Pathogenic Variants in GPC4 Cause Keipert Syndrome.

Glypicans are a family of cell-surface heparan sulfate proteoglycans that regulate growth-factor signaling during development and are thought to play a role in the regulation of morphogenesis. Whole-exome sequencing of the Australian family that defined Keipert syndrome (nasodigitoacoustic syndrome) identified a hemizygous truncating variant in the gene encoding glypican 4 (GPC4). This variant, located in the final exon of GPC4, results in premature termination of the protein 51 amino acid residues prior to the stop codon, and in concomitant loss of functionally important N-linked glycosylation (Asn514) and glycosylphosphatidylinositol (GPI) anchor (Ser529) sites. We subsequently identified seven affected males from five additional kindreds with novel and predicted pathogenic variants in GPC4. Segregation analysis and X-inactivation studies in carrier females provided supportive evidence that the GPC4 variants caused the condition. Furthermore, functional studies of recombinant protein suggested that the truncated proteins p.Gln506∗ and p.Glu496∗ were less stable than the wild type. Clinical features of Keipert syndrome included a prominent forehead, a flat midface, hypertelorism, a broad nose, downturned corners of mouth, and digital abnormalities, whereas cognitive impairment and deafness were variable features. Studies of Gpc4 knockout mice showed evidence of the two primary features of Keipert syndrome: craniofacial abnormalities and digital abnormalities. Phylogenetic analysis demonstrated that GPC4 is most closely related to GPC6, which is associated with a bone dysplasia that has a phenotypic overlap with Keipert syndrome. Overall, we have shown that pathogenic variants in GPC4 cause a loss of function that results in Keipert syndrome, making GPC4 the third human glypican to be linked to a genetic syndrome.

Myosin-X knockout is semi-lethal and demonstrates that myosin-X functions in neural tube closure, pigmentation, hyaloid vasculature regression, and filopodia formation.

Myosin-X (Myo10) is an unconventional myosin best known for its striking localization to the tips of filopodia. Despite the broad expression of Myo10 in vertebrate tissues, its functions at the organismal level remain largely unknown. We report here the generation of KO-first (Myo10 tm1a/tm1a ), floxed (Myo10 tm1c/tm1c ), and KO mice (Myo10 tm1d/tm1d ). Complete knockout of Myo10 is semi-lethal, with over half of homozygous KO embryos exhibiting exencephaly, a severe defect in neural tube closure. All Myo10 KO mice that survive birth exhibit a white belly spot, all have persistent fetal vasculature in the eye, and ~50% have webbed digits. Myo10 KO mice that survive birth can breed and produce litters of KO embryos, demonstrating that Myo10 is not absolutely essential for mitosis, meiosis, adult survival, or fertility. KO-first mice and an independent spontaneous deletion (Myo10 m1J/m1J ) exhibit the same core phenotypes. During retinal angiogenesis, KO mice exhibit a ~50% decrease in endothelial filopodia, demonstrating that Myo10 is required to form normal numbers of filopodia in vivo. The Myo10 mice generated here demonstrate that Myo10 has important functions in mammalian development and provide key tools for defining the functions of Myo10 in vivo.

Facial Nerve Recovery in KbDb and C1q Knockout Mice: A Role for Histocompatibility Complex 1.

BACKGROUND: Understanding the mechanisms in nerve damage can lead to better outcomes for neuronal rehabilitation. The purpose of our study was to assess the effect of major histocompatibility complex I deficiency and inhibition of the classical complement pathway (C1q) on functional recovery and cell survival in the facial motor nucleus (FMN) after crush injury in adult and juvenile mice. METHODS: A prospective blinded analysis of functional recovery and cell survival in the FMN after a unilateral facial nerve crush injury in juvenile and adult mice was undertaken between wild-type, C1q knockout (C1q-/-), and KbDb knockout (KbDb-/-) groups. Whisker function was quantified to assess functional recovery. Neuron counts were performed to determine neuron survival in the FMN after recovery. RESULTS: After facial nerve injury, all adult wild-type mice fully recovered. Juvenile mice recovered incompletely corresponding to a greater neuron loss in the FMN of juveniles compared with adults. The C1q-/- juvenile and adult groups did not differ from wild type. The KbDb-/- adults demonstrated 50% recovery of whisker movement and decreased cell survival in FMN. The KbDb-/- juvenile group did not demonstrate any difference from control group. CONCLUSION: Histocompatibility complex I plays a role for neuroprotection and enhanced facial nerve recovery in adult mice. Inhibition of the classical complement pathway alone does not affect functional recovery or neuronal survival. The alternative and mannose binding pathways pose alternative means for activating the final components of the pathway that may lead to acute nerve damage.

The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing.

The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.

Whirler mutant hair cells have less severe pathology than shaker 2 or double mutants.

MYOSIN XV is a motor protein that interacts with the PDZ domain-containing protein WHIRLIN and transports WHIRLIN to the tips of the stereocilia. Shaker 2 (sh2) mice have a mutation in the motor domain of MYOSIN XV and exhibit congenital deafness and circling behavior, probably because of abnormally short stereocilia. Whirler (wi) mice have a similar phenotype caused by a deletion in the third PDZ domain of WHIRLIN. We compared the morphology of Whrn (wi/wi) and Myo15 (sh2/sh2) sensory hair cells and found that Myo15 (sh2/sh2) have more frequent pathology at the base of inner hair cells than Whrn (wi/wi), and shorter outer hair cell stereocilia. Considering the functional and morphologic similarities in the phenotypes caused by mutations in Myo15 and Whrn, and the physical interaction between their encoded proteins, we used a genetic approach to test for functional overlap. Double heterozygotes (Myo15 (sh2/+), Whrn (wi/+)) have normal hearing and no increase in hearing loss compared to normal littermates. Single and double mutants (Myo15 (sh2/sh2), Whrn (wi/wi)) exhibit abnormal persistence of kinocilia and microvilli, and develop abnormal cytoskeletal architecture. Double mutants are also similar to the single mutants in viability, circling behavior, and lack of a Preyer reflex. The morphology of cochlear hair cell stereocilia in double mutants reflects a dominance of the more severe Myo15 (sh2/sh2) phenotype over the Whrn (wi/wi) phenotype. This suggests that MYOSIN XV may interact with other proteins besides WHIRLIN that are important for hair cell maturation.