Transthyretin amyloid polyneuropathy in France: A cross-sectional study with 413 patients and real-world tafamidis meglumine use (2009-2019).

OBJECTIVE: We aimed to describe characteristics of patients with ATTR variant polyneuropathy (ATTRv-PN) and ATTRv-mixed and assess the real-world use and safety profile of tafamidis meglumine 20mg. METHODS: Thirty-eight French hospitals were invited. Patient files were reviewed to identify clinical manifestations, diagnostic methods, and treatment compliance. RESULTS: Four hundred and thirteen patients (296 ATTRv-PN, 117 ATTRv-mixed) were analyzed. Patients were predominantly male (68.0%) with a mean age of 57.2±17.2 years. Interval between first symptom(s) and diagnosis was 3.4±4.3 years. First symptoms included sensory complaints (85.9%), dysautonomia (38.5%), motor deficits (26.4%), carpal tunnel syndrome (31.5%), shortness of breath (13.3%), and unexplained weight loss (16.0%). Mini-invasive accessory salivary gland or punch skin and nerve biopsies were most common, with a performance of 78.8-100%. TTR genetic sequencing, performed in all patients, revealed 31 TTR variants. Tafamidis meglumine was initiated in 156/214 (72.9%) ATTRv-PN patients at an early disease stage. Median treatment duration was 6.00 years in ATTRv-PN and 3.42 years in ATTRv-mixed patients. Tafamidis was well tolerated, with 20 adverse events likely related to study drug among the 336 patients. CONCLUSION: In France, ATTRv patients are usually identified early thanks to the national network and the help of diagnosis combining genetic testing and mini-invasive biopsies.

Upper limb onset of hereditary transthyretin amyloidosis is common in non-endemic areas.

BACKGROUND AND PURPOSE: The aim is to describe an uncommon phenotype of hereditary ATTR neuropathy with upper limb onset. METHODS: The French TTR Familial Amyloid Polyneuropathy database was used for a retrospective evaluation of 32 consecutive patients with upper limb onset of the neuropathy (study group) and they were compared to 31 Portuguese early-onset patients and 99 late-onset patients without upper limb onset. RESULTS: Initial upper limb symptoms were mostly sensory. Lower limb symptoms began 2.3 ± 3 years after upper limb symptoms. Twenty-four (75%) patients were initially misdiagnosed, with 15 different diagnoses. More patients in the study group had a Neuropathy Impairment Score upper limb/lower limb ratio > 1 compared to the late-onset patient group. The study group had significantly more pronounced axonal loss in the median and ulnar motor nerves and the ulnar sensory and sural nerves. On radial nerve biopsies (n = 11), epineurial vessels were abnormal in six cases, including amyloid deposits in vessel walls (3/11), with vessel occlusion in two cases. CONCLUSION: Upper limb onset of hereditary ATTR neuropathy is not rare in non-endemic areas. It is important to propose early TTR sequencing of patients with idiopathic upper limb neuropathies, as specific management and treatment are required.

NOBOX is a strong autosomal candidate gene in Tunisian patients with primary ovarian insufficiency.

Premature ovarian insufficiency (POI) affects approximately 1% of women before the age of 40. Genetic contribution is a significant component of POI. In this context, heterozygous mutations in NOBOX, BMP15 and GDF9 have been reported. The objective of our study was to evaluate the prevalence of these genes mutations in 125 unrelated Tunisian patients diagnosed with POI. The screening of NOBOX gene revealed three missense mutations (p.Arg117Trp; p.Gly91Trp and p.Pro619Leu) in eight patients. These mutations were not found in a 200 ethnically matched women without fertility problem. The sequencing of BMP15 and GDF9 gene revealed only previously reported variants. In contrast to previous studies, the prevalence of BMP15 variations is not higher than in the control population. Conversely, 6.4% of the cases present a NOBOX mutations; this high prevalence strengthens the consideration of NOBOX gene as strong autosomal candidate for POI.

Array comparative genomic hybridization analysis of small supernumerary marker chromosomes in human infertility.

Small supernumerary marker chromosomes (sSMC) are structurally abnormal chromosomes that cannot be unambiguously identified by conventional banding cytogenetics. This study describes four patients with sSMC in relation with infertility. Patient 1 had primary infertility. His brother, fertile, carried the same sSMC (patient 2). Patient 3 presented polycystic ovary syndrome and patient 4 primary ovarian insufficiency. Cytogenetic studies, array comparative genomic hybridization (CGH) and sperm analyses were compared with cases previously reported. sSMC corresponded to the 15q11.2 region (patients 1 and 2), the centromeric chromosome 15 region (patient 3) and the 21p11.2 region (patient 4). Array CGH showed 3.6-Mb gain for patients 1 and 2 and 0.266-Mb gain for patient 4. Sperm fluorescent in-situ hybridization analyses found ratios of 0.37 and 0.30 of sperm nuclei with sSMC(15) for patients 1 and 2, respectively (P < 0.001). An increase of sperm nuclei with disomy X, Y and 18 was noted for patient 1 compared with control and patient 2 (P < 0.001). Among the genes mapped in the unbalanced chromosomal regions, POTE B and BAGE are related to the testis and ovary, respectively. The implication of sSMC in infertility could be due to duplication, but also to mechanical effects perturbing meiosis.

Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain.

Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a lambda gt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.

Cytoplasmic-nuclear trafficking of steroid hormone receptors.

The nuclear localization of most steroid hormone receptors reflects a dynamic process: the receptor constantly diffuses out of the nucleus and is reimported by an active mechanism. The outward movement from the nucleus of the receptors and of other nuclear proteins is also mediated by the nuclear localization signals.

Effect of PML and PML-RAR on the transactivation properties and subcellular distribution of steroid hormone receptors.

PML (promyelocytic leukemia) is a protein involved in the t (15;17) translocation of promyelocytic leukemia and is mainly localized in nuclear bodies. Here we show that PML exerts a very powerful enhancing activity (up to 20-fold) on the transactivating properties of the progesterone receptor (PR) and has a similar effect on several other steroid hormone receptors. There is probably a direct or indirect interaction between PR and PML, because when the latter was expressed at high concentrations it shifted PR into the nuclear bodies. The use of deletion mutants showed that both activation functions (AF1 and AF2) of PR as well as the coiled coil and His-Cys-rich domains of PML were required for transcriptional enhancement. The fusion protein PML-RAR which is not localized in nuclear bodies, also enhanced the transactivating activity of PR, but this effect was totally suppressed by the administration of retinoic acid. PML, which is ubiquitously expressed, may thus be involved in the transactivation properties of steroid hormone receptors. This mechanism may also play a role in the oncogenic properties of PML-RAR and in their suppression by retinoic acid.

Mechanisms of progesterone receptor export from nuclei: role of nuclear localization signal, nuclear export signal, and ran guanosine triphosphate.

Steroid hormone receptors are, in most cases, mainly nuclear proteins that undergo a continuous nucleocytoplasmic shuttling. The mechanism of the nuclear export of these proteins remains largely unknown. To approach this problem experimentally in vivo, we have prepared cell lines permanently coexpressing the wild-type nuclear progesterone receptor (PR) and a cytoplasmic receptor mutant deleted of its nuclear localization signal (NLS) [(deltaNLS)PR]. Each receptor species was deleted from the epitope recognized by a specific monoclonal antibody, thus allowing separated observation of the two receptor forms in the same cells. Administration of hormone provoked formation of heterodimers during nucleocytoplasmic shuttling and import of (deltaNLS)PR into the nucleus. Washing out of the hormone allowed us to follow the export of (deltaNLS)PR into the cytoplasm. Microinjection of BSA coupled to a NLS inhibited the export of (deltaNLS)PR. On the contrary, microinjection of BSA coupled to a nuclear export signal (NES) was without effect. Moreover, leptomycin B, which inhibits NES-mediated export, was also without effect. tsBN2 cells contain a thermosensitive RCC1 protein (Ran GTP exchange protein). At the nonpermissive temperature, the nuclear export of (deltaNLS)PR could be observed, whereas the export of NES-BSA was suppressed. Microinjection of GTPgammaS confirmed that the export of (deltaNLS)PR was not dependent on GTP hydrolysis. These experiments show that the nuclear export of PR is not NES mediated but probably involves the NLS. It does not involve Ran GTP, and it is not dependent on the hydrolysis of GTP. The nucleocytoplasmic shuttling of steroid hormone receptors thus appears to utilize mechanisms different from those previously described for some viral, regulatory, and heterogeneous ribonuclear proteins.

Monoclonal antibodies against luteinizing hormone receptor. Immunochemical characterization of the receptor.

Human CG (hCG)-receptor complexes were solubilized from porcine testicular membranes. They were chromatographed on an immunomatrix of Affi-Gel 10-D1E8 anti beta-hCG monoclonal antibody (this antibody has been shown not to interfere with hCG binding to receptor). Elution was performed at alkaline pH, a condition in which hCG-receptor complexes are relatively stable. Immunization of a mouse with these partially (approximately 15%) purified hormone-receptor complexes allowed the preparation of 20 different hybridomas, each secreting antireceptor antibodies. The latter were used for receptor characterization. Immunoblot of testicular membrane extracts or of purified receptor preparations showed the presence of a major band at approximately 85,000 mol wt and minor bands at approximately 68,000 mol wt and approximately 48-45,000 mol wt. The width of all these bands suggested some microheterogeneity possibly due to glycosylation. The same approximately 85,000 mol wt receptor was seen in ovarian membranes, but no detectable antigen was observed in liver, muscle, and kidney membranes. An immunoaffinity method (using antibody LHR 38) was devised to purify the receptor in a single step. This demonstrated that the purified receptor preparation did not contain any protein component other than those detected by immunoblot. Comparison of receptors purified by immunoaffinity chromatography using either antireceptor or antihormone monoclonal antibodies showed in both cases the presence of the 85,000 mol wt and 48-45,000 mol wt species, but the absence, in the latter case, of the 68,000 mol wt species. This suggests that the 68,000 mol wt receptor cannot bind hormone and does not form oligomers with other receptor species.

Cytoplasmic-nuclear trafficking of progesterone receptor. In vivo study of the mechanism of action of antiprogestins.

The signal responsible for the nuclear localization of the progesterone receptor has been characterized. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between the nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. Preliminary evidence suggests that the same mechanism exists for estradiol and glucocorticoid receptors. Experiments designed to study the traffic of steroid hormone receptors have been applied to the determination of the molecular mechanism of action of antisteroids. Using these techniques, we have shown that two major antiprogestins, RU486 and ZK98299, act at the same point in the cell as the hormone.

The Ernst Schering Poster Award. Intracellular traffic of steroid hormone receptors.

The signal responsible for the nuclear localization of the progesterone receptor has been characterized. It is a complex signal. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. The same phenomenon exists for estradiol and glucocorticoid receptors. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement to the cytoplasm is not understood. We have grafted different nuclear localization signals (NLSs) onto beta-galactosidase and have studied the traffic of this protein using heterokaryons and microinjection experiments. We have demonstrated that the same NLSs are involved in both the inward and the outward movement of proteins through the nuclear membrane. These results suggest that the nucleocytoplasmic shuttling may be a general phenomenon for nuclear proteins that could possibly undergo modifications in the cytoplasm and exert some biological activities there. These conclusions also imply that at least part of the cellular machinery involved in the nuclear import of proteins may function bidirectionally. Using these techniques, we have shown that the two major antiprogestins, RU486 and ZK98299, act at the same distal level of hormone action.

Mechanisms of nuclear localization of the progesterone receptor.

Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638-642 and was shown to be constitutively active. When amino acids 638-642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or anti-hormone), it was almost fully active. A second putative nuclear localization signal is located in the DNA binding domain activated either through ligand binding or through production of constitutive receptor. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand the receptor was transferred into the nucleus as a monomer. After administration of hormone (or anti-hormone) a "cytoplasmic" monomer was transferred into the nucleus through interaction with a "nuclear" monomer. These interactions occurred through the steroid binding domains of both monomers.

Phe 84 deletion of the PMP22 gene associated with hereditary motor and sensory neuropathy HMSN III with multiple cranial neuropathy: clinical, neurophysiological and magnetic resonance imaging findings.

Hereditary motor and sensory neuropathy (HMSN) is a heterogeneous group of peripheral neuropathies which are diagnosed on the basis of clinical, electrophysiological and neuropathological findings. Among the hypertrophic demyelinating neuropathies, HMSN III is the most severe. It is often associated with de novo mutations in the genes encoding for peripheral myelin proteins. While peripheral nerve hypertrophy is an expected finding in HMSN III, cranial nerve hypertrophy is exceptional. Here we describe a mutation in the PMP22 gene in a 19-year-old man with infantile onset of sensory motor polyneuropathy without family history and multiple cranial nerve hypertrophy shown by cranial magnetic resonance imaging.

The range of chronic demyelinating neuropathy of infancy: a clinico-pathological and genetic study of 15 unrelated cases.

The concept of Dejerine-Sottas disease, which corresponds to presumed recessive demyelinating neuropathies with onset in infancy, remains controversial. To learn more on the subject, we performed a clinico-pathological and molecular genetic study in 15 unrelated patients with the Dejerine-Sottas phenotype seen over a 16 year period. There were 12 females and 3 males, born to asymptomatic parents. Study of the PMP22, P0 and Egr2 genes was performed in all cases and 14 underwent a nerve biopsy. First manifestations of neuropathy occurred before 3 years of age in all patients. An inherited disorder was suspected in 10 patients, because of their family history and/or disclosure of a molecular genetic defect in 4 of them. One patient had a recessively transmitted homozygous point mutation (Arg157Trp) of the PMP22 gene. A heterozygous duplication of the 17p11.2-12 segment was detected in one offspring of a consanguineous marriage. One patient carried a "de novo" heterozygous Ser72Leu substitution in the PMP22. A heterozygous double mutation of the P0 gene including a "de novo" Val42 deletion and an Ala221Thr substitution, maternally inherited, were found in an apparently sporadic case. No mutation of the Egr2 gene was identified. A neuropathy with focally folded myelin sheaths (CMT4B) was diagnosed in the nerve biopsy specimens of two patients. In five patients, the clinico-pathological findings along with the absence of an identified mutation suggested the diagnosis of chronic inflammatory demyelinating polyneuropathy of infantile onset. Our findings illustrate the genetic heterogeneity of cases with identified mutations, the scarcity of cases with "demonstrated" recessive transmission and the likelihood of early acquired chronic inflammatory demyelinating polyneuropathy in several patients.

[Intracellular traffic of the progesterone receptor]. / Trafic intracellulaire du récepteur de la progestérone.

The nuclear localization of the progesterone receptor is mediated by two signal sequences: one is constitutive and lies in the hinge region (between the DNA and steroid binding domains), the other is hormone-dependent and is localized in the second zinc finger of the DNA binding domain. The use of various inhibitors of energy synthesis in cells expressing permanently or transiently the wild-type receptor or a receptor mutated within the nuclear localization signals, demonstrated that the nuclear residency of the receptor reflects a dynamic situation: the receptor diffusing into the cytoplasm and being constantly and actively transported back into the nucleus. The existence of this nucleo-cytoplasmic shuttle mechanism was confirmed by receptor transfer from one nucleus to the other in heterokaryons. Preliminary evidence was obtained, using oestrogen receptor, that this phenomenon may be of general significance for steroid receptors.

[Specific modulators of estrogen action]. / Les modulateurs spécifiques de l'action des oestrogènes.

Selective estradiol receptor modulators (SERMs) are specific modulators of estradiol action. They are used in therapeutics to obtain an estrogenic effect on certain cells and an antiestrogenic effect on other cells. Recent progress in the knowledge of the mechanism of action of estradiols implies that new molecules could be designed. This progress involves the cloning of a new estradiol receptor, ER beta, the discovery of co-activators and the elucidation of their molecular mechanism of action, and the crystallization of the ligand binding domain in the presence of an agonist or an antagonist.

[Estrogen biosynthesis and receptors]. / Biosynthèse et récepteurs des estrogènes.

Most of the enzymes involved in steroidogenesis belong to the family of cytochrome P 450. Most of the corresponding genes have been cloned. The key enzyme for estradiol biosynthesis is P 450 arom. Several germline mutations have been described. These observations have lead to reconsider the role of estradiol. Estradiol plays a key role in bone growth and mineralisation and in gonadotrope regulation in male. Moreover, the recent discovery that an additional estrogen receptor (ER beta) is present in various tissues has advanced our understanding of the mechanisms underlying estrogen signalling. It suggests the existence of two previously unrecognized pathways of estrogen signalling: via the ER beta subtype in tissues exclusively expressing this subtype and via the formation of heterodimers in tissues expressing both ER subtypes. Various models have been suggested as explanations for the stricking cell and promoter-specific effects of estrogens and antiestrogens, all on the basis of the assumption that only a single ER exists. These models have to be reconsidered. Moreover, new antiestrogens with improved therapeutic profiles could be designed.

[Effect of PML and PML-RAR on the transactivation properties and subcellular localization of steroid hormone receptors]. / Effet de PML et PML-RAR sur les propriétés transactivatrices et la localisation sub-cellulaire des récepteurs stéroïdiens.

PML is a protein involved in the t (15, 17) translocation of promyelocytic leukemia and is mainly localized in nuclear bodies. Here we show that PML exerts a very powerful enhancing activity (up to 20-fold) on the transactivating properties of the progesterone receptor (PR) and has a similar effect on several other steroid hormone receptors. There is probably a direct or indirect interaction between PR and PML since when the latter was expressed at high concentrations it shifted PR into the nuclear bodies. Use of deletion mutants showed that both activation functions (AF1 and AF2) of PR as well as the coiled coil and His-Cys rich domains of PML were required for transcriptional enhancement. The fusion protein PML-RAR, which is not localized in nuclear bodies, also enhanced the transactivating activity of PR but this effect was totally suppressed by the administration of retinoic acid. PML, which is ubiquitously expressed, may thus be involved in the transactivation properties of steroid hormone receptors. This mechanism may also play a role in the oncogenic properties of PML-RAR and in their suppression by the retinoic acid.

[Regulation of the differentiation and proliferation of smooth muscle cells by the sex hormones]. / Régulation de la différenciation et de la prolifération des cellules musculaires lisses par les stéroïdes sexuels.

Steroids effects are mediated by their receptors. These proteins define the large family of steroid hormone receptors, characterized by the presence of 3 functional domains: a transactivation domain, a DNA-binding domain and a ligand-binding domain. Receptor activation induces the modulation of transcription of specific genes, and as a consequence, the modulation of production of specific proteins. Sex steroid receptors are located in the nucleus. This nuclear localization is in fact a dynamic situation, resulting from a continuous shuttling of the receptor between the cytoplasm and the nucleus. The recent discovery that an additional estrogen receptor is present in various tissues has advanced our understanding of the mechanism underlying estrogen signalling. Non genomic effects of steroids have also been described. Sex steroids inhibit proliferation of smooth muscle cells. On the contrary, they stimulate proliferation of tumoral muscle cells. The mechanisms of sex steroid effects on cellular proliferation are complex, and may involve transcriptional or non transcriptional phenomena.