Summary of reported patients harboring sequence variations and deletions affecting <i>MAMLD1</i> gene ^a.

<p>^a the variants were named according to NM_005491.4.</p><p>N: normal sequence; NA: not analyzed; ?/-: unknown; GD: gonadal dysgenesis; GOF: gain of function.</p><p>Summary of reported patients harboring sequence variations and deletions affecting <i>MAMLD1</i> gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142831#t003fn001" target="_blank">^a</a>.</p

Clinical, biochemical and genetic characteristics of the patients harboring mutations and polymorphisms in the <i>MAMLD1</i> gene.

<p>ND: not done. d: day(s), m: month(s), y: year(s).</p><p>Clinical, biochemical and genetic characteristics of the patients harboring mutations and polymorphisms in the <i>MAMLD1</i> gene.</p

Transactivation activity of MAMLD1 on the <i>Hes3</i> promoter.

<p>HEK293 cells were transiently transfected with wild-type (WT) and mutant MAMLD1 expression vectors and with a <i>Hes3</i> promoter luciferase reporter construct. Luciferase activity was measured with the Promega Dual Luciferase assay system. A. Comparison of the newly constructed MAMLD1 WT expression vector (WT (a), NM_005491.4) with the older WT (WT (b)), and ΔE5 (ΔE5 (b)) constructs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142831#pone.0142831.ref014" target="_blank">14</a>]. Similar transactivation activity on the <i>Hes3</i> promoter was found for all constructs. B. <i>Hes3</i> transactivation by WT and the 11 MAMLD1 mutants was assessed. Only the L210X MAMLD1 mutant showed an impaired activity on the <i>Hes3</i> promoter. Results are expressed in relative light units (RLU) and represent the mean and SEM of 3 independent experiments performed in duplicate. ΔE5: original WT (b) without exon 5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142831#pone.0142831.ref014" target="_blank">14</a>]; * <i>p</i>≤0.05.</p

MAMLD1 transcripts, reported mutations and tissue expression.

<p>A. Schemes of the 3 MAMLD1 human transcripts are shown (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>; <a href="http://www.ensembl.org" target="_blank">http://www.ensembl.org</a>). B. Scheme showing all reported <i>MAMLD1</i> gene mutations. Mutations described in this study are shown in red and the novel ones are marked with an asterisk. C. Assessment of MAMLD1 expression in human fetal and adult adrenal and testis. Semiquantitative RT-PCRs were performed using specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142831#pone.0142831.s005" target="_blank">S1 Table</a>). GAPDH was used as the internal control. A representative gel picture is shown (n = 3). For MAMLD1 the band at 581 bp corresponds to isoform 2 and the band at 506 bp to isoforms 1 and 3. Dashed red lines in A indicate the location of the PCR fragments amplified for the expression studies.</p

Effect of MAMLD1 on CYP17A1 promoter and enzyme activities.

<p>HEK293 cells or NCI-H295R cells were transiently transfected with MAMLD1 WT and mutant expression vectors. For promoter activation studies, the (-3.7kb) <i>CYP17A1</i> promoter luciferase reporter construct was co-transfected. A. <i>CYP17A1</i> promoter activation by MAMLD1 was assessed by the Promega Dual luciferase assay in HEK293 cells. Only for mutant MAMLD1 L210X and L724V an impaired <i>CYP17A1</i> activation was found. Results are expressed in RLU and represent the mean and SEM of 3 independent experiments performed in duplicate. B. The effect of WT and mutant MAMLD1 on CYP17A1 enzyme activity was assessed in transfected NCI-H295R, MA-10 and HEK293 cells by measuring the conversion of progesterone to 17-hydroxyprogesterone. Steroid production was labeled with [¹⁴C]progesterone for 60 min. Steroids were extracted and resolved by thin-layer chromatography, then quantified as % conversion. A representative steroid profile obtained from NCI-H295R cells is shown (n = 2). No effect of MAMLD1 on CYP17A1-hydroxylase activity was detected. P: progesterone; 17OHP: 17-hydroxyprogesterone; RLU: relative light units; Ve: empty vector; WT: wild type; NT: non-transfected; * <i>p</i>≤0.05.</p