Inhibin  -subunit (INHA) gene and locus changes in paediatric adrenocortical tumours from TP53 R337H mutation heterozygote carriers

Abstract

The R337H TP53 mutation is a low-penetrance molecular defect that predisposes to adrenocortical tumour (ACT) formation in Brazilian and possibly other populations. Additional genetic defects may be responsible for the variable expression of ACTs in these cases. The inhibin asubunit gene (INHA) on 2q33-qter has been implicated in mouse adrenocortical tumourigenesis. We studied 46 pediatric patients with ACTs from Brazil for INHA genetic alterations; 39 of these patients were heterozygous carriers of the R337H TP53 mutation. We first mapped the INHA gene by radiation hybrid analysis and determined 10 linked microsatellite markers in an area flanked by D2S1371 and D2S206 on 2q33-qter. These markers were then used for loss of heterozygozity (LOH) studies in nine paired germline and tumour DNA samples. Mapping placed the INHA gene in close proximity to D2S2848 (SHGC11864) with a log of odds (LOD) score of 5.84. LOH for at least one marker in the region was identified in 8/9 tumours (89%). Six patients were heterozygous for three INHA mutations: one in exon 1, 127C.G, and two in exon 2, 3998G.A and 4088G.A, all leading to amino acid substitutions (P43A, G227R, and A257T, respectively). A257T is located in a conserved INHA region, highly homologous to transforming growth factor-b; both G227R and A257T change polarity, and, in addition, G227R changes the pH. We conclude that these sequence alterations and the detected 2q allelic changes suggest that INHA may be one of the contributing factors needed for ACT formation in pediatric patient carriers of the R337H TP53 mutation.A drenocortical tumours (ACTs) are rare, with an estimated incidence in the US of 0.3 per million per year. 1 In southern Brazil, the incidence is 10-15 times greater, despite no evidence of occupational, endemic, or transmissable diseases that predispose to ACTs. Recently, a germline mutation of the TP53 gene (R337H) was described in 35 of 36 Brazilian children with ACTs. 2 The same mutation was then identified by Latronico and co-workers in adult, as well as additional paediatric, Brazilian patients with ACTs. 3 Furthermore, low-penetrance mutations of the TP53 gene were recently identified in other populations. 4 It appears likely that the tumourigenic effect of the low-penetrance R337H mutation is related to pH-induced conformational changes in the p53 protein 5 6 and/or other factors.In the case of ACTs in the Brazilian population, it has long been hypothesised that additional germline or somatic mutations of other genes with an established tumourigenic role may act in synergy, or may even be required for adrenocortical tumourigenesis.

Gene Expression Profiling of Childhood Adrenocortical Tumors

“…Deletion of inhibin-a by gene targeting in gonadectomized mice causes fully penetrant ACT by 4 to 5 weeks of age. Consistent with the mouse model, mutation of inhibin-a with loss of heterozygosity at chromosome 2q33 was commonly observed in human pediatric ACT (13). Comparative genomic hybridization analysis of pediatric ACT also showed recurrent chromosomal alterations, such as the amplification of chromosome 9q34 (14).…”

“…The t distribution was used to compute 95% confidence intervals for the difference of means of log signals; these intervals were transformed 12 www.stjude.org. 13 http://www.affymetrix.com/support/technical/manual/expression_manual.affx. 14 www.splus.com.…”

“…High-quality RNA, confirmed by UV spectrophotometry and an Agilent 2100 Bioanalyzer, was processed according to the Affymetrix one-cycle labeling protocol. 13 In brief, 5 to 10 Ag total RNA was annealed to an oligo-dT(24)-T7 primer to initiate cDNA synthesis. Purified double-stranded cDNA was used as a template to synthesize biotin-labeled cRNA using T7 RNA polymerase.…”

“…Animal studies implicate inhibin-a, a glycoprotein with homology to transforming growth factor-h, as a suppressor of ACT development (13). Deletion of inhibin-a by gene targeting in gonadectomized mice causes fully penetrant ACT by 4 to 5 weeks of age.…”

Uncovering p53 Mutations and Abnormal Gene Expression in Pediatric Adrenocortical Cancer

“…Overall structural functions of the p53 domains include maintaining stability ; shown in red), tetramerization domain (amino acids 326-355; shown in green), and the basic C-terminal domain (amino acids 363-393; shown in yellow). The protein is also comprised of five structurally conserved domains: I (amino acids [13][14][15][16][17][18][19][20][21][22][23], II (amino acids , III (amino acids [171][172][173][174][175][176][177][178][179][180][181], IV (amino acids 234-258), and V (amino acids 270-286). …”