Haploinsufficiency of Dmxl2, Encoding a Synaptic Protein, Causes Infertility Associated with a Loss of GnRH Neurons in Mouse

International audienceCharacterization of the genetic defects causing gonadotropic deficiency has made a major contribution to elucidation of the fundamental role of Kisspeptins and Neurokinin B in puberty onset and reproduction. The absence of puberty may also reveal neurodevelopmental disorders caused by molecular defects in various cellular pathways. Investigations of these neurodevelopmental disorders may provide information about the neuronal processes controlling puberty onset and reproductive capacity. We describe here a new syndrome observed in three brothers, which involves gonadotropic axis deficiency, central hypothyroidism, peripheral demyelinating sensorimotor polyneuropathy, mental retardation, and profound hypoglycemia, progressing to nonautoimmune insulin-dependent diabetes mellitus. High-throughput sequencing revealed a homozygous in-frame deletion of 15 nucleotides in DMXL2 in all three affected patients. This homozygous deletion was associated with lower DMXL2 mRNA levels in the blood lymphocytes of the patients. DMXL2 encodes the synaptic protein rabconnectin-3a, which has been identified as a putative scaffold protein for Rab3-GAP and Rab3-GEP, two regulators of the GTPase Rab3a. We found that rabconnectin-3a was expressed in exocytosis vesicles in gonadotropin-releasing hormone (GnRH) axonal extremities in the median eminence of the hypothalamus. It was also specifically expressed in cells expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) within the pituitary. The conditional heterozygous deletion of Dmxl2 from mouse neurons delayed puberty and resulted in very low fertility. This reproductive phenotype was associated with a lower number of GnRH neurons in the hypothalamus of adult mice. Finally, Dmxl2 knockdown in an insulin-secreting cell line showed that rabconnectin-3a controlled the constitutive and glucose-induced secretion of insulin. In conclusion, this study shows that low levels of DMXL2 expression cause a complex neurological phenotype, with abnormal glucose metabolism and gonadotropic axis deficiency due to a loss of GnRH neurons. Our findings identify rabconectin-3a as a key controller of neuronal and endocrine homeostatic processes

Molecular and clinical investigation of children with disorders of puberty: GPR54 gene and relevant genes

Background: Kisspeptin (KISS1)/GPR54 (KISSR) signaling complex and Neurokinin B (NKB)/ NKB receptor (TACR3) signaling have been proposed as an integral part of the network coordinating GnRH release. GPR54 (KISS1R) and TACR3 gene mutations have been described in cases of idiopathic hypogonadotrophic hypogonadism, while limited data exist on gain of function mutation in GPR54 (KISS1R) gene causing idiopathic central precocious puberty (ICPP). No data on TACR3 mutations in ICPP have been described so far. Aim of this study was to elucidate the possible impact of GPR54 (KISS1R) and TACR3 mutations in ICPP. Methods: PCR-amplified genomic DNA of 38 girls with ICPP was analysed for GPR54 and TACR3 genes mutations. Results: No GPR54 or TACR3 mutations were found. The A/G coding sequence SNP on the GPR54 gene (dbSNP ID: rs10407968) was found in 2 patients with ICPP.Conclusion: Our data indicate that GPR54 and TACR3 gene mutations are not a frequent cause of ICPP. The identified A/G synonymous SNP (dbSNP ID: rs10407968) located in exon 1 of the gene is not probable to have a pathogenic role in exon splicing and therefore in the premature initiation of puberty.Η έναρξη της εφηβείας είναι ένα σύνθετο φαινόμενο και οι μηχανισμοί που εμπλέκονται σε αυτήν παραμένουν εν πολλοίς άγνωστοι. Τα νευροπεπτίδια Kisspeptin και Neurokinin Β παίζουν κεντρικό ρόλο στην ενεργοποίηση των GnRH νευρώνων. Μελέτες σε ανθρώπους και πειραματόζωα έχουν δείξει ότι μεταλλάξεις στα γονίδια που κωδικοποιούν αυτά τα νευροπεπτίδια ή τους υποδοχείς τους (GPR54 και TACR3 αντίστοιχα) οδηγούν σε διαταραχές ενήβωσης.Σκοπός: Μελέτη της επίπτωσης μεταλλάξεων και πολυμορφισμών των γονιδίων GPR54 και TACR3 στην παθογένεια της ιδιοπαθούς κετρικής πρώιμης ήβης (ΙΚΠΗ).Μεθοδολογία: 38 θήλεα άτομα με διάγνωση ΙΚΠΗ και τακτική παρακολούθηση από παιδίατρο ενδοκρινολόγο στη Μονάδα Ενδοκρινολογίας, Μεταβολισμού και Διαβήτη της Α’ Πανεπιστημιακής Κλινικής, Νοσοκομείου Παίδων «Η Αγία Σοφία» μελετήθηκαν αναδρομικά. Το γενομικό DNA των ασθενών πολλαπλασιάστηκε με PCR και έγινε direct sequencing των γονιδίων GPR54 και TACR3. Η βάση δεδομένων Ensemble genome database χρησιμοποιήθηκε για των έλεγχο τυχών μεταλλάξεων και πολυμορφισμών.Αποτελέσματα: Δεν διαπιστώθηκαν μεταλλάξεις στα γονίδια GPR54 και TACR3. Η σιωπηλή παραλλαγή (SNP) A/G coding sequence στη θέση 857526 στο γονίδιο GPR54 (dbSNP ID: rs10407968) βρέθηκε σε 2 ασθενείς με ΙΚΠΗ.Συμπεράσματα: Mεταλλάξεις στα γονίδια GPR54 και TACR3 μάλλον δεν αποτελούν συχνά αίτια στην παθογένεια της ΙΚΠΗ

Cortisol response to adrenocorticotropin testing in non-classical congenital adrenal hyperplasia (NCCAH)

Background: The adequacy of cortisol response in non-classical congenital adrenal hyperplasia (NCCAH) has not been fully elucidated. The aim was to evaluate cortisol response to adrenocorticotropin (ACTH) stimulation test in children and adolescents with NCCAH and heterozygotes for CYP21A2 gene mutations. Methods: One hundred and forty-six children and adolescents, mean age 7.9 (0.7-17.5) years with clinical hyper-androgenism, were evaluated retrospectively. Thirty-one subjects had NCCAH, 30 were heterozygotes for CYP21A2 gene mutations, while 85 showed normal response to ACTH test. Results: Baseline cortisol levels did not differ among NCCAH, heterozygotes, and normal responders: 15.75 (5.83-59.6) mu g/dL vs. 14.67 (5.43-40.89) mu g/dL vs. 14.04 (2.97-34.8) mu g/dL, p = 0.721. However, NCCAH patients had lower peak cortisol compared to heterozygotes and control group: 28.34 (12.25-84.40) vs. 35.22 (17.47-52.37) mu g/dL vs. 34.92 (19.91-46.68) mu g/dL, respectively, p = 0.000. Peak cortisol was < 18 mu g/dL in 7/31 NCCAH patients and in one heterozygote. Conclusions: A percentage of 21.2% NCCAH patients showed inadequate cortisol response to ACTH stimulation. In these subjects, the discontinuation of treatment on completion of growth deserves consideration

Rbcn-3α is expressed in exocytosis vesicles in the external layer of the median eminence.

<p>(A) ISH with a mouse <i>Dmxl2</i> antisense probe (AS) and a sense probe (S). (B) Immunolabeling with an antibody against Rbcn-3α revealed high levels of <i>Dmxl2</i>/Rbcn-3α expression in the dentate gyrus, the CA1 and CA3 regions of the hippocampus, and the cortex (black arrowheads). Scale bars, 200 µm. (C and D) Rbcn-3α was found to be strongly expressed in the external layer of the ME (C) and the OVLT (D). Scale bars, 100 µm. (E) Confocal analysis with an antibody against Rbcn-3α showed punctate staining in the median eminence and staining of the long processes extending from the cell bodies lining the third ventricle. (F and G) Rbcn-3α immunoreactivity was observed in small clear vesicles and LDCVs at the extremities of the axons in the ME (white arrow). Scale bar, 0.2 µm.</p

<i>DMXL2</i> is mutated in affected patients.

<p>(A) Pedigree of the affected family. Closed symbols indicate affected individuals. (B) Linkage analysis delineated two candidate regions on chromosomes 13 and 15 with a LOD score of 2.5. (C) Next-generation sequencing characterized a deletion of 15 nucleotides (c.5824_5838del) in exon 24 of <i>DMXL2</i>. This deletion removes five amino acids (p.1942_1946del). Rbcn-3α is a protein with 17 WD domains (green box) and one Rav1p_C domain, which is involved in regulating the glucose-dependent assembly and disassembly of the V1 and V0 subunits of the vacuolar ATPase (purple box) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952-Seol1" target="_blank">[57]</a>. (D) Quantification, by RT-qPCR, of <i>DMXL2</i> mRNA levels relative to RNA 18S levels in blood lymphocytes. Error bars, SEM. * <i>p</i><0.05. Numerical data used to generate graph 1D may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s012" target="_blank">Table S5</a>.</p

<i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice displayed a partial gonadotropin deficiency.

<p>(A and B) Weights of testes and ovaries were low in <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (C) Histological analysis of ovaries showed a normal number of antral follicles but very few corpora lutea in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (D) Estradiol concentrations were normal in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (E) Plasma testosterone concentration was low in male <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (F) Plasma LH concentrations were moderately high in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (G) Despite their lower testosterone concentrations, male <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice had normal plasma LH concentrations. (H) The GnRH-induced increase in LH concentration was normal in <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (I) The administration of PMSG to young mice induced a normal increase in estradiol concentration in <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice, similar to that observed in their WT littermates (asterisks indicate significant differences: * <i>p</i><0.05, ** <i>p</i><0.001; ***<i>p</i><0.0001). Error bars: SEM. P, postnatal day. White bars, <i>Dmxl2^lox/wt</i>; black bars, <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt. Numerical data used to generate these graphs may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s012" target="_blank">Table S5</a>.</p

Summary of the clinical phenotype of the three affected patients with <i>DMXL2</i> mutations (see Figure 1 for patient numbering).

<p>SDS, standard deviation score.</p><p>Summary of the clinical phenotype of the three affected patients with <i>DMXL2</i> mutations (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio-1001952-g001" target="_blank">Figure 1</a> for patient numbering).</p

Hypothalamic GnRH mRNA and GnRH-IR neuron levels are lower in the hypothalamus of <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice.

<p>(A) Rbcn-3α is expressed in GnRH neurons in the median eminence. (B) Rbcn-3α is located in small clear vesicles and in LDCVs in GnRH neurons. White arrows indicate Rbcn-3α DAB staining; white arrow heads indicate GnRH nanogold staining. (C) <i>GnRH1</i> mRNA levels relative to RNA18S were lower in the hypothalamus of <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt male mice than in WT mice. (D) The total number of GnRH-ir neurons in the brain was lower in <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt male mice than in WT mice. (E) GnRH immunostaining in the OVLTs in <i>Dmxl2</i>^lox/wt and <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt male mice. (F) An analysis of the rostral–caudal distribution of GnRH-ir neurons in the hypothalamus revealed that <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt male mice had fewer GnRH-ir cell bodies in the OVLT (see inset) than their WT littermates. * <i>p</i><0.05, *** <i>p</i><0.0001. White bars, <i>Dmxl2^lox/wt</i>; black bars, <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt. Numerical data used to generate graphs 6C, 6D, 6F, and 6F inset may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s012" target="_blank">Table S5</a>.</p

Female <i>Nes-cre;Dmxl2</i>^–/wt mice displayed delayed puberty and were infertile.

<p>(A) Postnatal growth curve of female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice (black line, <i>Dmxl2^lox/wt</i>; hatched gray line, <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt). (B and C) VO and first estrus occurred significantly later in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice than in their WT littermates. (D) The interval from VO to first estrus was significantly longer in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice than in WT mice, suggesting a defect in maturation of the HPG axis. (E) Very few complete estrous cycles were observed in female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice. (F) Female <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt spent less time in high estradiol concentration (M, miestrus; E, estrus; D, diestrus; P, proestrus). (G) A significant difference in AGD was observed between male <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt mice and their WT littermates. Black line, <i>Dmxl2^lox/wt</i>; hatched gray line, <i>nes-Cre</i>;<i>Dmxl2</i>^-/wt. White bars, <i>Dmxl2^lox/wt</i>. Black bars, <i>nes-Cre</i>;<i>Dmxl2</i>^–/wt. Numerical data used to generate graphs 4B, 4C, 4D, 4E, 4F, or graphs 4A, 4G may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s012" target="_blank">Table S5</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001952#pbio.1001952.s013" target="_blank">Table S6</a>, respectively. Error bars are SEM. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p