A methodology to abridge microdosimetric distributions without a significant loss of the spectral information needed for the RBE computation in carbon ion therapy.

BACKGROUND: In order to compute the relative biological effectiveness (RBE) of ion radiation therapy with the Mayo Clinic Florida microdosimetric kinetic model (MCF MKM), it is necessary to process entire microdosimetric distributions. Therefore, a posteriori RBE recalculations (i.e., for a different cell line or another biological endpoint) would require whole spectral information. It is currently not practical to compute and store all this data for each clinical voxel. PURPOSE: To develop a methodology that allows to store a limited amount of physical information without losing accuracy in the RBE calculations nor the possibility of a posteriori RBE recalculations. METHODS: Computer simulations for four monoenergetic 12 C ion beams and a 12 C ion spread-out Bragg peak (SOBP) were performed to assess lineal energy distributions as a function of the depth within a water phantom. These distributions were used in combination with the MCF MKM to compute the in vitro clonogenic survival RBE for human salivary gland tumor cells (HSG cell line) and human skin fibroblasts (NB1RGB cell line). The RBE values were also calculated with a new abridged microdosimetric distribution methodology (AMDM) and compared with the reference RBE calculations using the entire distributions. RESULTS: The maximum relative deviation between the RBE values computed using the entire distributions and the AMDM was 0.61% (monoenergetic beams) and 0.49% (SOBP) for the HSG cell line, while 0.45% (monoenergetic beams) and 0.26% (SOBP) for the NB1RGB cell line. CONCLUSION: The excellent agreement between the RBE values computed using the entire lineal energy distributions and the AMDM represents a milestone for the clinical implementation of the MCF MKM.

Unveiling the pathogenic mechanisms of NPR2 missense variants: insights into the genotype-associated severity in acromesomelic dysplasia and short stature.

Introduction: Natriuretic peptide receptor 2 (NPR2 or NPR-B) plays a central role in growth development and bone morphogenesis and therefore loss-of-function variations in NPR2 gene have been reported to cause Acromesomelic Dysplasia, Maroteaux type 1 and short stature. While several hypotheses have been proposed to underlie the pathogenic mechanisms responsible for these conditions, the exact mechanisms, and functional characteristics of many of those variants and their correlations with the clinical manifestations have not been fully established. Methods: In this study, we examined eight NPR2 genetic missense variants (p.Leu51Pro, p.Gly123Val, p.Leu314Arg, p.Arg318Gly, p.Arg388Gln, p.Arg495Cys, p.Arg557His, and p.Arg932Cys) Acromesomelic Dysplasia, Maroteaux type 1 and short stature located on diverse domains and broadly classified as variants of uncertain significance. The evaluated variants are either reported in patients with acromesomelic dysplasia in the homozygous state or short stature in the heterozygous state. Our investigation included the evaluation of their expression, subcellular trafficking and localization, N-glycosylation profiles, and cyclic guanosine monophosphate (cGMP) production activity. Results and Discussion: Our results indicate that variants p.Leu51Pro, p.Gly123Val, p.Leu314Arg, p.Arg388Gln have defective cellular trafficking, being sequestered within the endoplasmic reticulum (ER), and consequently impaired cGMP production ability. Conversely, variants p.Arg318Gly, p.Arg495Cys, and p.Arg557His seem to display a non-statistically significant behavior that is slightly comparable to WT-NPR2. On the other hand, p.Arg932Cys which is located within the guanylyl cyclase active site displayed normal cellular trafficking profile albeit with defective cGMP. Collectively, our data highlights the genotype-phenotype relationship that might be responsible for the milder symptoms observed in short stature compared to acromesomelic dysplasia. This study enhances our understanding of the functional consequences of several NPR2 variants, shedding light on their mechanisms and roles in related genetic disorders which might also help in their pathogenicity re-classification.

Clinical Characteristics of Short-Stature Patients With an NPR2 Mutation and the Therapeutic Response to rhGH.

CONTEXT: The natriuretic peptide receptor 2 gene (NPR2) is a causative gene of idiopathic short stature (ISS) with an incidence rate of 2% to 6%. The clinical characteristics of patients with NPR2 heterozygous mutations are atypical, and data on the efficacy of recombinant human growth hormone (rhGH) treatment in patients with NPR2 mutations are limited. OBJECTIVES: This work reports 6 cases with NPR2 mutation and explores the characteristics of patients with an NPR2 mutation and their therapeutic response to rhGH. DESIGN, SETTINGS, AND PATIENTS: Six Chinese short-stature patients in our hospital with NPR2 mutations by whole-exome sequencing were included. We also searched all previously published NPR2 mutation cases as of August 10, 2020, and information about their medical history, mutations, and rhGH treatment were recorded and summarized. RESULTS: The clinical characteristics of patients with an NPR2 heterozygous mutation mainly included short stature, facial anomalies, and skeletal dysplasia. Skeletal dysplasia mainly included brachydactyly (56.2%), shortened metacarpals or metatarsals (particularly fourth to fifth; 26.1%), and clinodactyly (21.7%). rhGH treatment significantly improved the height SD score (SDS) of patients with NPR2 heterozygous mutations (median, -2.1 vs -2.9, P < .001), especially in girls. The height SDS change correlated negatively with initial age of treatment (r = -0.477; P = .034), and height SDS change of patients with NPR2 heterozygous mutations in the carboxyl-terminal guanylyl cyclase catalytic domain was significantly higher than that of the extracellular ligand-binding region domain (median, 1.9 vs 0.6, P = .019). CONCLUSIONS: ISS patients with skeletal deformities should be tested for an NPR2 mutation. rhGH treatment is beneficial for short-stature patients with NPR2 heterozygous mutations and needs further study.

A mild case of acromesomelic dysplasia, type Maroteaux with novel natriuretic peptide receptor B (NPR2) variants.

Acromesomelic dysplasia, type Maroteaux is caused by variants in NPR2. It is a severe chondrodysplasia resulting in shortening of the middle and distal segments of the limbs. Limb length at birth may be normal but decreased growth becomes obvious in the first 2 years of life. Here we present an 11-year-old male with mild but typical skeletal features of acromesomelic dysplasia, type Maroteaux. Whole exome sequencing has identified two likely pathogenic variants in NPR2 which have not previously been reported in individuals with acromesomelic dysplasia, type Maroteaux. Given these findings, a diagnosis of AMDM should be considered in individuals with characteristic radiological findings, even if stature is only modestly affected.

PICT1 is critical for regulating the Rps27a-Mdm2-p53 pathway by microtubule polymerization inhibitor against cervical cancer.

In our previous study, it showed that P-3F, a podophyllotoxin derivative, causes the increased level of p53 expression by enhancing p53 stability, resulting from blockage of the Mdm2-p53 feedback loop via nucleolus-to-nucleoplasm translocation of Rps27a in human cervical cancer HeLa cell line. However, the mechanism of regulating Rps27a localization remains to be studied. In the current study, it has been demonstrated that the level of protein interacting with carboxyl terminus 1 (PICT1), originally identified as a tumor suppressor, was decreased in a concentration-dependent manner in response to P-3F, leading to inhibition of human cervical cancer cell lines proliferation. Also remarkably, reduction of serine phosphorylation of STMN1 at position 16 induced by P-3F was required in the downregulation of PICT1, in which p53 activity was likely to be directly involved. Note as well that, PICT1 also played an important role in p53 stability enhancement by inhibiting Mdm2-mediated p53 ubiquitination due to Rps27a translocation from the nucleolus to the nucleoplasm to interact with Mdm2 following treatment with P-3F. Collectively, these findings indicated that P-3F, a microtubule polymerization inhibitor, promotes the decreased level of PICT1 expression, which is critical for regulating the Rps27a-Mdm2-p53 pathway against cervical cancer.

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression.

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo, representing a viable therapeutic strategy for cancer treatment. Using phage display techniques, we previously identified a potent peptide activator of P53, termed PMI (TSFAEYWNLLSP), with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range. Here we report an ultrahigh affinity, dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design. Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3 (LTFLEYWAQLMQ) that bound to MDM2 and MDMX with K d values in the low picomolar concentration range as verified by isothermal titration calorimetry. Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 Å resolution, respectively. Similar to PMI, PMI-M3 occupied the P53-binding pocket of MDM2/MDMX, which was dominated energetically by intermolecular interactions involving Phe3, Tyr6, Trp7, and Leu10. Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2, and Leu1 and Met11 with MDMX, collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins. By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake, we obtained modified peptides termed PMI-2K (KTSFAEYWNLLSPK) and M3-2K (KLTFLEYWAQLMQK). Compared with PMI-2K, M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner. This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become, in its own right, a powerful lead compound for anticancer drug development, and can aid molecular design of other classes of P53 activators as well for anticancer therapy.

A novel nonsense mutation in NPR2 gene causing Acromesomelic dysplasia, type Maroteaux in a consanguineous family in Southern Punjab (Pakistan).

BACKGROUND: Acromesomelic dysplasia, type Maroteaux (AMDM) is a rare skeletal dysplasia following autosomal recessive mode of inheritance and characterized by abnormal growth plates, short and abnormal bones in the extremities and spine. OBJECTIVE: Present study was designed to report the molecular basis of AMDM in enrolled consanguineous family from Pakistan. METHODS: A consanguineous family from Vehari District in Pakistan having multiple siblings suffering from AMDM was enrolled in present study. Whole exome sequencing (WES) approach was adopted to identify causative agent of AMDM. Human full length NPR2 gene and sequence with nonsense mutation was amplified by using Myc-tagged pXN vector and transformed in E. coli DH5α cells to confirm mutation. SDS-PAGE and Western blotting were done to confirm the production of truncated protein. Computational three dimensional structure generation through homology modeling approach was done to compare protein structure between patients and controls. RESULTS: WES reveled a nonsense mutation (c.613 C>T, p.R205X) in exon 1 of NPR2 gene leading to premature termination codon in mRNA of NPR2 gene resulting in a truncated protein with 204 amino acid residues that was confirmed by SDS-PAGE and Western blotting. Sanger sequencing confirmed that mutation in all subjects and mutation followed Mendalian pattern of inheritance. Multiple sequence alignment by ClustalW revealed that mutated domain of NPR2 is conserved region. Proetin structure comparison revealed a significant structural part of NPR2 was missing in truncated protein as compared to control. CONCLUSION: We are reporting that a novel nonsense mutation (c.613 C>T, p.R205X) in exon 1 of NPR2 gene is causing AMDM in a consanguineous Pakistani family.

Novel variants in natriuretic peptide receptor 2 in unrelated patients with acromesomelic dysplasia type Maroteaux.

Acromesomelic dysplasia are a heterogeneous group of disorders with variable spectrum and severity of skeletal anomalies in the affected individuals. Acromesomelic dysplasia type Maroteaux (AMDM) is characterized by extreme shortening of the forelimbs and disproportionate short stature. Several homozygous inactivating mutations in NPR2 have been identified in different AMDM patients. We report five novel variants in affected individuals in four different families. These include two nonsense and three missense variants. This study broadens the genotypic spectrum of NPR2 mutations in individuals with AMDM and also describes the intra- and inter-familial phenotypic variability due to NPR2 variants.

Molecular and in silico analyses validates pathogenicity of homozygous mutations in the NPR2 gene underlying variable phenotypes of Acromesomelic dysplasia, type Maroteaux.

Homozygous and/or heterozygous loss of function mutations in the natriuretic peptide receptor B (NPR2) have been reported in causing acromesomelic dysplasia, type Maroteaux with variable clinical features and idiopathic short stature with nonspecific skeletal deformities. On the other hand, gain of function mutations in the same gene result in overgrowth disorder suggesting that NPR2 and its ligand, natriuretic peptide precursor C (CNP), are the key players of endochondral bone growth. However, the precise mechanism behind phenotypic variability of the NPR2 mutations is not fully understood so far. In the present study, three consanguineous families of Pakistani origin (A, B, C) with variable phenotypes of acromesomelic dysplasia, type Maroteaux were evaluated at clinical and molecular levels. Linkage analysis followed by Sanger sequencing of the NPR2 gene revealed three homozygous mutations including p.(Leu314 Arg), p.(Arg371*), and p.(Arg1032*) in family A, B and C, respectively. In silico structural and functional analyses substantiated that a novel missense mutation [p.(Leu314 Arg)] in family A allosterically affects binding of NPR2 homodimer to its ligand (CNP) which ultimately results in defective guanylate cyclase activity. A nonsense mutation [p.(Arg371*)] in family B entirely removed the transmembrane domain, protein kinase domain and guanylate cyclase domains of the NPR2 resulting in abolishing its guanylate cyclase activity. Another novel mutation [p.(Arg1032*)], found in family C, deteriorated the guanylate cyclase domain of the protein and probably plundered its guanylate cyclase activity. These results suggest that guanylate cyclase activity is the most critical function of the NPR2 and phenotypic severity of the NPR2 mutations is proportional to the reduction in its guanylate cyclase activity.