Dominant negative variants in KIF5B cause osteogenesis imperfecta via down regulation of mTOR signaling.

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.

Osteogenesis imperfecta: an update on clinical features and therapies.

Osteogenesis imperfecta (OI) is an inherited skeletal dysplasia characterized by bone fragility and skeletal deformities. While the majority of cases are associated with pathogenic variants in COL1A1 and COL1A2, the genes encoding type I collagen, up to 25% of cases are associated with other genes that function within the collagen biosynthesis pathway or are involved in osteoblast differentiation and bone mineralization. Clinically, OI is heterogeneous in features and variable in severity. In addition to the skeletal findings, it can affect multiple systems including dental and craniofacial abnormalities, muscle weakness, hearing loss, respiratory and cardiovascular complications. A multi-disciplinary approach to care is recommended to address not only the fractures, reduced mobility, growth and bone pain but also other extra-skeletal manifestations. While bisphosphonates remain the mainstay of treatment in OI, new strategies are being explored, such as sclerostin inhibitory antibodies and TGF beta inhibition, to address not only the low bone mineral density but also the inherent bone fragility. Studies in animal models have expanded the understanding of pathomechanisms of OI and, along with ongoing clinical trials, will allow to develop better therapeutic approaches for these patients.

Osteogenesis imperfecta: advancements in genetics and treatment.

PURPOSE OF REVIEW: The purpose of this review is to outline the current understanding of the molecular mechanisms and natural history of osteogenesis imperfecta, and to describe the development of new treatments for this disorder. RECENT FINDINGS: The introduction of next-generation sequencing technology has led to better understanding of the genetic cause of osteogenesis imperfecta and enabled cost-effective and timely diagnosis via expanded gene panels and exome or genome sequencing. Clinically, despite genetic heterogeneity, different forms of osteogenesis imperfecta share similar features that include connective tissue and systemic manifestations in addition to bone fragility. Thus, the goals of treatment in osteogenesis imperfecta extend beyond decreasing the risk of fracture, to include the maximization of growth and mobility, and the management of extraskeletal complications. The standard of care in pediatric patients is bisphosphonates therapy. Ongoing preclinical studies in osteogenesis imperfecta mouse models and clinical studies in individuals with osteogenesis imperfecta have been instrumental in the development of new and targeted therapeutic approaches, such as sclerostin inhibition and transforming growth factor-ß inhibition. SUMMARY: Osteogenesis imperfecta is a skeletal dysplasia characterized by bone fragility and extraskeletal manifestations. Better understanding of the mechanisms of osteogenesis imperfecta will enable the development of much needed targeted therapies to improve the outcome in affected individuals.

Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration.

A set of glutamylases and deglutamylases controls levels of tubulin polyglutamylation, a prominent post-translational modification of neuronal microtubules. Defective tubulin polyglutamylation was first linked to neurodegeneration in the Purkinje cell degeneration (pcd) mouse, which lacks deglutamylase CCP1, displays massive cerebellar atrophy, and accumulates abnormally glutamylated tubulin in degenerating neurons. We found biallelic rare and damaging variants in the gene encoding CCP1 in 13 individuals with infantile-onset neurodegeneration and confirmed the absence of functional CCP1 along with dysregulated tubulin polyglutamylation. The human disease mainly affected the cerebellum, spinal motor neurons, and peripheral nerves. We also demonstrate previously unrecognized peripheral nerve and spinal motor neuron degeneration in pcd mice, which thus recapitulated key features of the human disease. Our findings link human neurodegeneration to tubulin polyglutamylation, entailing this post-translational modification as a potential target for drug development for neurodegenerative disorders.

De Novo Missense Variants in TRAF7 Cause Developmental Delay, Congenital Anomalies, and Dysmorphic Features.

TRAF7 is a multi-functional protein involved in diverse signaling pathways and cellular processes. The phenotypic consequence of germline TRAF7 variants remains unclear. Here we report missense variants in TRAF7 in seven unrelated individuals referred for clinical exome sequencing. The seven individuals share substantial phenotypic overlap, with developmental delay, congenital heart defects, limb and digital anomalies, and dysmorphic features emerging as key unifying features. The identified variants are de novo in six individuals and comprise four distinct missense changes, including a c.1964G>A (p.Arg655Gln) variant that is recurrent in four individuals. These variants affect evolutionarily conserved amino acids and are located in key functional domains. Gene-specific mutation rate analysis showed that the occurrence of the de novo variants in TRAF7 (p = 2.6 × 10-3) and the recurrent de novo c.1964G>A (p.Arg655Gln) variant (p = 1.9 × 10-8) in our exome cohort was unlikely to have occurred by chance. In vitro analyses of the observed TRAF7 mutations showed reduced ERK1/2 phosphorylation. Our findings suggest that missense mutations in TRAF7 are associated with a multisystem disorder and provide evidence of a role for TRAF7 in human development.

Neonatal fractures as a presenting feature of LMOD3-associated congenital myopathy.

Nemaline myopathy is a rare inherited disorder characterized by weakness, hypotonia, and depressed deep tendon reflexes. It is clinically and genetically heterogeneous, with the most severe phenotype presenting as perinatal akinesia, severe muscle weakness, feeding difficulties and respiratory failure, leading to early mortality. Pathogenic variants in 12 genes, encoding components of the sarcomere or factors related to myogenesis, have been reported in patients affected with the disorder. Here, we describe an early, lethal presentation of decreased fetal movements, hypotonia, muscle weakness, and neonatal respiratory failure requiring ventilator support in three siblings from a consanguineous family. All exhibited perinatal fractures, and thus, a skeletal dysplasia was considered as possibly contributing to the phenotype. However, whole exome sequencing revealed a homozygous, loss-of-function pathogenic variant in LMOD3, which has recently been associated with nemaline myopathy and, in a subset of patients, perinatal fractures. This case demonstrates the importance of considering congenital neuromuscular disorders in the differential diagnosis of perinatal fractures.

Milder clinical and biochemical phenotypes associated with the c.482G>A (p.Arg161Gln) pathogenic variant in cobalamin C disease: Implications for management and screening.

INTRODUCTION: Cobalamin C disease is a multisystemic disease with variable manifestations and age of onset. Genotype-phenotype correlations are well-recognized in this disorder. Here, we present a large cohort of individuals with cobalamin C disease, several of whom are heterozygous for the c.482G>A pathogenic variant (p.Arg161Gln). We compared clinical characteristics of individuals with this pathogenic variant to those who do not have this variant. To our knowledge, this study represents the largest single cohort of individuals with the c.482G>A (p.Arg161Gln) pathogenic variant. METHODS: A retrospective chart review of 27 individuals from 21 families with cobalamin C disease who are followed at our facility was conducted. RESULTS: 13 individuals (48%) are compound heterozygous with the c.482G>A (p.Arg161Gln) on one allele and a second pathogenic variant on the other allele. Individuals with the c.482G>A (p.Arg161Gln) pathogenic variant had later onset of symptoms and easier metabolic control. Moreover, they had milder biochemical abnormalities at presentation which likely contributed to the observation that 4 individuals (31%) in this group were missed by newborn screening. CONCLUSION: The c.482G>A (p.Arg161Gln) pathogenic variant is associated with milder disease. These individuals may not receive a timely diagnosis as they may not be identified on newborn screening or because of unrecognized, late onset symptoms. Despite the milder presentation, significant complications can occur, especially if treatment is delayed.

Pharmacological and biological therapeutic strategies for osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a connective tissue disorder characterized by bone fragility, low bone mass, and bone deformities. The majority of cases are caused by autosomal dominant pathogenic variants in the COL1A1 and COL1A2 genes that encode type I collagen, the major component of the bone matrix. The remaining cases are caused by autosomal recessively or dominantly inherited mutations in genes that are involved in the post-translational modification of type I collagen, act as type I collagen chaperones, or are members of the signaling pathways that regulate bone homeostasis. The main goals of treatment in OI are to decrease fracture incidence, relieve bone pain, and promote mobility and growth. This requires a multi-disciplinary approach, utilizing pharmacological interventions, physical therapy, orthopedic surgery, and monitoring nutrition with appropriate calcium and vitamin D supplementation. Bisphosphonate therapy, which has become the mainstay of treatment in OI, has proven beneficial in increasing bone mass, and to some extent reducing fracture risk. However, the response to treatment is not as robust as is seen in osteoporosis, and it seems less effective in certain types of OI, and in adult OI patients as compared to most pediatric cases. New pharmacological treatments are currently being developed, including anti-resorptive agents, anabolic treatment, and gene- and cell-therapy approaches. These therapies are under different stages of investigation from the bench-side, to pre-clinical and clinical trials. In this review, we will summarize the recent findings regarding the pharmacological and biological strategies for the treatment of patients with OI. © 2016 Wiley Periodicals, Inc.

Newborn screening: a review of history, recent advancements, and future perspectives in the era of next generation sequencing.

PURPOSE OF REVIEW: The purpose of this review is to summarize the development and recent advancements of newborn screening. RECENT FINDINGS: Early initiation of medical care has modified the outcome for many disorders that were previously associated with high morbidity (such as cystic fibrosis, primary immune deficiencies, and inborn errors of metabolism) or with significant neurodevelopmental disabilities (such as phenylketonuria and congenital hypothyroidism). The new era of mass spectrometry and next generation sequencing enables the expansion of the newborn screen panel, and will help to address technical issues such as turnaround time, and decreasing false-positive and false-negative rates for the testing. SUMMARY: The newborn screening program is a successful public health initiative that facilitates early diagnosis of treatable disorders to reduce long-term morbidity and mortality.

New Face for Chromatin-Related Mesenchymal Modulator: n-CHD9 Localizes to Nucleoli and Interacts With Ribosomal Genes.

Mesenchymal stem cells' differentiation into several lineages is coordinated by a complex of transcription factors and co-regulators which bind to specific gene promoters. The Chromatin-Related Mesenchymal Modulator, CHD9 demonstrated in vitro its ability for remodeling activity to reposition nucleosomes in an ATP-dependent manner. Epigenetically, CHD9 binds with modified H3-(K9me2/3 and K27me3). Previously, we presented a role for CHD9 with RNA Polymerase II (Pol II)-dependent transcription of tissue specific genes. Far less is known about CHD9 function in RNA Polymerase I (Pol I) related transcription of the ribosomal locus that also drives specific cell fate. We here describe a new form, the nucleolar CHD9 (n-CHD9) that is dynamically associated with Pol I, fibrillarin, and upstream binding factor (UBF) in the nucleoli, as shown by imaging and molecular approaches. Inhibitors of transcription disorganized the nucleolar compartment of transcription sites where rDNA is actively transcribed. Collectively, these findings link n-CHD9 with RNA pol I transcription in fibrillar centers. Using chromatin immunoprecipitation (ChIP) and tilling arrays (ChIP- chip), we find an association of n-CHD9 with Pol I related to rRNA biogenesis. Our new findings support the role for CHD9 in chromatin regulation and association with rDNA genes, in addition to its already known function in transcription control of tissue specific genes.