Biallelic KIF24 Variants Are Responsible for a Spectrum of Skeletal Disorders Ranging From Lethal Skeletal Ciliopathy to Severe Acromesomelic Dysplasia.

Skeletal dysplasias comprise a large spectrum of mostly monogenic disorders affecting bone growth, patterning, and homeostasis, and ranging in severity from lethal to mild phenotypes. This study aimed to underpin the genetic cause of skeletal dysplasia in three unrelated families with variable skeletal manifestations. The six affected individuals from three families had severe short stature with extreme shortening of forelimbs, short long-bones, and metatarsals, and brachydactyly (family 1); mild short stature, platyspondyly, and metaphyseal irregularities (family 2); or a prenatally lethal skeletal dysplasia with kidney features suggestive of a ciliopathy (family 3). Genetic studies by whole genome, whole exome, and ciliome panel sequencing identified in all affected individuals biallelic missense variants in KIF24, which encodes a kinesin family member controlling ciliogenesis. In families 1 and 3, with the more severe phenotype, the affected subjects harbored homozygous variants (c.1457A>G; p.(Ile486Val) and c.1565A>G; p.(Asn522Ser), respectively) in the motor domain which plays a crucial role in KIF24 function. In family 2, compound heterozygous variants (c.1697C>T; p.(Ser566Phe)/c.1811C>T; p.(Thr604Met)) were found C-terminal to the motor domain, in agreement with a genotype-phenotype correlation. In vitro experiments performed on amnioblasts of one affected fetus from family 3 showed that primary cilia assembly was severely impaired, and that cytokinesis was also affected. In conclusion, our study describes novel forms of skeletal dysplasia associated with biallelic variants in KIF24. To our knowledge this is the first report implicating KIF24 variants as the cause of a skeletal dysplasia, thereby extending the genetic heterogeneity and the phenotypic spectrum of rare bone disorders and underscoring the wide range of monogenetic skeletal ciliopathies. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Genome editing in stem cells for genetic neurodisorders.

The recent advent of genome editing techniques and their rapid improvement paved the way in establishing innovative human neurological disease models and in developing new therapeutic opportunities. Human pluripotent (both induced or naive) stem cells and neural stem cells represent versatile tools to be applied to multiple research needs and, together with genomic snip and fix tools, have recently made possible the creation of unique platforms to directly investigate several human neural affections. In this chapter, we will discuss genome engineering tools, and their recent improvements, applied to the stem cell field, focusing on how these two technologies may be pivotal instruments to deeply unravel molecular mechanisms underlying development and function, as well as disorders, of the human brain. We will review how these frontier technologies may be exploited to investigate or treat severe neurodevelopmental disorders, such as microcephaly, autism spectrum disorder, schizophrenia, as well as neurodegenerative conditions, including Parkinson's disease, Huntington's disease, Alzheimer's disease, and spinal muscular atrophy.

A Monolayer System for the Efficient Generation of Motor Neuron Progenitors and Functional Motor Neurons from Human Pluripotent Stem Cells.

Methods for the conversion of human induced pluripotent stem cells (hiPSCs) into motor neurons (MNs) have opened to the generation of patient-derived in vitro systems that can be exploited for MN disease modelling. However, the lack of simplified and consistent protocols and the fact that hiPSC-derived MNs are often functionally immature yet limit the opportunity to fully take advantage of this technology, especially in research aimed at revealing the disease phenotypes that are manifested in functionally mature cells. In this study, we present a robust, optimized monolayer procedure to rapidly convert hiPSCs into enriched populations of motor neuron progenitor cells (MNPCs) that can be further amplified to produce a large number of cells to cover many experimental needs. These MNPCs can be efficiently differentiated towards mature MNs exhibiting functional electrical and pharmacological neuronal properties. Finally, we report that MN cultures can be long-term maintained, thus offering the opportunity to study degenerative phenomena associated with pathologies involving MNs and their functional, networked activity. These results indicate that our optimized procedure enables the efficient and robust generation of large quantities of MNPCs and functional MNs, providing a valid tool for MNs disease modelling and for drug discovery applications.

CAMSAPs organize an acentrosomal microtubule network from basal varicosities in radial glial cells.

Neurons of the neocortex are generated by stem cells called radial glial cells. These polarized cells extend a short apical process toward the ventricular surface and a long basal fiber that acts as a scaffold for neuronal migration. How the microtubule cytoskeleton is organized in these cells to support long-range transport is unknown. Using subcellular live imaging within brain tissue, we show that microtubules in the apical process uniformly emanate for the pericentrosomal region, while microtubules in the basal fiber display a mixed polarity, reminiscent of the mammalian dendrite. We identify acentrosomal microtubule organizing centers localized in varicosities of the basal fiber. CAMSAP family members accumulate in these varicosities, where they control microtubule growth. Double knockdown of CAMSAP1 and 2 leads to a destabilization of the entire basal process. Finally, using live imaging of human fetal cortex, we reveal that this organization is conserved in basal radial glial cells, a related progenitor cell population associated with human brain size expansion.