Supplemental Table 2.xlsx

Supplemental Table: <i>in silico</i> prediction tools for c.570 C>A; p.S190R in <i>TBX3</i> gene (NM_005996,hg19 g.115118771) missense varian

Supplemental Table 1.xlsx

Supplemental Table 1: benign variants in <i>TBX3</i> 5' and 3'-UT

Recombinant BMP15 inhibition of progesterone secretion.

<p>Granulosa cells from small antral (1–3 mm in diameter) ovine follicles were cultured for 96 h in serum-free conditions. Cultures were performed in the absence (control) or in the presence of increasing dose (10, 50, 200ng/ml) of wild type recombinant human BMP15^wt or BMP15^Y235C mutant. Each treatment was tested in triplicate in each of 5 independent experiments. The results represent the amount of progesterone secreted by 50 000 cells between 48 h and 96 h of culture (mean ±SEM). Dose effect was analyzed by one-way ANOVA by comparing each dose to control (**, p<0.01; ***, p<0.001). Mutation effect was analyzed by Student t-test within each dose ($, p<0.05).</p

BMP15 positive selection pressure among mammals.

<p>Phylogenetic tree of BMP15 from 24 mammals species reconstructed using TreeBeST and rooted by minimizing with the number of duplications and losses. Bootstrap values are given when nodes are strongly supported (>80%). Branches or species that have a significant LRT for positive selection calculation are indicated in bold (threshold of <i>q</i>= 5% of false positives). The scale represents the substitution rate. </p

Functional analysis of amino acids under positive selection in human BMP15.

<p>A, Human BMP15 full sequence showing prodomain, cleavage site (underlined) and mature peptide (framed sequence); known human variants for BMP15 found in POI patients (framed grey highligthed); and identified amino acids under positive selection (framed colored highlighted): BMP15^F146, BMP15^L189, BMP15^Y235. B, <i>In </i><i>vitro</i> reporter luciferase assay from COV434 granulosa cells transiently transfected with empty vector +/- 100ng recombinant human BMP15 (mock +/- BMP15) or wild type human BMP15 expressing vector (WT) or the different BMP15 variants vectors obtained by directed-mutagenesis of residues under positive selection. Results are expressed as the mean (±SD) of 4 independent experiments. Differences between means were analyzed by one-way ANOVA by comparing each condition to WT (*, p<0.05; **, p<0;01; ***, p<0.001).</p

BMP15 evolution among TGFß/BMP family members.

<p>Phylogenetic tree of thirteen members of the TGFβ/BMP superfamily expressed by the mammalian ovary reconstructed using maximum likelihood. Bootstrap values are given when nodes are strongly supported (>80%). The scale represents the substitution rate.</p

Table_1_Genetic and phenotypic differences between sexes in congenital hypogonadotropic hypogonadism (CHH): Large cohort analysis from a single tertiary centre.docx

BackgroundCongenital hypogonadotropic hypogonadism (CHH) is a condition with a strong genetic background, caused by a deficient production, secretion, or action of gonadotropin-releasing hormone (GnRH). Published data on CHH cohorts indicate a male predominance, although this is not supported by our current understandings.AimsIn order to unravel the possible causes or contributors to such epidemiological sex difference, the aim of our study is to investigate differences in genetic background and clinical presentation between males and females in a large cohort of CHH patients.Materials and methodsWe enrolled 338 CHH patients with absent or arrested pubertal development, referred to our Center from 01/2016. Data collection included clinical assessment at diagnosis and genetic analysis performed by next generation sequencing (NGS), employing a custom panel of 28 candidate genes.ResultsAmong 338 patients 94 were female (F) and 244 male (M), with a ratio of 1:2.6. We found that 36.09% (122/338) of patients harbored potentially pathogenic rare genetic variants (RVs) with no significant differences between sexes; on the other hand, a significantly higher frequency of oligogenicity was observed in females (F 9,57% 9/94 vs M 3,69% 9/244, P = 0.034). The prevalence of non-reproductive phenotypic features was significantly higher (P = 0.01) in males (53/228, 23.2%) than in females (10/93, 10.8%): in particular, kidney abnormalities affected only male patients and midline defects had a significantly higher prevalence in males (P = 0.010). Finally, BMI SDS was -0.04 ± 1.09 in females and 0.69 ± 1.51 in males, with a statistically significant difference between groups (P = ConclusionOur data confirm the male predominance in CHH and identify some differences with regard to the clinical presentation between males and females that could indicate a variable expression of genetic rare variants and a dimorphic modulation of phenotype according to metabolic/behavioral factors, which will need to be substantiated and investigated by further studies.</p

Relative quantification of mitochondrial on nuclear DNA copy number (mtDNA/nDNA) in peripheral blood cells.

<p>Total DNA isolated from whole blood has been quantified by real-time quantitative PCR analysis using the RNAse P as an endogenous control. The PCR data were analyzed by using a comparative Ct method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042423#pone.0042423-Pfaffl1" target="_blank">[25]</a> and values are here expressed as the Logarithm of mtDNA/nDNA copy number. Mean value for each group is graphically indicated by a line. The grey area is indicating the range observed in the NR group. The significant differences detected by one-way ANOVA test are indicated in the figure (****p<0.0001; **p<0.01).</p

Anagraphical, clinical and biochemical parameters in the four groups of subjects.

a<p>Antral Follicle Count per ovary.</p>*<p><i>p</i><0.03 vs PR and NR.</p>**<p><i>p</i><0.05 vs NR.</p

Functional effects of <b><i>FecX^Gr</i></b><b> and </b><b><i>FecX^O</i></b><b> mutations on the BMP15 activity.</b>

<p><i>In vitro</i> reporter luciferase assay from COV434 granulosa cells transiently transfected with empty vector +/− 100 ng of recombinant human BMP15 (Control +/− rhBMP15) or wild-type human BMP15 expressing vector (WT) or the 2 different BMP15 variant vectors (<i>BMP15^T317I (FecX^Gr)</i>; <i>BMP15^N337H (FecX^O)</i>) obtained by directed-mutagenesis. Results are expressed as Means±SD of the relative light unit (RLU) from 3 independent experiments in triplicate for each condition. Pairwise statistical comparisons using a one-way ANOVA test between means were performed and results of statistic test are symbolized by stars: * = p<5E⁻⁰²; ** = p<1E⁻⁰² and *** = p<1E⁻⁰³.</p