MUTATIONAL ANALYSIS OF StAR GENE IN ADRENAL TUMORS
Antonio STIGLIANO1, Stefania CAIOLA2, Ester SINISCALCHI1, Enrico PAPINI3, Anna CRESCENZI3, Salvatore MONTI1, Giorgio ARNALDI4, Franco MANTERO5, Francesco SCIARRA1 and Vincenzo TOSCANO1*
1II Endocrinologia, Dipartimento di Fisiopatologia Medica Università La Sapienza, Rome, Italy
2Istituto Superiore di Sanità, Rome, Italy
3 Ospedale Regina Apostolorum, Albano Laziale, Italy
4Clinica di Endocrinologia Università di Ancona, Ancona, Italy
5 Clinica di Endocrinologia Università di Padova, Padova, Italy
Adrenal adenomas and carcinomas are mostly monoclonal, suggesting that a genetic alteration in a progenitor cell may contribute to their development. However, the molecular pathogenesis of these tumors still remains unclear. It has been already excluded that activating mutations of the ACTH recep- tor or of G protein stimulator alpha sub-units, affecting cAMP pathway, is involved in the tumorigenesis. Therefore, this work has been focused on post-transductional (ACTH) signal alter- ations and in particular on the mutational analysis of the Steroid Acute Regulatory protein (StAR) gene to verify whether so- matic mutations or genomic polymorphisms of this gene may be correlated with adrenal tumorigenesis. Tissue DNA was extracted from 40 functional and non-functional adrenocortical tumors that were removed from patients aged between 17 and 72 years (mean 43 + 4). Blood DNA was obtained from 24 patients (aged between 26 and 70 years) affected by adrenal tumors and from 100 healthy subjects without radiological and clinical evidence of adrenal masses, aged between 25-35 years (90 Caucasians and 10 Africans). The DNA was used as the template for the amplification of the StAR gene using the poly- merase chain reaction. The amplified DNA of each exon of the StAR gene was purified and sequenced in automatic sequencia- tor. With the exception of exon 5 showing in codon 203 an homozygous missense mutation, the sequence of the other exons of the StAR gene resulted normal in all tumors studied. The same homozygous mutation (Asp203Ala) was observed in the sequence of exon 5 performed on genomic DNA of the 24 affected patients and in the control subjects. The homozigous- ity of the mutation observed in all patients (either in tissue or blood samples) and in control subjects, independently of their ethnic origin, led us to suggest that the Asp203Ala cannot be considered as mutation or as polymorphism, but that it must be considered as a mistake in the sequence entered in the Gen- bank, which needs to be modified accordingly. These data, and those up to now reported in the literature, allow us to suggest that mutations of the gene coding for the protein involved in the initial step of the steroidogenesis could not be considered as a possible cause for the development of adrenal tumors. @ 2002 Wiley-Liss, Inc.
Key words: human mutation; StAR gene; adrenal neoplasia
Seventy to ninety percent of adrenal tumors are benign. When these tumors do not hypersecrete hormones, their discovery occurs by chance during radiological examinations performed for other clinical problems.1,2 A small percentage of adrenal tumors are malignant and only very few are diagnosed according to their hormonal hypersecre- tion, leading to their characteristic clinical manifestations.
Adrenal adenomas and carcinomas are mostly monoclonal,3 sug- gesting that a genetic alteration in a progenitor cell may contribute to tumorigenesis. However, the molecular pathogenesis of these tumors remains unclear.4 It has been already excluded that activating muta- tions of the ACTH receptors4 or of G protein stimulator alpha sub- units are involved in the tumorigenesis due to an impaired cAMP pathway.5 Therefore our interest has been focused on the possible alterations localized in the post-trasductional signals and in particular on mutational analysis of the Steroid Acute Regulatory protein (StAR) gene, which represents a rate limiting step in the steroidogenesis process, to verify if somatic mutations or genomic polymorphism of this gene may be correlated with adrenal tumorigenesis. The rational basis of our study on the mutational analysis of this gene is based on
the observation that an intrinsic alteration of steroidogenesis is de- scribed in most of the adrenal tumors,6 even in the absence of clinical symptoms. The observation that patients affected by 21-OH lase deficiency develop adrenal masses, more often described in homozi- gous compared to heterozigous,7-12 strongly supports the rationale of our study. Moreover recent studies reported the observation that subjects with Congenital Lipoid Adrenal Hyperplasia (CLAH) may develop testicular neoplasias and ovarian cysts.13,14 This is the reasons that in these patients the pediatrician’s therapeutic approach is mainly preventive, with a precise indication toward adrenalectomy.15 The StAR mutations that have been described until now are all correlated with Congenital Lipoid Adrenal Hyperplasia (CLAH) and are repre- sented by transitions and/or transversions and mostly localized in exon 5.16-20 Moreover a genetic polymorphism (Asp203Ala) in exon 5 that is not correlated with any clinical manifestation has been already described.21 In this article, we report the results of the muta- tional analysis of the StAR gene performed on 40 adrenal tumors, showing different histological types and the results of the study of the genomic polymorphism performed in the patients (when genomic DNA was available) and in 100 control subjects of various ethnic origins.
MATERIAL AND METHODS
Patients and tumor specimens
Tumor tissue was obtained at adrenalectomy. Tumors were frozen in liquid nitrogen immediately after surgical removal and stored at -80°℃ until analyzed. Diagnosis was established by clinical and hystological criteria. Forty adrenocortical tumors were studied: 3 non-functional adenomas, 2 non-functional carcinomas, 2 aldosterone-producing adenomas, 14 cortisol-producing adeno- mas, 13 cortisol-producing carcinomas, 4 androgen-producing car- cinomas and 2 macronodular cortisol-producing hyperplasias. The tumors were obtained from patients aged between 17 and 72 years (mean 43 ± 4); 94% of these were females and most affected by functional neoplasia (Table I). This casuistry reflects the incidence of adrenal neoplastic disease for age and gender.22-25
DNA extraction
The DNA from the tumors was extracted by using the Qiagen tissue kit (Qiagen, Hildesheim, Germany). Blood DNA obtained from 24 of the patients affected by adrenal tumors (aged between 26 and 70 years) was extracted using the Qiagen blood kit (Qiagen,
Grant sponsor: Il Endocrinologia, Dipartimento di Fisiopatologia Med- ica Università di Roman “La Sapienza;” Grant sponsor: Istituto Superiore di Santià.
*Correspondence to: II Endocrinologia, Dip Fisiopatologia Medica, Uni- versità La Sapienza, Rome, Italy. Fax: +39-06-445-5345 or +39-06- 49053. E-mail: i.s.g.s.h@agora.stm.it or vincenzo.toscano@uniroma1.it
| Tumor number | Age | Sex | Maximum diameter (mm) | Clinical and pathological features |
|---|---|---|---|---|
| 1 | 55 | F | 30 | Non-functional adenoma |
| 2 | 52 | F | 40 | Non-functional adenoma |
| 3 | 41 | F | 40 | Non-functional adenoma |
| 4 | 23 | F | 90 | Adrenocortical carcinoma (non functional) |
| 5 | 34 | M | 35 | Adrenocortical carcinoma (non functional) |
| 6 | 57 | F | 20 | Aldosterone producing adenoma |
| 7 | 39 | M | 30 | Aldosterone producing adenoma |
| 8 | 48 | F | 45 | Cortisol producing adenoma |
| 9 | 29 | F | 40 | Cortisol producing adenoma |
| 10 | 50 | M | 30 | Cortisol producing adenoma |
| 11 | 65 | F | 40 | Cortisol producing adenoma |
| 12 | 26 | F | 30 | Cortisol producing adenoma |
| 13 | 55 | F | 40 | Cortisol producing adenoma |
| 14 | 46 | F | 30 | Cortisol producing adenoma |
| 15 | 63 | F | 45 | Cortisol producing adenoma |
| 16 | 25 | F | 30 | Cortisol producing adenoma |
| 17 | 40 | F | 30 | Cortisol producing adenoma |
| 18 | 39 | F | 30 | Cortisol producing adenoma |
| 19 | 46 | F | 30 | Cortisol producing adenoma |
| 20 | 34 | F | 30 | Cortisol producing adenoma |
| 21 | 57 | F | 50 | Cortisol producing adenoma |
| 22 | 33 | M | 30 | Adrenocortical carcinoma (cortisol producing) |
| 23 | 70 | F | 45 | Adrenocortical carcinoma (cortisol producing) |
| 24 | 69 | F | 90 | Adrenocortical carcinoma (cortisol producing) |
| 25 | 42 | F | 120 | Adrenocortical carcinoma (cortisol producing) |
| 26 | 54 | M | 150 | Adrenocortical carcinoma (cortisol producing) |
| 27 | 43 | M | 110 | Adrenocortical carcinoma (cortisol producing) |
| 28 | 50 | F | 160 | Adrenocortical carcinoma (cortisol producing) |
| 29 | 59 | F | 110 | Adrenocortical carcinoma (cortisol producing) |
| 30 | 26 | F | 150 | Adrenocortical carcinoma (cortisol producing) |
| 31 | 25 | F | 80 | Adrenocortical carcinoma (cortisol producing) |
| 32 | 56 | F | 160 | Adrenocortical carcinoma (cortisol producing) |
| 33 | 18 | F | 50 | Adrenocortical carcinoma (cortisol producing) |
| 34 | 50 | F | 40 | Adrenocortical carcinoma (cortisol producing) |
| 35 | 50 | F | 70 | Adrenocortical carcinoma (androgen producing) |
| 36 | 17 | F | 60 | Adrenocortical carcinoma (androgen producing) |
| 37 | 27 | F | 100 | Adrenocortical carcinoma (androgen producing) |
| 38 | 27 | F | 35 | Adrenocortical carcinoma (androgen producing) |
| 39 | 54 | F | 48 | Macronodular hyperplasia (cortisol producing) |
| 40 | 50 | F | 27 | Macronodular hyperplasia (cortisol producing) |
| Exon I | ||||||||
| Sense | 5' - AGG | CTG | CAG | CTG | CGG | GAC | TCA | GAG G-3' |
| Antisense | 5' - TCG | CCT | CCT | TCC | CGC | AGC | GCT | CAC- 3' |
| Exon II | ||||||||
| Sense | 5' - AAC | AAG | GGT | TAT | TCC | CTT | CTG | CAG- 3' |
| Antisense | 5' - GAG | CCC | AGA | AGC | CTC | AGC | ACT | TAC- 3' |
| Exon III | ||||||||
| Sense | 5' - GTC | TCT | CCT | CGG | CTG | TGT | ATC | CAG- 3' |
| Antisense | 5' - CAC | AGG | CTT | CTC | CCC | GAC | ACT | TAC - 3' |
| Exon IV | ||||||||
| Sense | 5' - TCT | GGG | GGC | TCC | TTT | CTC | TGA | CAG- 3' |
| Antisense | 5' - CAC | CCG | CAC | CTG | GAC | TTT | GGT | CAC- 3' |
| Exon V | ||||||||
| Sense | 5'- TTC | TGG | TTC | CCC | ATG | GCC | TGG | TAG - 3' |
| Antisense | 5' - GTT | TGG | AGC | CTG | CTG | CCC | GTA | TTA C-3' |
| Exon VI | ||||||||
| Sense | 5' - GAC | TTG | ACT | TGC | TCC | ATT | TGC | CAG- 3' |
| Antisense | 5' - AGG | TCC | CCC | TCC | CAT | GCC | CTT | CAC- 3' |
| Exon VII | ||||||||
| Sense | 5' - AAA | TTC | TCC | TAC | CTC | CTA | CTG | CAG- 3' |
| Antisense | 5' - CCA | GTG | CAG | CTG | GGC | ACA | GTT | GG-3' |
Hildesheim, Germany). Blood DNA obtained from 100 healthy subjects, aged between 25 and 35 years, were used as controls. These subjects, 90 Caucasians and 10 Africans, according to data from ethnic studies, which demonstrated the prevalence of adrenal neoplastic disease in white subjects compared to blacks,26-28 had no radiological and clinical evidence of adrenal masses. The study was approved by local Ethical Committee.
PCR and sequencing analysis
The DNA was used as the template for the amplification of the intronless StAR gene by the polymerase chain reaction (PCR) (PCR System 9700 Perkin Elmer). The pair of flanking primers used (Life Technologies, GIBCO-BRL, Gaithersburg, MD) is re- ported in Table I. The PCR protocol comprised 5 min melting of
the strands at 94℃, then 30 sec of denaturation at 94℃, 30 sec of annealing at 60℃ and 30 sec of extension at 72℃, for 35 cycles. The final extension was performed for 10 min at 72℃, the reaction taking place in 20 ul volume containing Taq DNA polymerase (Promega Corp, Milan, Italy) with MgCl2 at 2.5 mM. The expected size bands on agarose gel electrophoresis were as follows: 203 bp for exon 1, 114 bp for exon 2, 126 bp for exon 3, 159 bp for exon 4, 185 bp for exon 5, 94 bp for exon 6, and 113 bp for exon 7. The amplified DNA of each exon of StAR gene was purified by filtra- tion through a Amicon membrane (Micropure-0.22 Separator, Amicon, Inc., Beverly, MA) and sequenced in automated DNA sequencer (Perkin-Elmer Cetus) with Dye-Deoxy® Rhodamine Terminator Cycle Sequencing kit (PE/ABI), using unlabeled PCR primers as sequencing primers, according to the manufacturer’s protocol. The base calling was determined automatically by ABI PRISM Sequencing Analysis 3.0 software.
RESULTS
Each exon of the StAR gene was successfully amplified by PCR and shown to have the expected size bands on agarose gel elec- trophoresis. With the exception of exon 5 that showed in codon 203 an homozygous missense mutation with the substitution of Asp with Ala respect to the wild-type sequence (GenBank acces- sion NM_000349 (gi:4507250) (Fig. 1), the automatic sequence of other exons of the StAR gene revealed a normal sequence in all tumors studied.
The same homozygous mutation (Asp203Ala) was observed in the sequence of exon 5 performed on genomic DNA of the 24 patients whose DNA was available.
To clarify if this mutation may be correlated with a silent polymorphism presented by patients developing adrenal tumors, exon 5 of the StAR gene from genomic DNA of control subjects was sequenced and in 100% of the cases, the same homozygous mutation (Asp203Ala) was observed.
DISCUSSION
This paper reports the results of the StAR gene mutational analysis performed on 40 adrenal tumors with different histolog- ical characteristics and in a group of 100 healthy subjects. In all cases an homozigous missense apparent mutation Asp203Ala (exon 5) of the StAR gene was shown. To our knowledge this is the first report in the literature that shows the results of mutational analysis of the StAR gene in adrenal tumors. Previous papers have demonstrated comparable expression of the StAR mRNA in normal and neoplastic adrenal tissues.29-32 These results, however, do not permit excluding that StAR gene mutation may be involved in adrenal tumorigenesis.
The StAR gene mutations, until now described, are mostly localized in exon 5 and all identified in patients affected by CLAH.16,33 Otherwise the possibility that an altered steroidogen- esis, even in the cases not associated with clinical hormonal abnormalities, may be involved in adrenal tumorigenesis has been
Esone five of StAR gene
V
GenBank gi:4507250
203
Gly Met Asp Asp Phe GGC ATG GAC ACA GAC TTC
Thr
Mutation
1
Gly
203 Ala
Met
Thr
Asp
Phe
GGCATGGCCACAGACTTC
already advanced.34-36 However, up to now no clear evidence for enzyme gene mutation along adrenal steroid pathways has been shown, even for the 21 hydroxylase gene37 whose mutation was strongly suggested by the high response of 17-hydroxyprogester- one after ACTH stimulation shown in 30-70% of the patients affected by adrenal incidentalomas.38,39
The observation that the StAR gene apparent mutation Asp203Ala was found in all subjects studied, both in controls and in those affected by adrenal tumor, led us to exclude that this apparent sequence change may be involved in adrenal tumorigen- esis.36 It is difficult to accept this apparent mutation as polymor- physm because if this is the case, it would be expected to find in some people the same sequence comparable to that entered in the Genbank, or at least some heterozygous subject. A most recent entry in the Genbank of 19 March 1999 (accession NM_ 000349; gi:4507250) for StAR gene sequence reported the GAC at the codon 203. The possibility that this apparent mutation may be a polymorphysm that predisposes to the development of adrenal tumors could be postulated. Considering however that the preva- lence of the adrenal tumors (including incidentalomas) cannot be
expected to be higher than 10%6 in the general population, we should expect the mutation in the same percentage of the cases in our controls, at least in homozygousity. Therefore the observation of the homozigousity of the apparent mutation in the 100% of patients and controls independently of their ethnic origin lead us to suggest that Asp203Ala has to be considered only as a mistake of the sequence entered in the Genbank, which could be modified accordingly.
On the basis of this body of evidence, we suggest that mutations of the gene coding for the protein involved in the initial step of the steroidogenesis cannot be considered involved in the development of functional or non-functional adrenal tumors, as well as exclud- ing the involvement of gene mutations in the mechanisms of the ACTH signal induction.
ACKNOWLEDGEMENT
This work was performed within a collaboration between Il Endocrinologia, Dipartimento di Fisiopatologia Medica Università di Roman “La Sapienza” and Istituto Superiore di Santià.
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