Antony Cougnoux

High incidence of unrecognized visceral/neurological late-onset Niemann-Pick disease, type C1, predicted by analysis of massively parallel sequencing data sets

Purpose:Niemann-Pick disease type C (NPC) is a recessive, neurodegenerative, lysosomal storage disease caused by mutations in either NPC1 or NPC2. The diagnosis is difficult and frequently delayed. Ascertainment is likely incomplete because of both these factors and because the full phenotypic spectrum may not have been fully delineated. Given the recent development of a blood-based diagnostic test and the development of potential therapies, understanding the incidence of NPC and defining at-risk patient populations are important.Method:We evaluated data from four large, massively parallel exome sequencing data sets. Variant sequences were identified and classified as pathogenic or nonpathogenic based on a combination of literature review and bioinformatic analysis. This methodology provided an unbiased approach to determining the allele frequency.Results:Our data suggest an incidence rate for NPC1 and NPC2 of 1/92,104 and 1/2,858,998, respectively. Evaluation of common NPC1 variants, however, suggests that there may be a late-onset NPC1 phenotype with a markedly higher incidence, on the order of 1/19,000-1/36,000.Conclusion:We determined a combined incidence of classical NPC of 1/89,229, or 1.12 affected patients per 100,000 conceptions, but predict incomplete ascertainment of a late-onset phenotype of NPC1. This finding strongly supports the need for increased screening of potential patients.Genet Med 18 1, 41–48.

Pituitary Adenoma With Paraganglioma/Pheochromocytoma (3PAs) and Succinate Dehydrogenase Defects in Humans and Mice

CONTEXT Germline mutations in genes coding succinate dehydrogenase (SDH) subunits A, B, C, and D have been identified in familial paragangliomas (PGLs)/pheochromocytomas (PHEOs) and other tumors. We described a GH-secreting pituitary adenoma (PA) caused by SDHD mutation in a patient with familial PGLs. Additional patients with PAs and SDHx defects have since been reported. DESIGN We studied 168 patients with unselected sporadic PA and with the association of PAs, PGLs, and/or pheochromocytomas, a condition we named the 3P association (3PAs) for SDHx germline mutations. We also studied the pituitary gland and hormonal profile of Sdhb(+/-) mice and their wild-type littermates at different ages. RESULTS No SDHx mutations were detected among sporadic PA, whereas three of four familial cases were positive for a mutation (75%). Most of the SDHx-deficient PAs were either prolactinomas or somatotropinomas. Pituitaries of Sdhb(+/-) mice older than 12 months had an increased number mainly of prolactin-secreting cells and several ultrastructural abnormalities such as intranuclear inclusions, altered chromatin nuclear pattern, and abnormal mitochondria. Igf-1 levels of mutant mice tended to be higher across age groups, whereas Prl and Gh levels varied according to age and sex. CONCLUSION The present study confirms the existence of a new association that we termed 3PAs. It is due mostly to germline SDHx defects, although sporadic cases of 3PAs without SDHx defects also exist. Using Sdhb(+/-) mice, we provide evidence that pituitary hyperplasia in SDHx-deficient cells may be the initial abnormality in the cascade of events leading to PA formation.

ClbP Is a Prototype of a Peptidase Subgroup Involved in Biosynthesis of Nonribosomal Peptides

The pks genomic island of Escherichia coli encodes polyketide (PK) and nonribosomal peptide (NRP) synthases that allow assembly of a putative hybrid PK-NRP compound named colibactin that induces DNA double-strand breaks in eukaryotic cells. The pks-encoded machinery harbors an atypical essential protein, ClbP. ClbP crystal structure and mutagenesis experiments revealed a serine-active site and original structural features compatible with peptidase activity, which was detected by biochemical assays. Ten ClbP homologs were identified in silico in NRP genomic islands of closely and distantly related bacterial species. All tested ClbP homologs were able to complement a clbP-deficient E. coli mutant. ClbP is therefore a prototype of a new subfamily of extracytoplasmic peptidases probably involved in the maturation of NRP compounds. Such peptidases will be powerful tools for the manipulation of NRP biosynthetic pathways.

FTY720/fingolimod increases NPC1 and NPC2 expression and reduces cholesterol and sphingolipid accumulation in Niemann-Pick type C mutant fibroblasts.

Niemann‐Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 or NPC2 with decreased functions leading to lysosomal accumulation of cholesterol and sphingolipids. FTY720/ fingolimod, used for treatment of multiple sclerosis, is phosphorylated by nuclear sphingosine kinase 2, and its active phosphorylated form (FTY720‐P) is an inhibitor of class I histone deacetylases. In this study, administration of clinically relevant doses of FTY720 to mice increased expression of NPC1 and −2 in brain and liver and decreased cholesterol in an SphK2‐dependent manner. FTY720 greatly increased expression of NPC1 and −2 in human NPC1 mutant fibroblasts that correlated with formation of FTY720‐P and significantly reduced the accumulation of cholesterol and glycosphingolipids. In agreement with this finding, FTY720 pretreatment of human NPC1 mutant fibroblasts restored transport of the cholera toxin B subunit, which binds ganglioside GM1, to the Golgi apparatus. Together, these findings suggest that FTY720 administration can ameliorate cholesterol and sphingolipid storage and trafficking defects in NPC1 mutant fibroblasts. Because neurodegeneration is the main clinical feature of NPC disease, and FTY720 accumulates in the CNS and has several advantages over available histone deacetylase inhibitors now in clinical trials, our work provides a potential opportunity for treatment of this incurable disease. —Newton, J., Hait, N. C., Maceyka, M., Colaco, A., Maczis, M., Wassif, C. A., Cougnoux, A., Porter, F. D., Milstien, S., Platt, N., Platt, F. M., Spiegel, S. FTY720/fingolimod increases NPC1 and NPC2 expression and reduces cholesterol and sphingolipid accumulation in Niemann‐Pick type C mutant fibroblasts. FASEB J. 31, 1719–1730 (2017) www.fasebj.org

Bortezomib Alone and in Combination With Salinosporamid A Induces Apoptosis and Promotes Pheochromocytoma Cell Death In Vitro and in Female Nude Mice

&NA; Proteasome inhibitors have been frequently used in treating hematologic and solid tumors. They are administered individually or in combination with other regimens, to prevent severe side effects and resistance development. Because they have been shown to be efficient and are pharmaceutically available, we tested the first Food and Drug Administration‐approved proteasome inhibitor bortezomib alone and in combination with another proteasome inhibitor, salinosporamid A, in pheochromocytoma cells. Pheochromocytomas/Paragangliomas (PHEOs/PGLs) are neuroendocrine tumors for which no definite cure is yet available. Therefore, drugs with a wide spectrum of mechanisms of action are being tested to identify suitable candidates for PHEO/PGL treatment. In the current study, we show that bortezomib induces PHEO cell death via the apoptotic pathway in vitro and in vivo. The combination of bortezomib with salinosporamid A exhibits additive effect on these cells and inhibits proliferation, cell migration and invasion, and angiogenesis more potently than bortezomib alone. Altogether, we suggest these proteasome inhibitors, especially bortezomib, could be potentially tested in PHEO/PGL patients who might benefit from treatment with either the inhibitors alone or in combination with other treatment options.

Noncovalent complexes of an inactive mutant of CTX-M-9 with the substrate piperacillin and the corresponding product.

ABSTRACT We determined the crystal structure of an inactive Ser70Gly mutant of CTX-M-9 in complex with the bulky penicillin piperacillin at precovalent and posthydrolytic stages in the catalytic process. The structures obtained at high resolution were compared with the corresponding structures for the small penicillin benzylpenicillin and the bulky cephalosporin cefotaxime. The findings highlight the key role of the configuration of the carbon adjacent to the acylamino group of the side chain of β-lactams in the precovalent recognition of substrates.

Characterization of the Inhibitor-Resistant SHV β-Lactamase SHV-107 in a Clinical Klebsiella pneumoniae Strain Coproducing GES-7 Enzyme

ABSTRACT The clinical Klebsiella pneumoniae INSRA6884 strain exhibited nonsusceptibility to all penicillins tested (MICs of 64 to >2,048 μg/ml). The MICs of penicillins were weakly reduced by clavulanate (from 2,048 to 512 μg/ml), and tazobactam restored piperacillin susceptibility. Molecular characterization identified the genes blaGES-7 and a new β-lactamase gene, blaSHV-107, which encoded an enzyme that differed from SHV-1 by the amino acid substitutions Leu35Gln and Thr235Ala. The SHV-107-producing Escherichia coli strain exhibited only a β-lactam resistance phenotype with respect to amoxicillin, ticarcillin, and amoxicillin-clavulanate combination. The kinetic parameters of the purified SHV-107 enzyme revealed a high affinity for penicillins. However, catalytic efficiency for these antibiotics was lower for SHV-107 than for SHV-1. No hydrolysis was detected against oxyimino-β-lactams. The 50% inhibitory concentration (IC50) for clavulanic acid was 9-fold higher for SHV-107 than for SHV-1, but the inhibitory effects of tazobactam were unchanged. Molecular dynamics simulation suggested that the Thr235Ala substitution affects the accommodation of clavulanate in the binding site and therefore its inhibitory activity.

The Role of Autophagy in the Degradation of Misfolded HLA-B27 Heavy Chains

To determine whether autophagy is involved in the degradation of misfolded HLA–B27 in experimental spondyloarthritis.

Association of NPC1 variant p.P237S with a pathogenic splice variant in two Niemann-Pick disease type C1 patients

Conflict of interest: The authors report no conflicts of interest. Grant sponsor: Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Correspondence to: Forbes D. Porter, M.D., Ph.D, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS, 10-CRC, Room 5-2571, 10 Center Drive, Bethesda, MD 20892. E-mail: fdporter@mail.nih.gov Article first published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/ajmg.a.38104 How to Cite this Article: Salman A, Cougnoux A, Farhat N, Wassif CA, Porter FD. 2017. Association of NPC1 variant p.P237S with a pathogenic splice variant in two Niemann–Pick disease type C1 patients.

Microglia activation in Niemann–Pick disease, type C1 is amendable to therapeutic intervention

Niemann-Pick disease, type C1 (NPC1) is a neurodegenerative disorder with limited treatment options. NPC1 is associated with neuroinflammation; however, attempts to therapeutically target neuroinflammation in NPC1 have had mixed success. We show here that NPC1 neuroinflammation is characterized by an atypical microglia activation phenotype. Specifically, Npc1-/- microglia demonstrated altered morphology, reduced levels of lineage markers and a shift toward glycolytic metabolism. Treatment with 2-hydroxypropyl-β-cyclodextrin (HPβCD), a drug currently being studied in a phase 2b/3 clinical trial, reversed all microglia-associated defects in Npc1-/- animals. In addition, impairing microglia mediated neuroinflammation by genetic deletion of IRF8 led to decreased symptoms and increased lifespan. We identified CD22 as a marker of dysregulated microglia in Npc1 mutant mice and subsequently demonstrated that elevated cerebrospinal fluid levels of CD22 in NPC1 patients responds to HPβCD administration. Collectively, these data provide the first in-depth analysis of microglia function in NPC1 and suggest possible new therapeutic approaches.