mamld1 mutant c.1503_1504dupcagcag  (GenScript corporation)

Figure Legend Snippet: A. Schemes of the 3 MAMLD1 human transcripts are shown ( http://www.ncbi.nlm.nih.gov/ ; http://www.ensembl.org ). B. Scheme showing all reported MAMLD1 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 . 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.

Techniques Used: Expressing, Control, Amplification

Figure Legend Snippet: Clinical, biochemical and genetic characteristics of the patients harboring mutations and polymorphisms in the MAMLD1 gene.

Techniques Used: Mutagenesis

Figure Legend Snippet: Study of polymorphisms in the MAMLD1 gene.

Techniques Used: Sequencing

Figure Legend Snippet: HEK293 cells were transiently transfected with wild-type (WT) and mutant MAMLD1 expression vectors and with a Hes3 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 . Similar transactivation activity on the Hes3 promoter was found for all constructs. B. Hes3 transactivation by WT and the 11 MAMLD1 mutants was assessed. Only the L210X MAMLD1 mutant showed an impaired activity on the Hes3 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 ; * p ≤0.05.

Techniques Used: Transfection, Mutagenesis, Expressing, Luciferase, Construct, Activity Assay, Comparison, Plasmid Preparation

Figure Legend Snippet: HEK293 cells or NCI-H295R cells were transiently transfected with MAMLD1 WT and mutant expression vectors. For promoter activation studies, the (-3.7kb) CYP17A1 promoter luciferase reporter construct was co-transfected. A. CYP17A1 promoter activation by MAMLD1 was assessed by the Promega Dual luciferase assay in HEK293 cells. Only for mutant MAMLD1 L210X and L724V an impaired CYP17A1 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 [ 14 C]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; * p ≤0.05.

Techniques Used: Transfection, Mutagenesis, Expressing, Activation Assay, Luciferase, Construct, Activity Assay, Labeling, Thin Layer Chromatography, Plasmid Preparation

Figure Legend Snippet: Mouse testis Leydig MA-10 cells were transiently transfected with Myc-tagged expression vectors for WT or mutant MAMLD1. Cells were lysed and Western blot (WB) was performed using anti-Myc antibody. B-actin was the control. A representative WB is shown. Two independent experiments were performed showing no significant variation in protein expression for wild-type (WT) or mutant MAMLD1. L210X gave a shorter protein. Ve: empty vector; WT: wild-type; WT(a): original WT construct; ΔE5(a): original construct lacking exon 5 .

Techniques Used: Transfection, Expressing, Mutagenesis, Western Blot, Control, Plasmid Preparation, Construct

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Figure Legend Snippet: Summary of reported patients harboring sequence variations and deletions affecting MAMLD1 gene a .}

Techniques Used: Sequencing, Mutagenesis, Functional Assay, Activity Assay, Expressing, Control, Isolation, Biomarker Discovery