Barbara Triggs-Raine

Key Points: A novel nonsense variant ( mcy ) in ankyrin repeat and sterile alpha motif domain-containing 6 ( Anks6 ) promotes rapid disease progression in the Han:SPRD- Cy rat carrying a missense variant in Anks6 . mcy ^−/− rats exhibit prenatal lethality characterized by laterality and cardiovascular abnormalities. These findings indicate that ANKS6 nonfunction in rats leads to prenatal lethality, whereas misfunction leads to polycystic kidney disease development. Background: Polycystic kidney disease (PKD) encompasses a group of genetic disorders characterized by the proliferation of fluid-filled renal cysts, leading to progressive renal failure and death. A key feature of PKD is its variable expressivity across patients, even when caused by the same variant, highlighting the importance of genetic background in PKD expression. Methods: We identified an ostensibly healthy Sprague Dawley rat line with a variant that modifies PKD expressivity in Han:SPRD- Cy rats (caused by a missense variant [p.Arg717Trp] in the ankyrin repeat and sterile alpha motif domain-containing 6 [ Anks6 ] gene), which we named mcy (modifier of Cy ). We used whole-genome sequencing and segregation analysis to identify the mcy variant, quantitative PCR and mRNA sequencing to evaluate its effects on gene expression, western blotting and immunohistochemistry to assess its protein consequences, and ultrasound and histology to examine its impact on rat embryonic development. Results: We identified a nonsense variant in the Anks6 gene as the genetic basis of the mcy phenotype (c.1126G>T [p.Glu376X]). Although mcy ^+/− rats are ostensibly healthy and do not develop PKD, mcy ^−/− rats exhibit laterality defects and die prenatally at E16.5 because of apparent perturbations in cardiovascular development. Notably, mcy ^+/− Cy ^+/− rats develop PKD much more rapidly than Cy ^+/− rats, and in a timeframe consistent with Cy ^−/− rats . Transcripts with the mcy variant allele seem to undergo nonsense-mediated decay, and no ANKS6 protein is detected. However, gene expression patterns in the kidneys did not differ significantly between age-matched mcy ^+/+ and mcy ^+/− rats, indicating that ANKS6 insufficiency does not cause PKD. Conclusions: We identified a novel nonsense variant in Anks6 . The findings indicate that the absence of wild-type ANKS6 accelerates PKD development in the Han:SPRD- Cy rat and that complete ANKS6 deficiency prevents normal embryonic development in rats.

The GM2 gangliosidoses are lysosomal storage disorders exhibiting a spectrum of neurological phenotypes ranging from childhood death to debilitating adult-onset neurological impairment. To date, no mouse model harbouring a specific human mutation causing GM2 gangliosidosis has been created. We used CRISPR/Cas9 to generate knockin (KI) mice with the common adult-onset Hexa Gly269Ser variant as well as knockout (KO) mice with Hexa mutations expected to cause complete HexA deficiency. We also created Neu3 KO alleles that combined with Hexa KO or KI alleles were expected to create acute and chronic models of GM2 gangliosidosis, respectively. However, both models accumulated GM2 ganglioside throughout the brain when compared to controls (CON), and exhibited progressive loss of reflexes, gait abnormalities, and premature death by 24 weeks of age. Although survival and behavioural phenotypes did not differ between KO and KI models, the KI model had substantial Hexa mRNA and evidence of GM2 turnover. This KI model will be useful for developing gene editing to correct the variant causing the Gly269Ser substitution and its novel biochemical phenotype suggests it may be suitable for testing therapies that treat partial β-hexosaminidase A deficiency.

Mitochondrial quality control is critical in muscle to ensure contractile and metabolic function. BNIP3L/Nix is a BCL2 member, a mitophagy receptor, and has been implicated in muscle atrophy. Human genome-wide association studies (GWAS) suggest altered BNIP3L expression could predispose to mitochondrial disease. To investigate BNIP3L function, we generated a muscle-specific knockout model. bnip3l knockout mice displayed a ragged-red fiber phenotype, along with accumulation of mitochondria and endo/sarcoplasmic reticulum with altered morphology. Intriguingly, bnip3l knockout mice were more insulin sensitive with a corresponding increase in glycogen-rich muscle fibers. Kinome and gene expression analyses revealed that bnip3l knockout impairs NFAT and MSTN (myostatin) signaling, with alterations in muscle fiber-type and evidence of regeneration. Mechanistic experiments demonstrated that BNIP3L modulates mitophagy, along with reticulophagy leading to altered nuclear calcium signaling. Collectively, these observations identify novel roles for BNIP3L coordinating selective autophagy, oxidative gene expression, and signaling pathways that maintain the muscle phenotype.

Hyaluronan (HA) is a large polysaccharide that is broadly distributed and highly abundant in the soft connective tissues and embryos of vertebrates. The constitutive turnover of HA is very high, estimated at 5 g per day in an average (70 kg) adult human, but HA turnover must also be tightly regulated in some processes. Six genes encoding homologues to bee venom hyaluronidase (HYAL1, HYAL2, HYAL3, HYAL4, HYAL6P/HYALP1, SPAM1/PH20), as well as genes encoding two unrelated G8-domain-containing proteins demonstrated to be involved in HA degradation (CEMIP/KIAA1199, CEMIP2/TMEM2), have been identified in humans. Of these, only deficiencies in HYAL1, HYAL2, HYAL3 and CEMIP have been identified as the cause or putative cause of human genetic disorders. The phenotypes of these disorders have been vital in determining the biological roles of these enzymes but there is much that is still not understood. Deficiencies in these HA-degrading proteins have been created in mice and/or other model organisms where phenotypes could be analyzed and probed to expand our understanding of HA degradation and function. This review will describe what has been found in human and animal models of hyaluronidase deficiency and discuss how this has advanced our understanding of HA’s role in health and disease.

Silver-Russell syndrome (SRS) is a heterogeneous disorder characterized by intrauterine and postnatal growth retardation. HMGA2 variants are a rare cause of SRS and its functional role in human linear growth is unclear. Patients with suspected SRS negative for 11p15LOM/mUPD7 underwent whole-exome and/or targeted-genome sequencing. Mutant HMGA2 protein expression and nuclear localization were assessed. Two Hmga2 -knockin mouse models were generated. Five clinical SRS patients harbored HMGA2 variants with differing functional impacts: 2 stop-gain nonsense variants (c.49G>T, c.52C>T), c.166A>G missense variant, and 2 frameshift variants (c.144delC, c.145delA) leading to an identical, extended-length protein. Phenotypic features were highly variable. Nuclear localization was reduced/absent for all variants except c.166A>G. Homozygous knockin mice recapitulating the c.166A>G variant ( Hmga2 ^K56E ) exhibited a growth-restricted phenotype. An Hmga2 ^Ter76 -knockin mouse model lacked detectable full-length Hmga2 protein, similarly to patient 3 and 5 variants. These mice were infertile, with a pygmy phenotype. We report a heterogeneous group of individuals with SRS harboring variants in HMGA2 and describe the first Hmga2 missense knockin mouse model ( Hmga2 ^K56E ) to our knowledge causing a growth-restricted phenotype. In patients with clinical features of SRS but negative genetic screening, HMGA2 should be included in next-generation sequencing testing approaches.

Mitochondrial quality control is critical in muscle to ensure both contractile and metabolic function. Nix is a BCL-2 family member, mitophagy receptor, and has been implicated in muscle atrophy. Human GWAS suggests altered Nix expression could predispose to manifestations of mitochondrial disease. To interrogate Nix function, we generated a muscle-specific knockout model. Nix knockout mice displayed a ragged-red fibre phenotype, along with accumulation of mitochondria and endo/sarcoplasmic reticulum with altered morphology. Intriguingly, Nix knockout mice were more insulin sensitive with a corresponding increase in glycogen-rich muscle fibres. Kinome- and gene expression analyses revealed that Nix knockout impairs NFAT and canonical myostatin signaling, with alterations in muscle fibre-type composition and evidence of regeneration. Mechanistic experiments demonstrated that Nix modulates mitophagy, along with ER-phagy leading to altered nuclear calcium signaling. Collectively, these observations identify novel roles for Nix coordinating selective autophagy, oxidative gene expression, and signaling pathways that maintain the muscle phenotype.

Chronic hypoxia leads to pathologic remodeling of the pulmonary vasculature and pulmonary hypertension (PH). The antioxidant enzyme extracellular superoxide dismutase (SOD3) protects against hypoxia-induced PH. Hyaluronan (HA), a ubiquitous glycosaminoglycan of the lung extracellular matrix, is rapidly recycled at sites of vessel injury and repair. We investigated the hypothesis that SOD3 preserves HA homeostasis by inhibiting oxidative and enzymatic hyaluronidase-mediated HA breakdown. In SOD3-deficient mice, hypoxia increased lung hyaluronidase expression and activity, hyaluronan fragmentation, and effacement of HA from the vessel wall of small pulmonary arteries. Hyaluronan fragmentation corresponded to hypoxic induction of the cell surface hyaluronidase-2 (Hyal2), which was localized in the vascular media. Human pulmonary artery smooth muscle cells (HPASMCs) demonstrated hypoxic induction of Hyal2 and SOD-suppressible hyaluronidase activity, congruent to our observations in vivo. Fragmentation of homeostatic high molecular weight HA promoted HPASMC proliferation in vitro, whereas pharmacologic inhibition of hyaluronidase activity prevented hypoxia- and oxidant-induced proliferation. Hypoxia initiates SOD3-dependent alterations in the structure and regulation of hyaluronan in the pulmonary vascular extracellular matrix. These changes occurred soon after hypoxia exposure, prior to appearance of PH, and may contribute to the early pathogenesis of this disease.

The glycosylphosphatidylinositol‐anchored sperm hyaluronidases (Hyals), sperm adhesion molecule 1 (SPAM1) and HYAL5, have long been believed to assist in sperm penetration through the cumulus‐oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we have shown that mouse sperm devoid of either Spam1 or Hyal5 are still capable of penetrating the COC and that the loss of either Spam1 or Hyal5 alone does not cause male infertility in mice. In the present study, we found that Spam1/Hyal5 double knockout (dKO) mice produced significantly fewer offspring compared with wild‐type (WT) mice, and this was due to defective COC dispersal. A comparative analysis between WT and Spam1/Hyal5 dKO epididymal sperm revealed that the absence of these 2 sperm Hyals resulted in a marked accumulation of sperm on the outside of the COC. This impaired sperm activity is likely due to the deficiency in the sperm Hyals, even though other somatic Hyals are expressed normally in the dKO mice. The fertilization ability of the Spam1/Hyal5 dKO sperm was restored by adding purified human sperm Hyal to the in vitro fertilization medium. Our results suggest that Hyal deficiency in sperm may be a significant risk factor for male sterility.—Park, S., Kim, Y.‐H., Jeong, P.‐S., Park, C., Lee, J.‐W., Kim, J.‐S., Wee, G., Song, B.‐S., Park, B.‐J., Kim, S.‐H., Sim, B.‐W., Kim, S.‐U., Triggs‐Raine, B., Baba, T., Lee, S.‐R., Kim, E. SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus‐oocyte complex penetration defect. FASEB J. 33, 14440‐14449 (2019). www.fasebj.org