The experience of diagnosis announcement in rare endocrine diseases: A survey of the French FIRENDO network.

CONTEXT: Diagnosis announcement of a chronic disease is a crucial moment for patients as well as for their families and an important step in the management of severe conditions such as rare endocrine diseases. Little is known of how diagnosis is communicated to patients and families. The FIRENDO network was created by the third French Plan for Rare Diseases, to promote autonomy, care and research on rare endocrine diseases. OBJECTIVES: The aim of this study was to characterize, for the first time, the experience and needs of patients and/or their parents around the announcement of diagnosis to ensure optimal quality of care. METHODS: A quantitative self-administered survey on diagnosis announcement procedures in rare endocrine diseases was launched in April 2017 by the ad hoc FIRENDO thematic working group in collaboration with its 11 partnering patient associations and support groups. The questionnaire was designed and revised by patient support group representatives, adult and pediatric endocrinologists, psychologists and biologists, all expert in rare endocrine diseases. It was made available on the FIRENDO network website and distributed mainly by email with electronic links on their respective websites to members of all affiliated patient support groups. RESULTS: Questionnaires were filled out by 391 patients and 223 parents (median age of patients: 39 years). The following conditions were associated with at least 30 answers: Addison's disease, classical forms of congenital adrenal hyperplasia (CAH), Russell-Silver syndrome, Cushing's syndrome, acromegaly and craniopharyngioma. Overall, some announcement modalities were judged favorably by patients: physician's empathy, availability and use of clear terms, and presence of family at the time of announcement. However, a lack of psychological care and information documents was reported, as well as some inadequate procedures such as postal mail announcements. CONCLUSION: This work suggests that better knowledge of the patient's experience is useful for improving the diagnosis announcement of rare endocrine disorders. The main recommendations derived from the survey were the need for several announcement visits, information on patient support groups and reference centers, imperatively avoiding impersonal announcement, and the usefulness of a written accompanying document.

Monogenic diabetes syndromes: Locus-specific databases for Alström, Wolfram, and Thiamine-responsive megaloblastic anemia.

We developed a variant database for diabetes syndrome genes, using the Leiden Open Variation Database platform, containing observed phenotypes matched to the genetic variations. We populated it with 628 published disease-associated variants (December 2016) for: WFS1 (n = 309), CISD2 (n = 3), ALMS1 (n = 268), and SLC19A2 (n = 48) for Wolfram type 1, Wolfram type 2, Alström, and Thiamine-responsive megaloblastic anemia syndromes, respectively; and included 23 previously unpublished novel germline variants in WFS1 and 17 variants in ALMS1. We then investigated genotype-phenotype relations for the WFS1 gene. The presence of biallelic loss-of-function variants predicted Wolfram syndrome defined by insulin-dependent diabetes and optic atrophy, with a sensitivity of 79% (95% CI 75%-83%) and specificity of 92% (83%-97%). The presence of minor loss-of-function variants in WFS1 predicted isolated diabetes, isolated deafness, or isolated congenital cataracts without development of the full syndrome (sensitivity 100% [93%-100%]; specificity 78% [73%-82%]). The ability to provide a prognostic prediction based on genotype will lead to improvements in patient care and counseling. The development of the database as a repository for monogenic diabetes gene variants will allow prognostic predictions for other diabetes syndromes as next-generation sequencing expands the repertoire of genotypes and phenotypes. The database is publicly available online at https://lovd.euro-wabb.org.

Structure-activity relationship of isoform selective inhibitors of Rac1/1b GTPase nucleotide binding.

The synthesis of a series of berberine, phenantridine and isoquinoline derivatives was realized to explore their Rho GTPase nucleotide inhibitory activity. The compounds were evaluated in a nucleotide binding competition assay against Rac1, Rac1b, Cdc42 and in a cellular Rac GTPase activation assay. The insertion of 19 AA in the splice variant Rac1b is shown to be sufficient to introduce a conformational difference that allows compounds 4, 21, 22, and 26 to exhibit selective inhibition of Rac 1b over Rac1.

Characterization of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases.

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that block Rho GTPase function indirectly, either by targeting enzymes involved in post-translational processing or downstream protein kinase effectors. We have reported the identification and characterization of the EHT 1864 small molecule as an inhibitor of Rac family small GTPases, placing Rac1 in an inert and inactive state and then impairing Rac1-mediated functions in vivo. Our work suggests that EHT 1864 selectively inhibits Rac1 downstream signaling and cellular transformation by a novel mechanism involving guanine nucleotide displacement. This chapter provides the details for some of the biochemical and biological methods used to characterize the mode of action of EHT 1864 on Rac1 and its impact on Rac1-dependent cellular functions.

Specificity and mechanism of action of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases.

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.

Identification of a tyrosine-based motif (YGSI) in the amino terminus of Nramp1 (Slc11a1) that is important for lysosomal targeting.

In macrophages, Nramp1 (Slc11a1) is expressed in lysosomes and restricts replication of intracellular pathogens by removing divalent metals (Mn2+ and Fe2+) from the phagolysosome. Nramp2 (DMT1, Slc11a2) is expressed both at the duodenal brush border where it mediates uptake of dietary iron and ubiquitously at the plasma membrane/recycling endosomes of many cell types where it transports transferrin-associated iron across the endosomal membrane. In Nramp2, a carboxyl-terminal cytoplasmic motif ((555)YLLNT(559)) is critical for internalization and recycling of the transporter from the plasma membrane. Here we studied the subcellular trafficking properties of Nramp1 and investigated the cis-acting sequences responsible for targeting to lysosomes. For this, we constructed and studied Nramp1/Nramp2 chimeric proteins where homologous domains of each protein were exchanged. Chimeras exchanging the amino-(upstream TM1) and carboxyl-terminal (downstream TM12) cytoplasmic segments of both transporters were stably expressed in porcine LLC-PK1 kidney cells and were studied with respect to expression, maturation, stability, cell surface targeting, transport activity, and subcellular localization. An Nramp2 isoform II chimera bearing the amino terminus of Nramp1 was not expressed at the cell surface but was targeted to lysosomes. This lysosomal targeting was abolished by single alanine substitutions at Tyr15 and Ile18 of a (15)YGSI(18) motif present in the amino terminus of Nramp1. These results identify YGSI as a tyrosine-based sorting signal responsible for lysosomal targeting of Nramp1.

RAC1 inhibition targets amyloid precursor protein processing by gamma-secretase and decreases Abeta production in vitro and in vivo.

beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.