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Background: Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown., Methods: To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy., Results: C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells., Conclusion: We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Marom et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a rare disorder of enteric smooth muscle function affecting the intestine and bladder. Patients with this severe phenotype are dependent on total parenteral nutrition and urinary catheterization. The cause of this syndrome has remained a mystery since Berdon's initial description in 1976. No genes have been clearly linked to MMIHS. We used whole-exome sequencing for gene discovery followed by targeted Sanger sequencing in a cohort of patients with MMIHS and intestinal pseudo-obstruction. We identified heterozygous ACTG2 missense variants in 15 unrelated subjects, ten being apparent de novo mutations. Ten unique variants were detected, of which six affected CpG dinucleotides and resulted in missense mutations at arginine residues, perhaps related to biased usage of CpG containing codons within actin genes. We also found some of the same heterozygous mutations that we observed as apparent de novo mutations in MMIHS segregating in families with intestinal pseudo-obstruction, suggesting that ACTG2 is responsible for a spectrum of smooth muscle disease. ACTG2 encodes γ2 enteric actin and is the first gene to be clearly associated with MMIHS, suggesting an important role for contractile proteins in enteric smooth muscle disease.

Mutations in CRTAP (coding for cartilage-associated protein), LEPRE1 (coding for prolyl 3-hydroxylase 1 [P3H1]) or PPIB (coding for Cyclophilin B [CYPB]) cause recessive forms of osteogenesis imperfecta and loss or decrease of type I collagen prolyl 3-hydroxylation. A comprehensive analysis of the phenotype of the Crtap-/- mice revealed multiple abnormalities of connective tissue, including in the lungs, kidneys, and skin, consistent with systemic dysregulation of collagen homeostasis within the extracellular matrix. Both Crtap-/- lung and kidney glomeruli showed increased cellular proliferation. Histologically, the lungs showed increased alveolar spacing, while the kidneys showed evidence of segmental glomerulosclerosis, with abnormal collagen deposition. The Crtap-/- skin had decreased mechanical integrity. In addition to the expected loss of proline 986 3-hydroxylation in alpha1(I) and alpha1(II) chains, there was also loss of 3Hyp at proline 986 in alpha2(V) chains. In contrast, at two of the known 3Hyp sites in alpha1(IV) chains from Crtap-/- kidneys there were normal levels of 3-hydroxylation. On a cellular level, loss of CRTAP in human OI fibroblasts led to a secondary loss of P3H1, and vice versa. These data suggest that both CRTAP and P3H1 are required to maintain a stable complex that 3-hydroxylates canonical proline sites within clade A (types I, II, and V) collagen chains. Loss of this activity leads to a multi-systemic connective tissue disease that affects bone, cartilage, lung, kidney, and skin.