blind sterile (bs) is a spontaneous autosomal-recessive mouse mutation discovered more than 30 years ago. Phenotypically, bs mice exhibit nuclear cataracts and male infertility; genetic analyses assigned the bs locus to mouse chromosome 2. In this study, we first positionally cloned the bs locus and identified a putative causative mutation in the Tbc1d20 gene. Functional analysis established the mouse TBC1D20 protein as a GTPase-activating protein (GAP) for RAB1 and RAB2, and bs as a TBC1D20 loss-of-function mutation. Evaluation of bs mouse embryonic fibroblasts (mEFs) identified enlarged Golgi morphology and aberrant lipid droplet (LD) formation. Based on the function of TBC1D20 as a RABGAP and the bs cataract and testicular phenotypes, we hypothesized that mutations in TBC1D20 may contribute to Warburg micro syndrome (WARBM); WARBM constitutes a spectrum of disorders characterized by eye, brain, and endocrine abnormalities caused by mutations in RAB3GAP1, RAB3GAP2, and RAB18. Sequence analysis of a cohort of 77 families affected by WARBM identified five distinct TBC1D20 loss-of-function mutations, thereby establishing these mutations as causative of WARBM. Evaluation of human fibroblasts deficient in TBC1D20 function identified aberrant LDs similar to those identified in the bs mEFs. Additionally, our results show that human fibroblasts deficient in RAB18 and RAB3GAP1 function also exhibit aberrant LD formation. These findings collectively indicate that a defect in LD formation/metabolism may be a common cellular abnormality associated with WARBM, although it remains unclear whether abnormalities in LD metabolism are contributing to WARBM disease pathology.
Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations are associated with de novo heterozygous mutations in tubulin genes. However, the absolute requirement for many of these genes in development and disease has not been previously tested in genetic loss of function models. Here we directly test the requirement for Tuba1a , Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tubb2a or Tubb2b does not impair survival but does lead to relatively mild cortical malformation phenotypes. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. We also present a novel mouse ENU allele of Tuba1a with phenotypes similar to the null allele. This demonstrates the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases.
Blind sterile 2 (bs2) is a spontaneous autosomal recessive mouse mutation exhibiting cataracts and male sterility. Detailed clinical and histological evaluation revealed that bs2 mice have cataracts resulting from severely disrupted lens fiber cells. Analysis of bs2 testes revealed the absence of mature sperm and the presence of large multinucleate cells within the lumens of seminiferous tubules. Linkage analysis mapped the bs2 locus to mouse chromosome 2, approximately 45cM distal from the centromere. Fine mapping established a 3.1Mb bs2 critical region containing 19 candidate genes. Sequence analysis of alkylglycerone-phosphate synthase (Agps), a gene within the bs2 critical region, revealed a G to A substitution at the +5 position of intron 14. This mutation results in two abundantly expressed aberrantly spliced Agps transcripts: Agps Δexon14 lacking exon 14 or Agps exonΔ13-14 lacking both exons 13 and 14 as well as full-length Agps transcript. Agps is a peroxisomal enzyme which catalyzes the formation of the ether bond during the synthesis of ether lipids. Both aberrantly spliced Agps Δexon14 and Agps exonΔ13-14 transcripts led to frame shift, premature stop and putative proteins lacking the enzymatic FAD domain. We present evidence that bs2 mice have significantly decreased levels of ether lipids. Human mutations in Agps result in rhizomelic chondrodysplasia punctata type 3 (RCDP3), a disease for which bs2 is the only genetic model. Thus, bs2 is a hypomorphic mutation in Agps, and represents a useful model for investigation of the tissue specificity of ether lipid requirements which will be particularly valuable for elucidating the mechanism of disease phenotypes resulting from ether lipid depletion.
The waved with open eyes (woe) locus is a spontaneous recessive mouse mutation that exhibits wavy fur, eyelids open at birth, and enlarged heart and esophagus. In this study, we confirmed the previously identified woe phenotypes and additionally identified anterior eye segment defects, absence of the meibomian glands, and defects in the semilunar cardiac valves. Positional cloning identified a C794T substitution in the Adam17 gene that ablates a putative exonic splicing enhancer (ESE) sequence in exon 7 resulting in aberrant Adam17 splicing. The predominant woe transcript, Adam17 Dexon7, lacks exon 7 resulting in an in-frame deletion of 90 bp and a putative Adam17 D252-281 protein lacking residues 252-281 from the metalloprotease domain. Western blot analysis in woe identified only the precursor form of Adam17 D252-281 protein. Absence of cleavage of the prodomain renders Adam17 D252-281 functionally inactive; however, constitutive and stimulated shedding of Adam17 substrates was detected in woe at significantly reduced levels. This residual Adam17 shedding activity in woe most likely originates from full-length Adam17 T265M encoded by the Adam17 C794T transcript identified expressed at severely reduced levels. These results show that even small amounts of functional Adam17 allow woe mice to survive into adulthood. In contrast to Adam17 À/À mice that die at birth, the viability of woe mice provides an excellent opportunity for studying the role of Adam17 throughout postnatal development and homeostasis.
Rhizomelic chondrodysplasia punctata (RCDP) is a genetically heterogeneous autosomal recessive syndrome characterized by congenital cataracts, shortening of the proximal limbs, neurological abnormalities, seizures, growth delays, and severe intellectual disability. Most RCDP children die in the first decade of life due to respiratory complications. Mutations in alkylglycerone phosphate synthase (AGPS) cause RCDP type 3 (RCDP3). We've previously established that cataracts and male infertility in blind sterile 2 (bs2) mice are caused by a spontaneous hypomorphic mutation in Agps. As a part of this study, we set out to further explore the bs2 phenotypes and how they correlate to the clinical presentations of RCDP3 patients. Our results show that ~ 50% bs2 mice die embryonically and surviving bs2 mice exhibit growth delays that they overcome by adulthood. The X-ray analysis of adult bs2 mice revealed significant humeral, but not femoral shortening. Clinical and histological eye evaluations revealed that bs2 lenses undergo normal development with first opacities developing at P21 that by P28 rapidly progress to mature cataracts. Evaluation of testes determined that infertility in bs2 mice is due to the aberrant formation of multicellular cellular clusters that undergo apoptosis. Given that the bs2 locus is a hypomorphic Agps mutation, we set out to generate Agps knockout mice utilizing the Knockout Mouse Project (KOMP) resource. Our results showed that ~ 85% of Agps knock-out mice die embryonically whereas surviving adult Agps knock-out mice phenotypically exhibit cataracts and testicular abnormalities similar to those observed in bs2 mice. Given that the majority of Agps knock-out mice die embryonically, this presented a challenge for further analyses of Agps deficiency in mouse models. Although not done as a part of this study, Agps-KOMP mice or ES cells can be further modified with FLP recombinase to generate mice suitable for subsequent matings with a transgenic Cre strain of choice, thereby providing an opportunity to study conditional Agps deficiency in a specific tissue or desired developmental time points without Agps deficiency-mediated embryonic lethality.
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