes syndrome is associated with RNF216 and is characterised by hypogonadism and cerebellar ataxia. Rnf216^{tm1b(EUCOMM)Wtsi} homozygous null male mice are infertile. Histopathology images at 20x magnification show seminiferous tubule degeneration and atrophy with Leydig cell hyperplasia (Fa) and epididymal aspermia (Fb) in null mice compared to unaffected seminiferous tubules (Fc) and epididymis (Fd) in control mice.

Figure 3. Psph​

Phosphoserine phosphatase deficiency (OMIM: 614023) is an autosomal recessive disorder characterised by prenatal and postnatal growth retardation, psychomotor retardation and facial dysmorphologies with the severity of the symptoms requiring medical support for survival. Complete preweaning lethality was observed in Psph^{tm1.1(KOMP)Vlcg} homozygous null mice. Pup number, genotypes and sex ratios of heterozygous intercrosses were set to generate cohorts for phenotyping. No homozygous pups were observed whereas respectively 66% (54/82) and 34% (28/82) were produced (A; # of pups, asterisks indicate no surviving homozygotes). LacZ reporter expression regulated by the Psph promoter in asymptomatic heterozygous E12.5 embryos shows extensive gene expression (B; bar 1mm). Gross images of E15.5 homozygous mutant embryos confirmed growth retardation, haemorrhage, and facial dysmorphologies (C; bar 5mm). Imaging of E15.5 embryos by microCT showed significant growth retardation, as well as facial dysmorphologies consistent with the human Mendelian disorder (D).

Figure 4. Novel Mouse Models of Disease –

Over 40% of IMPC strains are for genes that lack experimental evidence for function according to the Gene Ontology Consortium (A in grey). Fam53b - Fam53b^{tm1b(EUCOMM)Hmgu} homozygous mutant mice had significantly decreased red blood cell counts (B; box plot representations throughout represent first and 3rd quartiles with the line indicating the median and whiskers representing the min and max values and asterisks indicating a significant difference between mutant and same sex controls using the mixed model with a p < 0.0000. Female control=597, female homozygous=8, male control=635, male homozygous=8; linear mixed-effects model without Weight; p=2.81E-11), and enlarged erythrocytes (C; Female control=598, female homozygous=8, male control=634, male homozygous=9; linear mixed-effects model without Weight; p=0), consistent with Diamond-Blackfan Anemia (DBA, OMIM: 105560). Dnajc5b - Dnajc5b^{tm1b(EUCOMM)Hmgu} homozygous mutants displayed significantly shortened QT interval as measured by electrocardiogram (D; Female control=7, female homozygous=6, male control=7, male homozygous=8; generalized least squares without weight; p=7.41E-08), supporting a role for DNAJC5b variants associated with human variability to statin effects on cardiovascular incident frequency.