Osteopathia striata with cranial sclerosis as a cancer predisposition syndrome: The first report of neuroblastoma and review of all cancers in OSCS.

Osteopathia Striata with Cranial Sclerosis (OSCS) is a rare genetic condition primarily characterized by metaphyseal striations of long bones, bone sclerosis, macrocephaly, and other congenital anomalies. It is caused by pathogenic variants in AMER1, a tumor suppressor and a WNT signaling repressor gene with key roles in tissue regeneration, neurodevelopment, tumorigenesis, and other developmental processes. While somatic AMER1 pathogenic variants have frequently been identified in several tumor types (e.g., Wilms tumor and colorectal cancer), whether OSCS (i.e., with AMER1 germline variants) is a tumor predisposition syndrome is not clear, with only nine cases reported with tumors. We here report the first case of neuroblastoma diagnosed in a male child with OSCS, review all previously reported tumors diagnosed in individuals with OSCS, and discuss potential tumorigenic mechanisms of AMER1. Our report adds to the accumulating evidence suggesting OSCS is a tumor predisposition condition, highlighting the importance of maintaining a high index of suspicion for the associated tumors when evaluating patients with OSCS. Importantly, Wilms tumor stands out as the most commonly observed tumor in OSCS patients, underscoring the need for regular surveillance.

Cancer-associated mutations in the ribosomal protein L5 gene dysregulate the HDM2/p53-mediated ribosome biogenesis checkpoint.

Perturbations in ribosome biogenesis have been associated with cancer. Such aberrations activate p53 through the RPL5/RPL11/5S rRNA complex-mediated inhibition of HDM2. Studies using animal models have suggested that this signaling pathway might constitute an important anticancer barrier. To gain a deeper insight into this issue in humans, here we analyze somatic mutations in RPL5 and RPL11 coding regions, reported in The Cancer Genome Atlas and International Cancer Genome Consortium databases. Using a combined computational and statistical approach, complemented by a range of biochemical and functional analyses in human cancer cell models, we demonstrate the existence of several mechanisms by which RPL5 mutations may impair wild-type p53 upregulation and ribosome biogenesis. Unexpectedly, the same approach provides only modest evidence for a similar role of RPL11, suggesting that RPL5 represents a preferred target during human tumorigenesis in cancers with wild-type p53. Furthermore, we find that several functional cancer-associated RPL5 somatic mutations occur as rare germline variants in general population. Our results shed light on the so-far enigmatic role of cancer-associated mutations in genes encoding ribosomal proteins, with implications for our understanding of the tumor suppressive role of the RPL5/RPL11/5S rRNA complex in human malignancies.

A novel FAM20C mutation causes a rare form of neonatal lethal Raine syndrome.

Raine syndrome is a rare, autosomal recessive, osteosclerotic bone dysplasia due to pathogenic variants in FAM20C. The clinical phenotype is characterized by generalized osteosclerosis affecting all bones, cerebral calcifications, and craniofacial dysmorphism. Most cases present during the neonatal period with early lethality due to pulmonary hypoplasia and respiratory compromise while only few affected individuals have been reported to survive into adulthood. FAM20C is a ubiquitously expressed protein kinase that contains five functional domains including a catalytic domain, a binding pocket for FAM20A and three distinct N-glycosylation sites. We report a newborn infant with a history of prenatal onset fractures, generalized osteosclerosis, and craniofacial dysmorphism and early lethality. The clinical presentation was highly suggestive of Raine syndrome. A homozygous, novel missense variant in exon 5 of FAM20C (c.1007T>G; p.Met336Arg) was identified by targeted Sanger sequencing. Following in silico analysis and mapping of the variant on a three-dimensional (3D) model of FAM20C it is predicted to be deleterious and to affect N-glycosylation, protein folding, and subsequent secretion of FAM20C. In addition, we reviewed all published FAM20C mutations and observed that most pathogenic variants affect functional regions within the protein establishing evidence for an emerging genotype-phenotype correlation.

Utility of rapid whole-exome sequencing in the diagnosis of Niemann-Pick disease type C presenting with fetal hydrops and acute liver failure.

Rapid whole-exome sequencing (rWES) is used in critically ill newborn infants to inform about diagnosis, clinical management, and prognosis. Here we report a male newborn infant with hydrops, pancytopenia, and acute liver failure who was listed for liver transplantation. Given the acuity of the presentation, the procedure-related morbidity and mortality, and lack of diagnosis, we used rWES in the proband and both parents with a turnaround time of 10 business days. rWES returned one maternally inherited, likely pathogenic and one paternally inherited, likely pathogenic variant in NPC1, suggestive of a diagnosis of Niemann-Pick disease type C (NPC). Interestingly, a diagnosis of NPC was entertained prior to rWES, but deemed unlikely in light of absent cholesterol storage on liver biopsy and near-normal oxysterol levels in dried blood. The diagnosis of NPC was confirmed on filipin stain in fibroblasts demonstrating defective cholesterol trafficking. NPC is a slowly progressive neurodegenerative disorder that may also affect the liver with overall poor prognosis. It was decided to take the infant off the transplant list and transfer to palliative care, where he died after 4 wk. This case highlights the utility of rWES in an acute clinical setting for several domains of precision medicine including (1) diagnosis, (2) prognosis and outcome, (3) management and therapy, and (4) utilization of resources.

Active site coupling in PDE:PKA complexes promotes resetting of mammalian cAMP signaling.

Cyclic 3'5' adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.

WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification.

Wnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/ß-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding ß-barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.

Manifestation of hawkinsinuria in a patient compound heterozygous for hawkinsinuria and tyrosinemia III.

Mutations in the gene for 4-hydroxyphenylpyruvic acid dioxygenase (HPD) cause either autosomal recessive tyrosinemia type III or autosomal dominant hawkinsinuria. We report a 6-month-old Indian infant who is compound heterozygous for both alleles and who has hawkinsinuria but not tyrosinemia type III based on biochemical investigations. The HPD gene was directly sequenced in the proband and both parents. The mechanistic model of the enzymatic function was built using the known structure of rat HPD. We identified a novel hawkinsinuria mutation, Asn241Ser, and a known tyrosinemia type III mutation, Ile335Met, in trans configuration. The structural analysis of the active site revealed that the IIe335Met mutation is situated in the close vicinity of one of the two highly conserved Phe rings which stack with the phenol ring of the substrate. The Asn241Ser mutation is situated further away from the 4-hydroxyphenylpyruvate binding pocket. Assuming that Asn241Ser causes hawkinsinuria, we propose positioning the dioxygen molecule in the HPD-catalyzed reaction as a novel role for the Asn residue. The IIe335Met allele is equivalent to a null mutation while the Asn241Ser allele results in a partially active enzyme with an uncoupled turnover causing hawkinsinuria.

Role of transmembrane domain/transmembrane domain interfaces of P-glycoprotein (ABCB1) in solute transport. Convergent information from photoaffinity labeling, site directed mutagenesis and in silico importance prediction.

Human P-glycoprotein (P-gp, ABCB1) plays an important role in the development of resistance to anticancer therapy. This ABC-transporter (ATP-binding cassette transporter) intercepts drugs at the level of the plasma membrane and effluxes them before they are able to reach their intracellular target structures. Inhibition of P-gp by low molecular weight compounds has been advocated as a concept for resensitization of cells to anticancer agents and several clinical studies in oncological patients have advanced to phase III. Even more importantly, P-glycoprotein also represents an antitarget. Its expression in cells lining the intestinal tract, the canalicular side of hepatocytes, renal tubuli and the blood brain barrier lead to interference with pharmacokinetics of compounds that are recognized as pump substrates. An early prediction of ADMET (Absorption-Distribution-Metabolism-Excretion-Toxicity) properties is important during drug development, since interference of a compound with P-gp might compromise its future development into a drug. Despite considerable efforts, the mechanism by which P-gp binds and transports its solutes remains unclear. Generation of homology models of the protein allowed integration of data obtained by photoaffinity labeling, in silico prediction of functional importance by evolutionary tracing and site directed mutagenesis. An integral view of data indicates that these three lines of evidence converge to indicate two pseudosymmetric P-gp drug binding pockets located at the two transmembrane domain interfaces.