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Original Studies |
Institute of Medical Technology and University Hospital, University of Tampere (P.P., H.H., K.J.E.K.), Tampere 33101, Finland; Institute of General and Molecular Pathology (K.R., R.U.), University of Tartu, Tartu 51014, Estonia; and Departments of Obstetrics and Gynecology (I.C.) and Medicine (A.P.W.), University of Sheffield, Sheffield S10 2RX, United Kingdom
Address all correspondence and requests for reprints to: Dr. Koit Reimand, Department of Immunology, University of Tartu, Ravila 19, Tartu 51014, Estonia. E-mail: reimand{at}ut.ee
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Abstract |
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-hydroxylase (P450c17), and side-chain
cleavage enzyme (P450scc) in POF patients and patient groups with
autoimmune polyendocrinopathy syndromes type 1 and 2 (APS1 and -2),
isolated Addison’s disease, type 1 diabetes, and healthy controls. The
SCA were found in 2 of 48 POF, 11 of 15 APS1, and 1 of 9 APS2, and
autoantibodies to in vitro translated 3ßHSD protein
were detected in 1 POF serum associated with Addison’s disease and 3
APS1 sera. All 3ßHSD precipitating sera were also positive for SCA.
However, no SCA or 3ßHSD autoantibodies were found in 38 Addison’s
disease, 28 type 1 diabetes, and 71 healthy control sera. In analysis
of autoantibodies to P450c17 and P450scc, antibodies to these enzymes
were not found in POF sera, but were found in 10 and 12 APS1 patient
sera, respectively, and 1 APS2 patient serum contained anti-P450c17
antibodies. Our results show that autoantibodies to 3ßHSD in POF
patients are rare and are also found in patients with APS1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The molecular targets of SCA have been described as two steroidogenic
enzymes, 17-hydroxylase (P450c17) and side-chain cleavage enzyme
(P450scc). Autoantibodies to P450c17 and P450scc as well as to
21-hydroxylase (P450c21), an enzyme expressed only in adrenal cortex
(9, 10, 11), are found in patients’ sera with Addison’s disease in two
autoimmune polyglandular syndromes (APS). The patients with type 1
syndrome (APS1) have autoantibodies to all the three enzymes (9, 12, 13), whereas patients with type 2 syndrome (APS2) and isolated
Addison’s disease have autoantibodies mostly to P450c21. The SCA found
in POF associated with Addison’s disease and the close association of
these two diseases support the idea of a shared autoimmune response in
ovarian and adrenal autoimmunity. However, the molecular nature of
autoantigen(s) in POF unassociated with Addison’s disease (idiopathic
POF) has remained unclear.
The 3ß-hydroxysteroid dehydrogenase (3ßHSD) enzyme was recently characterized as an autoantigen in POF by adrenal complementary DNA (cDNA) library screening (14). 3ßHSD is involved in the steroid metabolic pathway and is expressed in all tissues recognized by SCA (15, 16). To further study this reactivity we have analyzed series of patients with POF, APS1, APS2, isolated Addison’s disease, and type 1 diabetes and healthy controls for SCA by immunofluorescence assay and for 3ßHSD by immunoprecipitation with in vitro translated antigen. We also studied the reactivity of POF sera to two other steroid cell specific autoantigens, P450c17 and P450scc.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum samples from 48 consecutive Caucasian patients from Northern England (mean age ± SD, 36 ± 11 yr; range, 17–67 yr; 8 >40 yr old) with POF were collected during 1992–1996. POF was defined as hypergonadotropic amenorrhea, with serum LH and FSH more than 10 IU/L on at least 2 occasions and amenorrhea for at least 6 months. In 30 patients there was no apparent cause of POF or any associated disease; 7 had an iatrogenic or genetic (Turner’s syndrome) etiology, and 11 had an associated disease (Addison’s disease in 6, autoimmune thyroid disease in 5). In addition, sera from 15 Finnish patients with APS1, 9 Finnish patients with APS2, 33 Caucasians from Northern England and 5 Finnish patients with isolated Addison’s disease, and 28 Finnish patients with newly diagnosed type 1 diabetes were used. The clinical criteria of these patients have been previously published (9, 17). As a control material, serum samples from 48 Caucasians of Northern England and 23 Finnish healthy volunteers were used. Informed consent was obtained from all patients. The sera were stored frozen at -20 C.
Immunofluorescence assay
Indirect immunofluorescence assay for SCA was performed as previously described (9). Human adrenal, placenta, ovary at follicular stage, and monkey (Macaca fascicularis) testis were used as antigenic substrate. Cryostat sections (6 µm) were prepared and incubated with test sera at 1:5 to 1:100 dilutions and were visualized by fluorescent secondary antibody (fluorescein isothiocyanate-conjugated rabbit antihuman IgG, DAKO Corp., Copenhagen, Denmark).
cDNA cloning and in vitro translation
The human 3ßHSD cDNA encoding 287 C-terminal amino acids from 373 in pIBI25 vector was obtained as a gift from Dr. B. Murry, Southwestern Medical Center, University of Texas (Dallas, TX). This fragment was cleaved with restriction enzyme KpnI and ligated with the N-terminal encoding part of the human 3ßHSD cDNA fragment (18), amplified from human placental cDNA by PCR with 3ßHSD-specific primers and digested with EcoRI and KpnI restriction enzymes, yielding a full-length 3ßHSD cDNA in pGEM3 vector. Both constructs, pGEM3ßHSD (full-length cDNA) and pGEM3ßHSD-C (containing 287 amino acids from the C-terminus encoding cDNA), were sequenced with T7 primer to confirm the correct orientation and reading frame of the cDNAs. The cloning of P450c17 and P450scc has been described previously (9). The pGEM3ßHSD and pGEM3ßHSD-C as well as pJEX17 and pJEXscc were transcribed with T7 ribonucleic acid polymerase and translated into [35S]cysteine (Amersham International, Aylesbury, UK)-labeled protein with an in vitro transcription/translation kit (TNT-kit, Promega Corp., Madison, WI) according to the manufacturer’s instructions. The translation products were further purified by Sephadex G-50 column chromatography and were analyzed by SDS-PAGE and autoradiography.
Radioimmunoprecipitation assay
In each assay the translation mixture of the labeled protein (20,000–50,000 cpm) was suspended in 50 µl RIP buffer [20 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 0.1% Triton X-100, and 10 µg/ml aprotinin] and incubated with diluted serum (1:10 in RIP buffer) for 1 h at room temperature. Fifty microliters of Sepharose Fast Flow protein G (Pharmacia Biotech, Uppsala, Sweden) diluted 1:5 in RIP buffer was added and incubated for 1 h at room temperature with shaking. The immune complexes were washed four times by centrifugation and analyzed by SDS-PAGE, followed by autoradiography. Rabbit IgG-enriched antiserum against human 3ßHSD was obtained from Dr. Ian Mason (University of Edinburgh, Edinburgh, UK) and has been proved to be specific for 3ßHSD (19).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The in vitro translated and
[35S]cysteine-labeled full-length (pGEM3ßHSD)
and C-terminal (pGEM3ßHSD-C) 3ßHSD products appeared as the
expected 42- and 31-kDa bands, respectively, in SDS-PAGE and
autoradiography (Fig. 1
). As a positive
control, rabbit antiserum against human 3ßHSD clearly recognized both
35S-labeled 3ßHSD protein fragments in an
immunoprecipitation assay. During immunoprecipitation with human sera,
1 SCA-positive POF serum precipitated the full-length 3ßHSD product
(1 of 41 idiopathic POF; prevalence, 2.4%; 95% confidence interval,
0–7.2%). Another SCA-positive serum as well as the rest of the 46 POF
sera remained negative. In addition, 3 of 15 APS1 sera that were all
positive for SCA reacted with this product (prevalence, 20%; 95%
confidence interval, 0–40.2%). None of the 9 APS2, 38 isolated
Addison’s disease, 28 type 1 diabetes, or 71 healthy control sera was
able to precipitate the 3ßHSD product. When the 287-amino acid long
C-terminal part of the 3ßHSD protein (pGEM3ßHSD-C) was used in
immunoprecipitation, no positive band was found by autoradiography,
indicating that the epitope(s) needed for autoantibody binding locates
within 86 amino acids from the N-terminal part of the protein. As 10
APS1 and 1 APS2 sera were positive for anti-P450c17, and 12 APS1 sera
were positive for anti-P450scc antibodies, POF patient sera and serum
samples from other studied patient series were analyzed for the
presence of these autoantibodies. However, no antibody reactivity with
these serum samples was observed with P450c17 and P450scc
autoantigens.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The previously reported finding of autoantibodies to steroidogenic
enzyme 3ßHSD not belonging to the P450 cytochrome family in a
relatively large proportion (21%) of POF cases (14) was intriguing and
suggested a pathogenesis for idiopathic autoimmune ovarian failure that
is distinct from that seen in APS1 and Addison’s disease. Our results,
instead, indicate that the presence of autoantibodies to 3ßHSD is
rare (2%) among POF patients. In contrast, we found anti-3ßHSD
antibodies at an even higher frequency (20%) among the APS1 patients
studied. Interestingly, all four sera positive for anti-3ßHSD
antibodies precipitated the full-length protein, but not the C-terminal
polypeptide (last 287 amino acids), demonstrating an immunodominant
epitope to locate most likely in the N-terminal region of 3ßHSD that
contains a long stretch (amino acids 25–75) of the predicted
-helix. The shared epitope region in one POF and three APS1 cases
may, in turn, reflect the partially common autoimmune mechanism in POF
and APS1. Although having Addison’s disease, the 3ßHSD
autoantibody-positive POF patient could not belong to the group of APS1
or APS2 because she had no signs of candidiasis and hypoparathyroidism
or type 1 diabetes and autoimmune hypothyroidism. Furthermore, no
mutations were found in her DNA in the two most common positions (R257X
and 1094–1106del) of the APS1-causing AIRE (autoimmune regulator) gene
that together cover more than 80% of the British APS1 chromosomes
(22).
In the previous study (14), none of the POF cases positive for anti-3ßHSD antibodies had associated Addison’s disease, and only 3 of the 48 had autoimmune thyroid disease, compared to 6 and 5, respectively, in the current series. This seems unlikely to explain the difference found in 3ßHSD autoantibody frequency, as 2 of the 3 hypothyroid POF patients in the first study had 3ßHSD antibodies, and therefore the higher proportion of patients in our study with associated autoimmune disorders should, if anything, have increased the chances of finding 3ßHSD antibodies. We, in addition, included 7 POF patients with iatrogenic or genetic etiology, because we and others have previously found ovarian antibodies by enzyme-linked immunosorbent assay in such patients (20, 23), presumably arising secondary to ovarian damage, and we wondered whether these ovarian antibodies were directed against 3ßHSD. We were not able to find 3ßHSD autoantibodies in our patients with type 1 diabetes, although a large number (23%) of such patients was reported in the first study (14).
As the P450c17 and P450scc enzymes are known to be targets of SCA, we studied the reactivity of POF sera to these two enzymes. We have shown previously that the SCA seen in APS1 is mainly, if not entirely, due to antibody reactivity toward these two steroidogenic enzymes (9, 17). All three APS1 patients with 3ßHSD autoantibodies also had autoantibodies to P450c17 and P450scc, but none of the POF sera precipitated these two autoantigens. The lack of P450c17 and P450scc autoantibodies in POF and APS2 and the evidence of shared immune response in ovarian and adrenal autoimmunity (24, 25) suggest the presence of other autoantigens in steroid cells. In our patient series another SCA-positive, but 3ßHSD autoantibody-negative, POF serum had no reactivity to P450scc and P450c17, suggesting the existence of still uncharacterized SCA targets.
In conclusion, our findings indicate that 3ßHSD autoantibodies are rare in patients with POF; therefore, their significance as a diagnostic marker remains low. However, due to the discordance of the results, the prevalence and significance of 3ßHSD antibodies remain unclear and need further examination in view of their role in the diagnosis and evaluation of autoimmune POF.
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Footnotes |
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Received June 28, 1999.
Revised December 29, 1999.
Accepted February 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase. Lancet. 339:770–773.[CrossRef][Medline]
5-4-isomerase
in human adrenals and gonads. Mol Endocrinol. 5:1147–1157.[Abstract]
5–4isomerase from placenta:
expression in nonsteroidogenic cells of a protein that catalyzes the
dehydrogenation/isomerization of C21 and C19 steroids. Endocrinology. 126:2493–2498.[Abstract]
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