ABCB4 variants in adult patients with cholestatic disease are frequent and underdiagnosed.

BACKGROUND: Heterozygous ABCB4 variants are not routinely tested in adults with cholestasis because of their supposed rarity and high costs. METHODS: Nineteen adult patients presenting with unexplained cholestasis, and/or recurrent gallstones were included; genotyping was not done in five due to lack of health insurance approval. RESULTS: heterozygous ABCB4 variants were identified in seven patients, followed by cascade testing of 12 family members: one patient underwent liver transplantation at age 40 for end-stage liver disease; one had compensated cirrhosis; all symptomatic adults had gallstones, including four with low phospholipid-associated cholelithiasis; four had intrahepatic cholestasis of pregnancy; all children and one 54-year old female were asymptomatic. Genotype: Families A and C: c.2211G>A (p.Ala737=) combined with c.959C>T (p.Ser320Phe) in one subject; Family B: c.1130T>C (p.Ile377Thr); Family D: large deletion removing ABCB4 exons 1-4 plus ABCB1, RUNDC3B, SLC25A40, DBF4, ADAM22 exons 1-3; Family E: c.1565T>C (p.Phe522Ser) ; Family F: c.1356+2T>C combined with c.217C>G (p.Leu73Val). All patients responded to ursodeoxycholic acid. CONCLUSIONS: We found ABCB4 variants in half of the adults with unexplained cholestasis and/or recurrent gallstones presenting at our center, suggesting that this condition is underdiagnosed and undertreated, with serious consequences not only for the patients and their families, but also in terms of healthcare costs.

Neuron-Derived Neurotrophic Factor Is Mutated in Congenital Hypogonadotropic Hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by infertility and the absence of puberty. Defects in GnRH neuron migration or altered GnRH secretion and/or action lead to a severe gonadotropin-releasing hormone (GnRH) deficiency. Given the close developmental association of GnRH neurons with the olfactory primary axons, CHH is often associated with anosmia or hyposmia, in which case it is defined as Kallmann syndrome (KS). The genetics of CHH are heterogeneous, and >40 genes are involved either alone or in combination. Several CHH-related genes controlling GnRH ontogeny encode proteins containing fibronectin-3 (FN3) domains, which are important for brain and neural development. Therefore, we hypothesized that defects in other FN3-superfamily genes would underlie CHH. Next-generation sequencing was performed for 240 CHH unrelated probands and filtered for rare, protein-truncating variants (PTVs) in FN3-superfamily genes. Compared to gnomAD controls the CHH cohort was statistically enriched for PTVs in neuron-derived neurotrophic factor (NDNF) (p = 1.40 × 10-6). Three heterozygous PTVs (p.Lys62∗, p.Tyr128Thrfs∗55, and p.Trp469∗, all absent from the gnomAD database) and an additional heterozygous missense mutation (p.Thr201Ser) were found in four KS probands. Notably, NDNF is expressed along the GnRH neuron migratory route in both mouse embryos and human fetuses and enhances GnRH neuron migration. Further, knock down of the zebrafish ortholog of NDNF resulted in altered GnRH migration. Finally, mice lacking Ndnf showed delayed GnRH neuron migration and altered olfactory axonal projections to the olfactory bulb; both results are consistent with a role of NDNF in GnRH neuron development. Altogether, our results highlight NDNF as a gene involved in the GnRH neuron migration implicated in KS.

Defective AMH signaling disrupts GnRH neuron development and function and contributes to hypogonadotropic hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) is a condition characterized by absent puberty and infertility due to gonadotropin releasing hormone (GnRH) deficiency, which is often associated with anosmia (Kallmann syndrome, KS). We identified loss-of-function heterozygous mutations in anti-Müllerian hormone (AMH) and its receptor, AMHR2, in 3% of CHH probands using whole-exome sequencing. We showed that during embryonic development, AMH is expressed in migratory GnRH neurons in both mouse and human fetuses and unconvered a novel function of AMH as a pro-motility factor for GnRH neurons. Pathohistological analysis of Amhr2-deficient mice showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in reduced fertility in adults. Our findings highlight a novel role for AMH in the development and function of GnRH neurons and indicate that AMH signaling insufficiency contributes to the pathogenesis of CHH in humans.

A novel CHD7 mutation in an adolescent presenting with growth and pubertal delay.

Mutations in the CHD7 gene, encoding for the chromodomain helicase DNA-binding protein 7, are found in approximately 60% of individuals with CHARGE syndrome (coloboma, heart defects, choanal atresia, retarded growth and development, genital hypoplasia, ear abnormalities and/or hearing loss). Herein, we present a clinical case of a 14-year-old male presenting for evaluation of poor growth and pubertal delay highlighting the diagnostic challenges of CHARGE syndrome. The patient was born full term and underwent surgery at 5 days of life for bilateral choanal atresia. Developmental milestones were normally achieved. At age 14 his height and weight were -2.04 and -1.74 standard deviation score respectively. He had anosmia as well as prepubertal testes and micropenis (4 cm×1 cm). The biological profile showed low basal serum testosterone and gonadotropins (testosterone, 0.2 nmol/L; luteinizing hormone, 0.5 U/L; follicle-stimulating hormone, 1.3 U/L), and otherwise normal pituitary function and normal imaging of the hypothalamic-pituitary area. The constellation of choanal atresia, anosmia, mild dysmorphic features, micropenis and delayed puberty were suggestive of CHARGE syndrome. Targeted genetic testing of CHD7 was performed revealing a de novo heterozygous CHD7 mutation (c.4234T>G [p.Tyr1412Asp]). Further paraclinical investigations confirmed CHARGE syndrome. Despite the presence of suggestive features, CHARGE syndrome remained undiagnosed in this patient until adolescence. Genetic testing helps clarify the phenotypic and genotypic spectrum to facilitate diagnosis, thus promoting optimal follow-up, treatment, and appropriate genetic counselling.

Congenital hypogonadotropic hypogonadism and constitutional delay of growth and puberty have distinct genetic architectures.

OBJECTIVE: Congenital hypogonadotropic hypogonadism (CHH) and constitutional delay of growth and puberty (CDGP) represent rare and common forms of GnRH deficiency, respectively. Both CDGP and CHH present with delayed puberty, and the distinction between these two entities during early adolescence is challenging. More than 30 genes have been implicated in CHH, while the genetic basis of CDGP is poorly understood. DESIGN: We characterized and compared the genetic architectures of CHH and CDGP, to test the hypothesis of a shared genetic basis between these disorders. METHODS: Exome sequencing data were used to identify rare variants in known genes in CHH (n = 116), CDGP (n = 72) and control cohorts (n = 36 874 ExAC and n = 405 CoLaus). RESULTS: Mutations in at least one CHH gene were found in 51% of CHH probands, which is significantly higher than in CDGP (7%, P = 7.6 × 10-11) or controls (18%, P = 5.5 × 10-12). Similarly, oligogenicity (defined as mutations in more than one gene) was common in CHH patients (15%) relative to CDGP (1.4%, P = 0.002) and controls (2%, P = 6.4 × 10-7). CONCLUSIONS: Our data suggest that CDGP and CHH have distinct genetic profiles, and this finding may facilitate the differential diagnosis in patients presenting with delayed puberty.

DCC/NTN1 complex mutations in patients with congenital hypogonadotropic hypogonadism impair GnRH neuron development.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease characterized by absent puberty and infertility due to GnRH deficiency, and is often associated with anosmia [Kallmann syndrome (KS)]. The genetic etiology of CHH is heterogeneous, and more than 30 genes have been implicated in approximately 50% of patients with CHH. We hypothesized that genes encoding axon-guidance proteins containing fibronectin type-III (FN3) domains (similar to ANOS1, the first gene associated with KS), are mutated in CHH. We performed whole-exome sequencing in a cohort of 133 CHH probands to test this hypothesis, and identified rare sequence variants (RSVs) in genes encoding for the FN3-domain encoding protein deleted in colorectal cancer (DCC) and its ligand Netrin-1 (NTN1). In vitro studies of these RSVs revealed altered intracellular signaling associated with defects in cell morphology, and confirmed five heterozygous DCC mutations in 6 probands-5 of which presented as KS. Two KS probands carry heterozygous mutations in both DCC and NTN1 consistent with oligogenic inheritance. Further, we show that Netrin-1 promotes migration in immortalized GnRH neurons (GN11 cells). This study implicates DCC and NTN1 mutations in the pathophysiology of CHH consistent with the role of these two genes in the ontogeny of GnRH neurons in mice.

Evaluating CHARGE syndrome in congenital hypogonadotropic hypogonadism patients harboring CHD7 variants.

PURPOSE: Congenital hypogonadotropic hypogonadism (CHH), a rare genetic disease caused by gonadotropin-releasing hormone deficiency, can also be part of complex syndromes (e.g., CHARGE syndrome). CHD7 mutations were reported in 60% of patients with CHARGE syndrome, and in 6% of CHH patients. However, the definition of CHD7 mutations was variable, and the associated CHARGE signs in CHH were not systematically examined. METHODS: Rare sequencing variants (RSVs) in CHD7 were identified through exome sequencing in 116 CHH probands, and were interpreted according to American College of Medical Genetics and Genomics guidelines. Detailed phenotyping was performed in CHH probands who were positive for CHD7 RSVs, and genotype-phenotype correlations were evaluated. RESULTS: Of the CHH probands, 16% (18/116) were found to harbor heterozygous CHD7 RSVs, and detailed phenotyping was performed in 17 of them. Of CHH patients with pathogenic or likely pathogenic CHD7 variants, 80% (4/5) were found to exhibit multiple CHARGE features, and 3 of these patients were reclassified as having CHARGE syndrome. In contrast, only 8% (1/12) of CHH patients with nonpathogenic CHD7 variants exhibited multiple CHARGE features (P = 0.01). CONCLUSION: Pathogenic or likely pathogenic CHD7 variants rarely cause isolated CHH. Therefore a detailed clinical investigation is indicated to clarify the diagnosis (CHH versus CHARGE) and to optimize clinical management.

KLB, encoding ß-Klotho, is mutated in patients with congenital hypogonadotropic hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic form of isolated gonadotropin-releasing hormone (GnRH) deficiency caused by mutations in > 30 genes. Fibroblast growth factor receptor 1 (FGFR1) is the most frequently mutated gene in CHH and is implicated in GnRH neuron development and maintenance. We note that a CHH FGFR1 mutation (p.L342S) decreases signaling of the metabolic regulator FGF21 by impairing the association of FGFR1 with ß-Klotho (KLB), the obligate co-receptor for FGF21. We thus hypothesized that the metabolic FGF21/KLB/FGFR1 pathway is involved in CHH Genetic screening of 334 CHH patients identified seven heterozygous loss-of-function KLB mutations in 13 patients (4%). Most patients with KLB mutations (9/13) exhibited metabolic defects. In mice, lack of Klb led to delayed puberty, altered estrous cyclicity, and subfertility due to a hypothalamic defect associated with inability of GnRH neurons to release GnRH in response to FGF21. Peripheral FGF21 administration could indeed reach GnRH neurons through circumventricular organs in the hypothalamus. We conclude that FGF21/KLB/FGFR1 signaling plays an essential role in GnRH biology, potentially linking metabolism with reproduction.

Dysfunctional SEMA3E signaling underlies gonadotropin-releasing hormone neuron deficiency in Kallmann syndrome.

Individuals with an inherited deficiency in gonadotropin-releasing hormone (GnRH) have impaired sexual reproduction. Previous genetic linkage studies and sequencing of plausible gene candidates have identified mutations associated with inherited GnRH deficiency, but the small number of affected families and limited success in validating candidates have impeded genetic diagnoses for most patients. Using a combination of exome sequencing and computational modeling, we have identified a shared point mutation in semaphorin 3E (SEMA3E) in 2 brothers with Kallmann syndrome (KS), which causes inherited GnRH deficiency. Recombinant wild-type SEMA3E protected maturing GnRH neurons from cell death by triggering a plexin D1-dependent (PLXND1-dependent) activation of PI3K-mediated survival signaling. In contrast, recombinant SEMA3E carrying the KS-associated mutation did not protect GnRH neurons from death. In murine models, lack of either SEMA3E or PLXND1 increased apoptosis of GnRH neurons in the developing brain, reducing innervation of the adult median eminence by GnRH-positive neurites. GnRH neuron deficiency in male mice was accompanied by impaired testes growth, a characteristic feature of KS. Together, these results identify SEMA3E as an essential gene for GnRH neuron development, uncover a neurotrophic function for SEMA3E in the developing brain, and elucidate SEMA3E/PLXND1/PI3K signaling as a mechanism that prevents GnRH neuron deficiency.

Male infertility and copy number variants (CNVs) in the dog: a two-pronged approach using Computer Assisted Sperm Analysis (CASA) and Fluorescent In Situ Hybridization (FISH).

BACKGROUND: Infertility affects ~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors. RESULTS: We identified two genomic regions hybridized by BACs CH82-321J09 and CH82-509B23 showing duplication patterns in all samples except for an azoospermic dog. These two regions harbor two important genes for spermatogenesis: DNM2 and TEKT1. The genomic region encompassed by the BAC clone CH82-324I01 showed a single-copy pattern in all samples except for one dog, assessed with low-quality sperm, displaying a marked duplication pattern. This genomic region harbors SOX8, a key gene for testis development. CONCLUSION: We present the first study involving functional and genetic analyses in male infertility. We set up an extremely reliable analysis on dog sperm cells with a highly consistent statistical significance, and we succeeded in conducting FISH experiments on sperm cells using BAC clones as probes. We found copy number differences in infertile compared with fertile dogs for genomic regions encompassing TEKT1, DNM2, and SOX8, suggesting those genes could have a role if deleted or duplicated with respect to the reference copy number in fertility biology. This method is of particular interest in the dog due to the recognized role of this species as an animal model for the study of human genetic diseases and could be useful for other species of economic interest and for endangered animal species.