Pathological spectrum of hereditary transthyretin renal amyloidosis and clinicopathologic correlation: a French observational study.

BACKGROUND: Cardiac and neurological involvements are the main clinical features of hereditary transthyretin (ATTRv) amyloidosis. Few data are available about ATTRv amyloid nephropathy (ATTRvN). METHODS: We retrospectively included 30 patients with biopsy-proven ATTRvN [V30M (26/30) including two domino liver recipients, S77Y (2/30), V122I (1/30) and S50R (1/30) variants] from two French reference centers. We described the pathological features by comparing amyloid deposits distribution to patients with AL or AA amyloidosis, and sought to determine clinicopathological correlation with known disease-modifying factors such as TTR variant, gender and age at diagnosis. RESULTS: In comparison with AL and AA amyloidosis, ATTRv patients had similar glomerular, arteriolar and arterial amyloid deposits, but more cortical and medullary tubulointerstitial (33%, 44%, 77%, P = .03) involvement. While the presence of glomerular deposits is associated with the range of proteinuria, some patients with abundant glomerular ATTRv amyloidosis had no significant proteinuria. V30M patients had more glomerular (100% and 25%, odds ratio = 114, 95% confidence interval 3.85-3395.00, P = .001) deposits, and higher estimated glomerular filtration rate [50 (interquartile range 44-82) and 27 (interquartile range 6-31) mL/min/1.73 m², P = .004] than non-V30M patients. We did not find difference in amyloid deposition according to gender or age at diagnosis. CONCLUSION: ATTRvN affects all kidney compartments, but compared with AL/AA amyloidosis, ATTRvN seems to involve more frequently tubulointerstitial areas. V30M patients represents the dominant face of the disease with a higher risk of glomerular/arteriolar involvement. ATTRvN should thus be considered in patients, and potential relatives, with ATTRv amyloidosis and kidney dysfunction, regardless of proteinuria level.

A SPIDR homozygous nonsense pathogenic variant in isolated primary ovarian insufficiency with chromosomal instability.

Primary ovarian insufficiency (POI), affecting 1% of women under 40 years is a public health problem. Genes involved in meiosis/DNA repair were recently shown to be the leading family of associated causal genes, some of them also cause tumors/cancers. Here, using targeted next-generation sequencing in an Indian POI patient with primary amenorrhea and streak ovaries, we identified a novel homozygous nonsense variant in exon 7 of SPIDR (KIAA0146) c.814C > T, R272*, predicted to lead a nonsense-mediated mRNA decay. SPIDR was recently identified by in vitro assays as an auxiliary protein interacting with RAD51 and BLM, two major proteins involved in genome stability. Consistent with alteration of the RAD51 pathway, we observed a strong increase in mitomycin C-induced DNA breaks and aberrant metaphases in the patient's cells compared to a control. However, sister chromatid exchanges were normal in contrast to the sharp increase characteristic of the BLM pathway. This is the first evidence of chromosomal instability associated with a SPIDR molecular defect, which supports the role of SPIDR in double-stranded DNA damage repair in vivo in humans and its causal role in POI. Our study increases knowledge on the SPIDR function and has broad implications in the management of such patients.

The Roles of Luteinizing Hormone, Follicle-Stimulating Hormone and Testosterone in Spermatogenesis and Folliculogenesis Revisited.

Spermatogenesis and folliculogenesis involve cell-cell interactions and gene expression orchestrated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). FSH regulates the proliferation and maturation of germ cells independently and in combination with LH. In humans, the requirement for high intratesticular testosterone (T) concentration in spermatogenesis remains both a dogma and an enigma, as it greatly exceeds the requirement for androgen receptor (AR) activation. Several data have challenged this dogma. Here we report our findings on a man with mutant LH beta subunit (LHß) that markedly reduced T production to 1-2% of normal., but despite this minimal LH stimulation, T production by scarce mature Leydig cells was sufficient to initiate and maintain complete spermatogenesis. Also, in the LH receptor (LHR) knockout (LuRKO) mice, low-dose T supplementation was able to maintain spermatogenesis. In addition, in antiandrogen-treated LuRKO mice, devoid of T action, the transgenic expression of a constitutively activating follicle stimulating hormone receptor (FSHR) mutant was able to rescue spermatogenesis and fertility. Based on rodent models, it is believed that gonadotropin-dependent follicular growth begins at the antral stage, but models of FSHR inactivation in women contradict this claim. The complete loss of FSHR function results in the complete early blockage of folliculogenesis at the primary stage, with a high density of follicles of the prepubertal type. These results should prompt the reassessment of the role of gonadotropins in spermatogenesis, folliculogenesis and therapeutic applications in human hypogonadism and infertility.

Position statement on the diagnosis and management of premature/primary ovarian insufficiency (except Turner Syndrome).

Premature ovarian insufficiency (POI) is a rare pathology affecting 1-2% of under-40 year-old women, 1 in 1000 under-30 year-olds and 1 in 10,000 under-20 year-olds. There are multiple etiologies, which can be classified as primary (chromosomal, genetic, auto-immune) and secondary or iatrogenic (surgical, or secondary to chemotherapy and/or radiotherapy). Despite important progress in genetics, more than 60% of cases of primary POI still have no identifiable etiology; these cases are known as idiopathic POI. POI is defined by the association of 1 clinical and 1 biological criterion: primary or secondary amenorrhea or spaniomenorrhea of>4 months with onset before 40 year of age, and elevated follicle-stimulating hormone (FSH)>25IU/L on 2 assays at>4 weeks' interval. Estradiol level is low, and anti-Müllerian hormone (AMH) levels have usually collapsed. Initial etiological work-up comprises auto-immune assessment, karyotype, FMR1 premutation screening and gene-panel study. If all of these are normal, the patient and parents may be offered genome-wide analysis under the "France Génomique" project. The term ovarian insufficiency suggests that the dysfunction is not necessarily definitive. In some cases, ovarian function may fluctuate, and spontaneous pregnancy is possible in around 6% of cases. In confirmed POI, hormone replacement therapy is to be recommended at least up to the physiological menopause age of 51 years. Management in a rare diseases center may be proposed.

Homozygous hypomorphic BRCA2 variant in primary ovarian insufficiency without cancer or Fanconi anaemia trait.

BACKGROUND: Primary ovarian insufficiency (POI) affects 1% of women under 40 years and is a public health problem. The genetic causes of POI are highly heterogeneous with isolated or syndromic forms. Recently, variants in genes involved in DNA repair have been shown to cause POI. Notably, syndromic POI with Fanconi anaemia (FA) traits related to biallelic BRCA2 truncated variants has been reported. Here, we report a novel phenotype of isolated POI with a BRCA2 variant in a consanguineous Turkish family. METHODS: Exome sequencing (ES) was performed in the patient. We also performed functional studies, including a homologous recombination (HR) test, cell proliferation, radiation-induced RAD51 foci formation assays and chromosome breakage studies in primary and lymphoblastoid immortalised cells. The expression of BRCA2 in human foetal ovaries was studied. RESULTS: ES identified a homozygous missense c.8524C>T/p.R2842C-BRCA2 variant. BRCA2 defects induce cancer predisposition and FA. Remarkably, neither the patient nor her family exhibited somatic pathologies. The patient's cells showed intermediate levels of chromosomal breaks, cell proliferation and radiation-induced RAD51 foci formation compared with controls and FA cells. R2842C-BRCA2 only partially complemented HR efficiency compared with wild type-BRCA2. BRCA2 is expressed in human foetal ovaries in pachytene stage oocytes, when meiotic HR occurs. CONCLUSION: We describe the functional assessment of a homozygous hypomorphic BRCA2 variant in a patient with POI without cancer or FA trait. Our findings extend the phenotype of BRCA2 biallelic alterations to fully isolated POI. This study has a major impact on the management and genetic counselling of patients with POI.

A Novel Phenotype Combining Primary Ovarian Insufficiency Growth Retardation and Pilomatricomas With MCM8 Mutation.

CONTEXT: Primary Ovarian insufficiency (POI) affects 1% of women aged <40 years and leads most often to definitive infertility with adverse health outcomes. Very recently, genes involved in deoxyribonucleic acid (DNA) repair have been shown to cause POI. OBJECTIVE: To identify the cause of a familial POI in a consanguineous Turkish family. DESIGN: Exome sequencing was performed in the proposita and her mother. Chromosomal breaks were studied in lymphoblastoid cell lines treated with mitomycin (MMC). SETTING AND PATIENTS: The proposita presented intrauterine and postnatal growth retardation, multiple pilomatricomas in childhood, and primary amenorrhea. She was treated with growth hormone (GH) from age 14 to 18 years. RESULTS: We identified a novel nonsense variant in exon 9 of the minichromosome maintenance complex component 8 gene (MCM8) NM_001281522.1: c0.925C > T/p.R309* yielding either a truncated protein or nonsense-mediated messenger ribonucleic acid decay.The variant was homozygous in the daughter and heterozygous in the mother. MMC induced DNA breaks and aberrant metaphases in the patient's lymphoblastoid cells. The mother's cells had intermediate but significantly higher chromosomal breaks compared with a control. CONCLUSION: We describe a novel phenotype of syndromic POI related to a novel truncating MCM8 variant. We show for the first time that spontaneous tumors (pilomatricomas) are associated with an MCM8 genetic defect, making the screening of this gene necessary before starting GH therapy in patients with POI with short stature, especially in a familial or consanguineous context. Appropriate familial monitoring in the long term is necessary, and fertility preservation should be considered in heterozygous siblings to avoid rapid follicular atresia.

Novel STAG3 mutations in a Caucasian family with primary ovarian insufficiency.

Primary ovarian insufficiency (POI) affects ~ 1-3, 7% of women under forty and is a public health problem. Most causes are unknown, but an increasing number of genetic causes have been identified recently. The identification of such causes is essential for genetic and therapeutic counseling in patients and their families. We performed whole exome sequencing in two Caucasian sisters displaying non syndromic POI and their unaffected mother. We identified two novel pathogenic variants in STAG3 encoding a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion: a c.3052delC truncating mutation in exon 28 yielding p.Arg1018Aspfs*14, and a c.659T > G substitution in exon seven yielding p.Leu220Arg. Leu220, highly conserved throughout species, belongs to the STAG domain conserved with other mitotic subunits of the cohesion complex STAG1 and 2. In silico analysis reveals that this substitution markedly impacts the structure of this domain. The truncation removes the last 206 C-terminal residues, not conserved in STAG1 and 2, supporting an important specific role in STAG3, especially meiosis. This is the first occurrence of STAG3 mutations in a Caucasian family. Very little is known about the function of STAG proteins domains. The "knock out-like" phenotype described here supports the crucial role of a single residue in the STAG domain and of the C-terminal region in STAG3 function. In conclusion, this observation shows the necessity to perform the genetic study of POI worldwide including STAG3. This could lead to appropriate genetic counseling and long term follow-up since these patients may develop ovarian tumors.

Advances in the Molecular Pathophysiology, Genetics, and Treatment of Primary Ovarian Insufficiency.

Primary ovarian insufficiency (POI) affects ∼1% of women before 40 years of age. The recent leap in genetic knowledge obtained by next generation sequencing (NGS) together with animal models has further elucidated its molecular pathogenesis, identifying novel genes/pathways. Mutations of >60 genes emphasize high genetic heterogeneity. Genome-wide association studies have revealed a shared genetic background between POI and reproductive aging. NGS will provide a genetic diagnosis leading to genetic/therapeutic counseling: first, defects in meiosis or DNA repair genes may predispose to tumors; and second, specific gene defects may predict the risk of rapid loss of a persistent ovarian reserve, an important determinant in fertility preservation. Indeed, a recent innovative treatment of POI by in vitro activation of dormant follicles proved to be successful.

A common African variant of human connexin 37 is associated with Caucasian primary ovarian insufficiency and has a deleterious effect in vitro.

Folliculogenesis requires communication between granulosa cells and oocytes, mediated by connexin-based gap junctions. Connexin 37 (Cx37)-deficient female mice are infertile. The present study assessed Cx37 deficiency in patients with primary ovarian insufficiency (POI). A candidate gene study was performed in patients and controls from the National Genotyping Center (Evry, France) including 58 Caucasian patients with idiopathic isolated POI and 142 Caucasian controls. Direct genomic sequencing of the coding regions of the GJA4 gene (encoding Cx37) was performed with the aim to identify a deleterious variant associated with POI and absent in ethnically matched controls. A single Cx37 variant absent in the control population was identified, namely a c.946G>A heterozygous substitution leading to a p.Gly316Ser variant that was present in two POI patients. This variant was absent in all Caucasian controls from various databases, and has been observed exclusively in African populations. This variant was identified to have a dominant negative effect in HeLa cells in vitro to alter connexon function (by 67.2±7.17%), as determined by Gap-fluorescence recovery after photobleaching. The alteration principally resulted from a decrease of cell surface connexons due to altered trafficking (by 47.73±8.59%). In marked contrast to this observation, a p.Pro258Ser variant frequent in all ethnic populations in databases had no functional effect in vitro. In conclusion, the present study reported on a Cx37 variant in two Caucasian POI patients, which was absent in control Caucasian populations, and which had a deleterious effect in vitro. It is therefore suggested that in the genetic context of the Caucasian population, this variant may contribute to POI.

A homozygous FANCM mutation underlies a familial case of non-syndromic primary ovarian insufficiency.

Primary Ovarian Insufficiency (POI) affects ~1% of women under forty. Exome sequencing of two Finnish sisters with non-syndromic POI revealed a homozygous mutation in FANCM, leading to a truncated protein (p.Gln1701*). FANCM is a DNA-damage response gene whose heterozygous mutations predispose to breast cancer. Compared to the mother's cells, the patients' lymphocytes displayed higher levels of basal and mitomycin C (MMC)-induced chromosomal abnormalities. Their lymphoblasts were hypersensitive to MMC and MMC-induced monoubiquitination of FANCD2 was impaired. Genetic complementation of patient's cells with wild-type FANCM improved their resistance to MMC re-establishing FANCD2 monoubiquitination. FANCM was more strongly expressed in human fetal germ cells than in somatic cells. FANCM protein was preferentially expressed along the chromosomes in pachytene cells, which undergo meiotic recombination. This mutation may provoke meiotic defects leading to a depleted follicular stock, as in Fancm-/- mice. Our findings document the first Mendelian phenotype due to a biallelic FANCM mutation.

ß-Klotho sustains postnatal GnRH biology and spins the thread of puberty.

Hypogonadotropic hypogonadism is a syndrome found to be isolated (IHH) or associated with anosmia, corresponding to the Kallmann syndrome (KS). It comprises a defect in gonadotropin-releasing hormone (GnRH) secretion and absent or delayed puberty. Genetic causes have been identified with a high genetic heterogeneity. Fibroblast growth factor receptor 1 (FGFR1), a tyrosine kinase receptor, was one of the first genes whose mutations were identified as causative in KS FGFR1 is responsible for the formation of the GnRH neuron system. Studying patients has not only allowed the identification of new etiologies for this syndrome but also helped to unravel the signaling pathways involved in the development of GnRH neurons and in GnRH control and function. The FGF21/FGFR1/Klotho B (KLB) signaling pathway mediates the response to starvation and other metabolic stresses. Preventing reproduction during nutritional deprivation is an adaptive process that is essential for the survival of species. In this work, Xu et al (2017), using a candidate gene approach, provide a description of the essential role played by this pathway in GnRH biology and in the pathogenesis of IHH and KS They establish a novel link between metabolism and reproduction in humans.

Ovarian-like differentiation in eutopic and ectopic endometrioses with aberrant FSH receptor, INSL3 and GATA4/6 expression.

Endometriosis, the hormone-dependent extrauterine dissemination of endometrial tissue outside the uterus, affects 5-15% of women of reproductive age. Pathogenesis remains poorly understood as well as the estrogen production by endometriotic tissue yielding autocrine growth. Estrogens (E2) are normally produced by the ovaries. We investigated whether aberrant "ovarian-like" differentiation occurred in endometriosis. 69 women, with (n = 38) and without (n = 31) histologically proven endometriosis were recruited. Comparative RT-qPCR was performed on 20 genes in paired eutopic and ectopic lesions, together with immunohistochemistry. Functional studies were performed in primary cultures of epithelial endometriotic cells (EEC). A broaden ovarian-like differentiation was found in half eutopic and all ectopic endometriosis with aberrant expression of transcripts and protein for the transcription factors GATA4 and GATA6 triggering ovarian differentiation, for the FSH receptor (FSHR) and the ovarian hormone INSL3. Like in ovaries the FSHR induced aromatase, the key enzyme in E2 production, and vascular factors in EEC. The LH receptor (LHR) was also aberrantly expressed in a subset of ectopic endometriosis (21%) and induced strongly androgen-synthesizing enzymes and INSL3 in EEC, as in ovaries, as well as endometriotic cell growth. The ERK pathway mediates signaling by both hormones. A positive feedback loop occurred through FSHR and LHR-dependent induction of GATA4/6 in EEC, as in ovaries, enhancing the production of the steroidogenic cascade. This work highlights a novel pathophysiological mechanism with a broadly ovarian pattern of differentiation in half eutopic and all ectopic endometriosis. This study provides new tools that might improve the diagnosis of endometriosis in the future.

Identification of Variants of Hepatitis C Virus (HCV) Entry Factors in Patients Highly Exposed to HCV but Remaining Uninfected: An ANRS Case-Control Study.

Hepatitis C virus (HCV) causes persistent infection in 75% of cases and is a major public health problem worldwide. More than 92% of intravenous drug users (IDU) infected by human immunodeficiency virus type 1 (HIV-1) are seropositive for HCV, and it is conceivable that some HIV-1-infected IDU who remain uninfected by HCV may be genetically resistant.Here we conducted a case-control study to identify mutations in HCV entry coreceptors in HIV-infected IDU who remained uninfected by HCV. We recruited 138 patients, comprising 22 HIV+ HCV- case IDU and 116 HIV+ HCV+ control IDU. We focused on coreceptors in which point mutations are known to abolish HCV infectivity in vitro. Our previous study of the Claudin-1 gene revealed no specific variants in the same case population. Here we performed direct genomic sequencing of the Claudin-6, Claudin-9, Occludin and Scavenger receptor-B1 (SCARB1) gene coding regions. Most HIV+ HCV- IDU had no mutations in HCV coreceptors. However, two HIV+ HCV- patients harbored a total of four specific mutations/variants of HCV entry factors that were not found in the HIV+ HCV+ controls. One case patient harbored heterozygous variants of both Claudin-6 and Occludin, and the other case patient harbored two heterozygous variants of SCARB1. This suggests that HCV resistance might involve complex genetic events and factors other than coreceptors, a situation similar to that reported for HIV-1 resistance.

Claudin-6 and Occludin Natural Variants Found in a Patient Highly Exposed but Not Infected with Hepatitis C Virus (HCV) Do Not Confer HCV Resistance In Vitro.

The clinical course of Hepatitis C Virus (HCV) infection is highly variable between infected individual hosts: up to 80% of acutely HCV infected patients develop a chronic infection while 20% clear infection spontaneously. Spontaneous clearance of HCV infection can be predicted by several factors, including symptomatic acute infection, favorable IFNL3 polymorphisms and gender. In our study, we explored the possibility that variants in HCV cell entry factors might be involved in resistance to HCV infection. In a same case patient highly exposed but not infected by HCV, we previously identified one mutation in claudin-6 (CLDN6) and a rare variant in occludin (OCLN), two tight junction proteins involved in HCV entry into hepatocytes. Here, we conducted an extensive functional study to characterize the ability of these two natural variants to prevent HCV entry. We used lentiviral vectors to express Wildtype or mutated CLDN6 and OCLN in different cell lines and primary human hepatocytes. HCV infection was then investigated using cell culture produced HCV particles (HCVcc) as well as HCV pseudoparticles (HCVpp) expressing envelope proteins from different genotypes. Our results show that variants of CLDN6 and OCLN expressed separately or in combination did not affect HCV infection nor cell-to-cell transmission. Hence, our study highlights the complexity of HCV resistance mechanisms supporting the fact that this process probably not primarily involves HCV entry factors and that other unknown host factors may be implicated.