Hypoxia-inducible factor (HIF), an αβ dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxiainducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αβ dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression.

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Proper deposition of the extracellular matrix and its major components, the collagens, is essential for endochondral ossification and bone mass accrual. Collagen prolyl 4‐hydroxylases (C‐P4Hs) hydroxylate proline residues in the ‐X‐Pro‐Gly‐ repeats of all known collagen types. Their product, 4‐hydroxyproline, is essential for correct folding and thermal stability of the triple‐helical collagen molecules in physiological body temperatures. We have previously shown that inactivation of the mouse P4ha1 gene, which codes for the catalytic α subunit of the major C‐P4H isoform, is embryonic lethal, whereas inactivation of the P4ha2 gene produced only a minor phenotype. Instead, mice with a haploinsufficiency of the P4ha1 gene combined with a homozygous deletion of the P4ha2 gene present with a moderate chondrodysplasia due to transient cell death of the growth plate chondrocytes. Here, to further characterize the bone phenotype of the P4ha1+/−; P4ha2−/− mice, we have carried out gene expression analyses at whole‐tissue and single‐cell levels, biochemical analyses, microcomputed tomography, histomorphometric analyses, and second harmonic generation microscopy to show that C‐P4H α subunit expression peaks early and that the C‐P4H deficiency leads to reduced collagen amount, a reduced rate of bone formation, and a loss of trabecular and cortical bone volume in the long bones. The total osteoblast number in the proximal P4ha1+/−; P4ha2−/− tibia and the C‐P4H activity in primary P4ha1+/−; P4ha2−/− osteoblasts were reduced, whereas the population of osteoprogenitor colony‐forming unit fibroblasts was increased in the P4ha1+/−; P4ha2−/− marrow. Thus, the P4ha1+/−; P4ha2−/− mouse model recapitulates key aspects of a recently recognized congenital connective tissue disorder with short stature and bone dysplasia caused by biallelic variants of the human P4HA1 gene. Altogether, the data demonstrate the allele dose‐dependent importance of the C‐P4Hs to the developing organism and a threshold effect of C‐P4H activity in the proper production of bone matrix. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Background Medulloblastomas (MBs) are a heterogeneous group of childhood brain tumors with four consensus subgroups, namely MBSHH, MBWNT, MBGroup 3, and MBGroup 4, representing the second most common type of pediatric brain cancer after high‐grade gliomas. They suffer from a high prevalence of genetic predisposition with up to 20% of MBSHH caused by germline mutations in only six genes. However, the spectrum of germline mutations in MBSHH remains incomplete. Methods Comprehensive Next‐Generation Sequencing panels of both tumor and patient blood samples were performed as molecular genetic characterization. The panels cover genes that are known to predispose to cancer. Results Here, we report on a patient with a pathogenic germline PTEN variant resulting in an early stop codon p.(Glu7Argfs*4) (ClinVar ID: 480383). The patient developed macrocephaly and MBSHH, but reached remission with current treatment protocols. Conclusions We propose that pathogenic PTEN variants may predispose to medulloblastoma, and show that remission was reached with current treatment protocols. The PTEN gene should be included in the genetic testing provided to patients who develop medulloblastoma at an early age. We recommend brain magnetic resonance imaging upon an unexpected acceleration of growth of head circumference for pediatric patients harboring pathogenic germline PTEN variants.

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