| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Department of Pediatrics, University of Illinois College of Medicine (L.Z., Y.N., Y.T.C., S.P.), Chicago, Illinois 60612; Department of Pediatrics, Baylor College of Medicine (K.C.C.), Houston, Texas 77030; Department of Pediatrics, Winthrop University Hospital, State University of New York (M.C.M.), Long Island, New York 11501; and Department of Reproductive and Developmental Sciences (Clinical Biochemistry), University of Edinburgh (J.I.M.), EH3 9YW Edinburgh, Scotland
Address all correspondence and requests for reprints to: Dr. Songya Pang, Department of Pediatrics (M/C 856), University of Illinois, 840 South Wood Street, Chicago, Illinois 60612.
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
5-3ß-hydroxysteroids such
as pregnenolone (5-P), 17-hydroxypregnenolone
(5-17P), and dehydroepiandrosterone (DHEA)
to the corresponding 4-3ß-ketosteroids progesterone
(P), 17-hydroxyprogesterone (17-OHP), and androstenedione
(4-A), respectively, in intra- and extraadrenal and
gonadal tissues. Thus, 3ßHSD activity is essential for biosynthesis
of aldosterone (Aldo) and cortisol (F) in the adrenal cortex and of
testosterone (T) and estradiol (E2) in the gonads. In
humans, the type I and II 3ßHSD genes encode for extra- and
intraadrenal/gonadal 3ßHSD, respectively; hence, type II 3ßHSD
enzyme is essential for adrenal/gonadal steroid biosynthesis (1, 2).
3ßHSD deficiency congenital adrenal hyperplasia (CAH) results from
either absent or decreased type II 3ßHSD activity due to a
deleterious mutation in the type II 3ßHSD gene whose locus is on
chromosome 1 (3) and is transmitted by a recessive trait (4, 5, 6, 7, 8). Severe adrenal 3ßHSD deficiency in the affected males and females causes either salt-wasting or nonsalt-wasting disorder, and fetal T deficiency due to testicular 3ßHSD deficiency results in genital ambiguity in males, whereas metabolites of excess fetal adrenal DHEA secreted in genetic females may cause mild virilization of external genitalia (9, 10). Studies of type II 3ßHSD genotype and characterization of mutant type II 3ßHSD genes from 3ßHSD deficiency CAH patients have helped to increase understanding of the function and structure relationship of the human type II 3ßHSD gene (3, 4, 5, 6, 7, 8). In this report we characterized two additional novel homozygous point mutations in the type II 3ßHSD gene resulting in nonsalt-wasting and salt-wasting 3ßHSD deficiency disorders and male pseudohermaphroditism and elaborated a possible mechanism for altered 3ßHSD activity due to the novel point mutations.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The patient of family 1 (case 1) is a Pakistanian first child
born at term and offspring of consanguineous marriage in whom the
patient’s parents as well as great grandfathers were first cousins
(Fig. 1
). At birth the infant had a more
male-appearing, but ambiguous genitalia with hyperpigmented and bifid
scrotum, third degree hypospadias, 3-cm phallus, and palpable gonads
bilaterally in the scrotum. Laboratory evaluation of the infant on day
7 revealed 46,XY karyotype, normal serum electrolytes, blood urea
nitrogen (BUN), and glucose levels; age-appropriate levels of random
serum LH, FSH, DHEA, 4-A, 17-OHP,
and Aldo; high normal F level compared to the reported normal values
(11, 12, 13); as well as age-appropriate PRA level (Table 1). However, plasma ACTH and serum
5-17P levels were inappropriately elevated,
whereas the serum T level was low for that of a young male infant
(Table 1) compared to reported normal values (11, 13, 14). On day 15,
hCG treatment (1000 IU 1 mol/L daily for 4 days) revealed adequate
rises in serum 4-A and T levels. However, the
serum DHEA level was inappropriately elevated (Table 1)
compared to reported normal values (15). On day 21, the infant’s serum
electrolytes revealed low normal serum sodium level and upper normal
serum potassium level, with normal BUN and glucose levels (Table 1).
Clinically, the infant had no signs or symptoms of adrenal
insufficiency. Basal and ACTH-stimulated
5-17-P levels were unequivocally elevated,
whereas the normal basal 17-OHP level rose moderately in response to
ACTH stimulation compared to reported control values (14). The basal
DHEA level was slightly elevated for age, but
ACTH-stimulated DHEA and 4-A
levels (Table 1) were in the normal infant control level (11, 13, 14).
After these evaluations, the infant was initially treated with
hydrocortisone (1 mg, orally, three times daily) and Florinef
acetate (0.05 mg, orally, three times daily; Apothecon, Bristol-Myers
Squibb Co., Princeton, NJ) until age 2 yr. The child never had clinical
salt loss, maintaining normal electrolytes and growth. Subsequently at
age 2 yr, both hydrocortisone and Florinef acetate were discontinued
for 6 weeks under close supervision to reassess the phenotype of
3ßHSD deficiency CAH in the child. The child remained asymptomatic,
with no clinical or biochemical evidence of adrenal insufficiency on no
hormonal replacement therapy. Serum electrolytes and PRA remained
normal. Basal and ACTH-stimulated hormonal findings at this time were
similar to the levels in infancy. Thus, nonsalt-wasting 3ßHSD
deficiency CAH was established in the patient (case 1, Table 1). The
family history is pertinent for consanguineous marriage (Fig. 1),
although no other family members were reported to have genital
ambiguity. Blood samples were obtained from the patient and the parents
of the patient for DNA analysis.
|
|
). The
karyotype was 46,XY. The infant was initially thought to have partial
androgen insensitivity syndrome and was given a female sex assignment.
At 3 weeks of age, however, the infant was admitted to a hospital due
to symptoms of lethargy and shock. Serum electrolytes at this time
revealed hyponatremia, hyperkalemia, elevated BUN, and low glucose
levels (Table 1) that necessitated fluid, electrolyte, and hormonal
replacement therapy. The serum DHEA level at the time was
highly elevated (Table 1) compared to reported normal values (11), and
the infant was diagnosed to have male pseudohermaphoditism due to
severe salt-wasting 3ßHSD deficiency CAH. At age 2 yr, serum
DHEA and PRA levels were elevated compared to the reported
age-matched normal values (12, 13), after discontinuation of the
patient’s maintenance hydrocortisone and Florinef acetate therapy for
2 days (Table 1). At age 6 yr, the child had phallic corporectomy and
bilateral gonadectomy. At age 17 yr, the patient’s random hormonal
levels were reevaluated after discontinuation of maintenance prednisone
therapy (5 mg, orally, twice daily) for 1 week while continuing
Florinef (0.1 mg, orally, daily) and oral contraceptives. At this time,
plasma ACTH and serum 5-17P and
DHEA levels were exceedingly high compared to the reported
normal male or female values (16), whereas serum 17-OHP and
4-A levels were slightly elevated for
gonadectomized subjects (Table 1), presumably due to peripheral
conversion of elevated 5-17P and
DHEA concentrations, respectively. The serum F level was
low, and the serum Aldo level was detectable, although the specificity
of the Aldo assay was uncertain, and PRA was at a high normal level
(Table 1). Serum LH and FSH levels were appropriately elevated (Table 1). At age 21 yr, basal and ACTH-stimulated hormonal profiles after
discontinuation of genocorticoid therapy for 7 days revealed similar
hormonal findings as the levels at 17 yr of age. These data confirmed
severe salt-wasting 3ßHSD deficiency CAH in this genetic male,
resulting in severely ambiguous genitalia more compatible with a
feminized phenotype. The unrelated parents of the patient were healthy
and born in Taiwan. There was no family history of ambiguous genitalia
or sudden death. A blood sample was obtained for DNA analysis from the
family after obtaining informed consent. The study was approved by the
institutional review board at the University of Illinois at
Chicago. Laboratory methods
PCR and sequencing of the type II 3ßHSD gene. The genomic DNA of all subjects were extracted from peripheral white blood cells. PCR was performed using primers previously reported (8, 14) to amplify the regions of a putative promoter from -1053 base; exons I, II, III, and IV; and exon and intron boundaries of the type II 3ßHSD gene as reported previously (8, 14). The PCR products were verified on an agarose gel for DNA size and purified with a Gene Clean kit (Biology Life Science Institute, La Jolla, CA). Direct sequencing of the PCR products was performed with a fmol DNA sequencing system (Promega Corp., Madison, WI) using primers for PCR and nine additional primers for sequencing as reported previously (8, 14). Thermocycling for sequencing was performed at 95 C for 0.5 min, 42 C for 0.5 min, and 72 C for 1 min, for a total of 30 cycles as previously reported (8, 14).
Site-directed mutagenesis. Full-length wild-type (WT) type II 3ßHSD complementary DNA (cDNA) cloned in a pcDNA-1/neo vector was cut by XbaI and XhoI and ligated in the pcDNA-3.1 vector (Stratagene, La Jolla, CA). The technique of site-directed mutagenesis by Quickchange site-directed mutagenesis kit (Stratagene) was employed to generate the L6F, T259M, and T259R mutations using an oligonucleotide primer of 5'-pGGGCTGGAGCTGCTTTGTGACAGGAGCAGG-3', 5'-pCATCTCAGATGACATGC-CTCACCAAAGC-3', and 5'-pCATCTCAGATGACAGGCCTCACCAAAGC-3', respectively, which contained a mutation on the target sequence site of the reported type II 3ßHSD gene structure (17). A double stranded target plasmid was heat denatured, and oligonucleotide primers containing the desired mutation were annealed to the target site, each complementary to opposite strands of the vector, extended during temperature cycling by means of Pfu DNA polymerase, and incorporated the mutagenic primer, resulting in nicked circular strands. The DNA products were treated with DpnI to digest the nonmutated parental DNA template. Mutated plasmid containing nicked circular strands was transfected into XL I blue supercompetent cells, which repaired the nicks in the mutated plasmid. Midi-preparation was performed to prepare the plasmid. The plasmids containing L6F, T259M, and T259R mutations were confirmed by double strand DNA sequence analysis using a thermocycle sequencing method.
Expression of WT type II 3ßHSD and mutant L6F, T259M, and T259R cDNAs. A pcDNA-3.1 3ßHSD expression vector containing the entire type II 3ßHSD-coding region of WT or mutant cDNA was purified for transfection by QIAGEN plasmid Midi kit (QIAGEN). COS-1 or COS-7 cells were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) plus 10% FBS and 1% antibacterial antibiotic solution (Sigma, St. Louis, MO) and transfected with 1 µg plasmid DNAs using Lipofectamine (Life Technologies, Inc.). Transfection efficiency was tested by a ß-galactosidase activity assay using a ß-galactosidase/vector (cytomegalovirus-ß) plasmid in three separate experiments. Type II 3ßHSD messenger ribonucleic acid (mRNA) from the harvested cells transfected for 48 h with WT and mutant type II 3ßHSD cDNAs was determined by Northern blot analysis as previously described (18). The amount of translated WT and mutant type II 3ßHSD proteins was assessed by Western immunoblot analysis by harvesting the cells after enzyme activity study. Cells were first washed and collected in a PBS, then lysed by three freeze-thaw cycles. The cell debris was pelleted, and the total protein concentration of the supernatant was determined by bicinchoninic acid protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Ten to 25 µg total protein were resolved on a 12% SDS-PAGE gel and electroblotted to an Immobilon-P membrane (Millipore Corp., Bedford, MA). The blot was probed with a rabbit polyclonal anti-3ßHSD primary antibody, followed by horseradish peroxidase-conjugated sheep antirabbit IgG as the second antibody with chemiluminescence detection (Pierce Chemical Co., Rockford, IL) as described previously (19).
Assay of WT and mutant type II 3ßHSD enzyme activity.
Seventy-two hours after transfection, 3ßHSD activities in intact
transfected cells were determined by adding 1 µmol/L 5-P or
DHEA to the medium. In our initial experiments, aliquots
of medium were collected at various times up to 6 h for studies of
conversion of 5-P to P. In subsequent
experiments for studies of conversion of DHEA to
4-A, aliquots of medium were collected at
various times, including 6 and 12 h incubation samples to
determine whether the enzyme activity improves with a longer incubation
time. Steroids in the medium were extracted into 4 vol diethyl ether,
and the organic phase was evaporated. For the measurement of P, the
dried residue was reconstituted with 0.1 mL dichloroethane and
subjected to thin layer chromatography. For the determination of
4-A, the dried residue was reconstituted with
saturated isooctane and subjected to Celite chromatography as reported
previously (11, 14, 15). After the chromatographic procedures, all
samples were reconstituted with phosphate buffer solution, and steroid
concentrations were measured by RIA. Cell homogenates were prepared by
harvesting transfected cells into PBS/20% glycerol (vol/vol) using a
rubber policeman, snap-frozen in a CO2/ethanol
bath, and subjected to three freeze-thaw cycles. Samples were sonicated
and centrifuged at 800 rpm for 1 min to pellet cellular debris. The
protein content of the supernatant was determined by the method of
Bradford (protein assay, Bio-Rad Laboratories, Inc.) using
BSA as standard. 3ßHSD activity was determined for
5-P and DHEA at various
concentrations. Assays were performed at 37 C in a total reaction
volume of 0.5 mL consisting of 100 µg total protein, 50 mmol/L Tris,
1 mmol/L NAD+, radiolabeled substrate (0.025 mCi
[3H]pregnenolone, 0.005 mCi
[3H]pregnenolone, or 0.005 mCi
[14C]DHEA), and the desired
concentration of the relevant unlabeled substrate. The reaction was
stopped by the addition of dichloromethane, and after extraction,
steroids were separated by thin layer chromatography and quantified by
a liquid scintillation spectrometry.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
and 2)
Direct sequencing of PCR products of the genetic male with
nonsalt-wasting 3ßHSD deficiency CAH and mild genital ambiguity from
family 1 revealed a single nucleotide substitution mutation from C to T
at nucleotide 286 of the gene in exon II in both alleles, resulting in
a homozygous missense mutation in codon 6 from CTT (Leu) to TTT (Phe;
Fig. 1). Remaining sequences of all exons, exon, and intron boundaries
as well as sequences of the putative promoter region were normal. The
possibility that the mutation found in exon II of both alleles in the
patient was caused by PCR artifacts was excluded by confirmation of the
identical findings by repeat PCR and sequencing as well as by the
findings of single strand conformation polymorphism analysis of exon
II. Direct sequencing of exon II PCR products from the patient’s
parents, who were clinically normal, revealed the L6F mutation in one
allele of both parents, verifying the carrier state of the L6F mutation
(Fig. 1). The remaining sequences of the affected exon II allele as
well as the sequences of the other exon II allele from both parents
were normal (Fig. 1). These data confirmed that the homozygous L6F
mutation in the patient was inherited by a recessive trait from both
parents who were first cousins (Fig. 1). It was not possible to
determine the origin of the L6F mutant chromosome in the family
pedigree, whether it originated from the maternal/paternal great
grandfather or maternal/paternal great great grandfather (Fig. 1).
|
). The remaining sequences of all exons and
exon and intron boundaries as well as sequences of regions of the
putative promoter were normal. A repeat PCR product and direct
sequencing as well as sequencing of the subcloned PCR product revealed
identical sequences, confirming a genuine mutation in the exon of the
patient. Sequencing of exon IV PCR product from the patient’s mother
revealed the T259M in one allele, confirming the carrier state of the
T259M mutation. The remaining sequences of the affected exon IV allele
as well as sequencing of the other exon IV allele from the mother were
normal. The father’s DNA was unavailable for analysis.
Northern blot analysis of the cells transfected with WT and mutant
type II 3ßHSD cDNAs (Fig. 3)
The transfection efficiency determined by ß-galactocemia
activity was 18 ± 6% (±SD) in three separate
experiments. The expression of the mutated recombinant type II L6F and
T259M cDNA as well as the mutant recombinant T259R cDNA was compared by
the amount of type II 3ßHSD mRNA of WT to the amount of mutant type
II 3ßHSD L6F, T259M, and T259R generated by site-directed
mutagenesis. As depicted in Fig. 3, an autoradiogram of the Northern
blot revealed a similar band intensity of type II 3ßHSD mRNA from
cells transfected with WT cDNA and three mutant type II cDNAs, whereas
control vector containing no type II 3ßHSD cDNA generated no mRNA. A
slight smearing effect of 18S RNA in the Northern blot suggested the
presence of the multiple hybridizing species of 3ßHSD sequence from
the transfected COS cells. These findings demonstrated the presence of
transfected WT and all three mutant type II 3ßHSD cDNAs in the COS-1
cells.
|
, A and
B)
An autoradiogram of immunoblotting of type II 3ßHSD protein
translated by WT type II 3ßHSD cDNA in COS-1 cells revealed a 43-kDa
3ßHSD protein (Fig. 4A). The level of sensitivity for detection of
3ßHSD protein is estimated to be approximately 5% (
1.25 µg
protein) that of WT protein (25 µg protein COS-1 or COS-7 cells). The
mutant type II L6F cDNA generated by a site-directed mutagenesis
revealed a similar amount of type II 3ßHSD protein compared to the
wild type protein in COS-1 cells (Fig. 4A), but none was detected in
COS-7 cells (Fig. 4B). In the case of T259M and T259R cDNAs, the mutant
proteins were undetectable in all instances (Fig. 4, A and B). The
control vector containing no type II 3ßHSD cDNA or an aromatase P450
cDNA revealed no detectable type II 3ßHSD protein.
|
)
The time course and amount of formation of P from
5-P and of 4-A from
DHEA in intact COS-1 cells are depicted in Fig. 5. The
percentages of P formed after 60, 120, 180, 240, and 360 min of
incubation with 5-P substrate in cells
transfected with pcDNA wild-type II 3ßHSD cDNA were 22%, 23%, 24%,
30%, and 46%, respectively, above the values of the control pcDNA
vector. In cells transfected with pcDNA L6F, the percentages of P
formed at the same incubation time points as in the wild-type with
5-P substrate were 1.8%, 6.5%, 12%, 15%,
and 22%, respectively, above the values of the control vector. In
cells transfected with pcDNA T259M, the percentages were 0%, 0%, 0%,
2%, and 8%, respectively, above the values of the control vector. No
enzyme activity was detected at any time point in cells transfected
with pcDNA T259R. The percentages of 4-A
formed after 300, 360, 540, and 720 min of incubation with
DHEA substrate in cells transfected with pcDNA3 wild-type
type II 3ßHSD were 61%, 89%, 100%, and 100%, respectively, above
the values of the control pcDNA vector, demonstrating no difference in
the activity between 6 and 12 h incubation points. In cells
transfected with pcDNA L6F, the percentages of
4-A formed at the same incubation time point
with DHEA substrate as in the wild-type were 25%, 35%,
63%, and 77%, respectively, above the control vector values. In cells
transfected with pcDNA T259M, the percentages of
4-A formed were 0%, 5.6%, 6.4%, and 9.4%,
respectively, above the values of the control vector, whereas the
percentages of 4-A formed for cells
transfected with pcDNA T259R were 0%, 1.5%, 1.8%, and 4% above
control values, respectively. Similar results were obtained when
3ßHSD activities were assayed in intact COS-7 cells after
transfection with the various plasmids as described above (data not
shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
To date, the functional significance of Leu6 in
the type II 3ßHSD enzyme has not been defined; however, the
importance of Leu6 in the gene is evident by the
fact that Leu6 residue in the family of 3ßHSD
genes is conserved throughout all mammalian and other vertebrate
species (Fig. 6) (20). In
vitro, the Phe6 mutant type II 3ßHSD
enzyme was detectable and demonstrated decreased, but nonetheless
measurable, 3ßHSD activity for both substrates of
5-P and DHEA in intact cells.
These findings corroborated with a less severe clinical and biochemical
phenotype of 3ßHSD deficiency CAH in the affected patient, as
demonstrated by relatively normal serum electrolytes and PRA during the
early neonatal period as well as at age 2 yr without glucocorticoid or
Florinef acetate replacement therapy. In addition, the genital
ambiguity was less prominent than that in the patient with salt-wasting
3ßHSD deficiency described in this report and in other reports with
genotype-proven 3ßHSD deficiency CAH (4, 5, 6, 21, 22, 23, 24). The patient’s
in vivo hormonal data suggested a greater impairment of
3ßHSD activity in the C21 steroid pathway than
in the C19 steroid pathway, as evidenced by
highly increased 5-17P level and an age
appropriately increased DHEA level after ACTH
administration during neonatal age. The L6F mutant enzyme in intact
cells, however, demonstrated a similar degree of decreased 3ßHSD
activity for both substrates of 5-P and
DHEA. The very low activity of the
Phe6 mutant enzyme in homogenates and
undetectable L6F enzyme in COS-7 cell immunoblots may result from a
conformational modification of this region that confers relative
instability of the enzyme. Thus, the nonsalt-wasting 3ßHSD-deficient
phenotype of the affected individual may in large part arise from the
expression of a relatively unstable enzyme rather than a catalytically
compromised enzyme.
|
The critical importance of Thr259 in the type II
3ßHSD enzyme has been predicted by the demonstration of a
substrate-binding domain comprising amino acids
Gly256 to Lys274 and
Asn176 to Arg186 of type I
3ßHSD by affinity radiolabeling of the enzyme with
2
-bromo-[2'-14C]acetoxyprogesterone (26).
Thus, Thr259 of type II 3ßHSD enzyme is
predicted to be involved in a putative substrate-binding domain. In
addition, the importance of the Thr259 residue in
the family of 3ßHSD isoenzymes is further substantiated by the
conservation of the Thr259 residue throughout all
mammalian and vertebrate species (Fig. 6) (20). Consequently, a
structural change involving this putative substrate-binding domain is
likely to cause a seriously deleterious effect. The mutant alleles
involving an amino acid residue change in this domain, such as Y253N
(6) and T259R (27) in the type II 3ßHSD gene, were associated with
the salt-wasting phenotype (6, 27) and male pseudohermaphroditism (6),
and in vitro Y253N (6) and Y254D (28) mutant enzymes
demonstrated no detectable 3ßHSD activity (6, 28)
The present report further elucidates both in vivo and
in vitro evidence of detrimental outcome resulting from
T259M and T259R mutation in a putative substrate-binding domain of the
type II 3ßHSD gene. The patient with homozygous T259M mutation in the
gene had severely undervirilized external genitalia leading to female
sex assignment at birth and developed profound salt-wasting disorder
and hypoglycemia during neonatal period, indicating severe adrenal and
testicular type II 3ßHSD deficiency. The markedly elevated
5 precursor steroid levels after short term
discontinuation of glucocorticoid therapy in later life also
substantiated profound adrenal type II 3ßHSD deficiency. Furthermore,
both T259M and T259R mutant enzymes demonstrated very little or no
enzyme activity in vitro, further substantiating that the
Thr259 residue is critical in the
substrate-binding domain of the gene. Undetectable T259M or T259R
mutant protein in the homogenates of the transfected cells on Western
immunoblotting, despite detectable type II 3ßHSD mRNA, suggests that
these mutant enzymes are unstable, which would also account for the
lack of enzyme activity. A similar observation has recently been
proposed by Moisan et al. using human fetal kidney 293 cells
regarding T259M, A10E, and G294V mutant enzymes (24). The nucleotide
sequences of type I and type II 3ßHSD genes at codons 6 and 259 are
identical. Thus, it is unlikely that mutant amino acid residues
Phe6, Met259, and
Arg259 were products of gene conversion between
the two types of 3ßHSD genes. However, at this time, we cannot
exclude the small possibility of gene conversion resulting in the codon
6 and 259 mutations between the human type II 3ßHSD gene and human
3ßHSD pseudogenes whose sequence information is not available
(29).
In conclusion, these in vitro findings of the mutant type II 3ßHSD enzyme activities correlated with a less severe clinical phenotype of nonsalt-wasting and a lesser degree of genital ambiguity in the patient with homozygous L6F mutation compared to a more severe clinical phenotype of salt-wasting and severe degree of genital ambiguity in the patient with homozygous T259M mutation in the gene. The presence of L6F enzyme activity in intact cells and undetectable enzyme activity in homogenates with inconsistent L6F immunoblots in vitro suggests a relative instability of this enzyme attributable to the nonsalt-wasting disorder. The two different missense mutations involving codon 259 that reveal very little enzyme activity further substantiate the deleterious effect of a structural change in this predicted putative steroid-binding domain of the gene. The presence of type II 3ßHSD mRNA expression with undetectable type II 3ßHSD proteins in vitro by the T259M and T259R mutations further suggests the very labile nature of these mutant proteins, resulting in very little enzyme activity and thus causing severe 3ßHSD deficiency disorder.
|
Footnotes |
|---|
Received April 26, 1999.
Revised December 3, 1999.
Accepted December 23, 1999.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
5-4-isomerase in human adrenals and
gonads. Mol Endocrinol. 5:1147–1157.[Abstract]
4
5 isomerase. Mol
Endocrinol. 4:1850–1855.[Abstract]
5-steroid levels. J
Clin Endocrinol Metab. 81:3961–3965.-androstanediol glucuronide
concentrations in women with various causes of hirsutism and its
correlation with degree of hirsutism and androgen levels. J Clin
Endocrinol Metab. 75:243–248.[Abstract]
5-4 isomerase (3ßHSD) gene: adrenal
and gonadal specificity. DNA Cell Biol. 10:701–711.[Medline]
AA in type II 3ß-hydroxysteroid dehydrogenase (HSD
3ß2) in three male patients of Afghan/Pakistani origin. Hum Mol
Genet. 3:327–330.5-steroid dehydrogenase. J Biol Chem. 268:18507–18512.5-4 isomerase:
characterization of three additional related genes [Abstract 1499].
Proc of the 74th Annual Meet of The Endocr Soc. 1992; 426.
This article has been cited by other articles:
 |
|
 |
|
  J. Simard, M.-L. Ricketts, S. Gingras, P. Soucy, F. A. Feltus, and M. H. Melner Molecular Biology of the 3{beta}-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4 Isomerase Gene Family Endocr. Rev., June 1, 2005; 26(4): 525 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Mermejo, L. L. K. Elias, S. Marui, A. C. Moreira, B. B. Mendonca, and M. de Castro Refining Hormonal Diagnosis of Type II 3{beta}-Hydroxysteroid Dehydrogenase Deficiency in Patients with Premature Pubarche and Hirsutism Based on HSD3B2 Genotyping J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1287 - 1293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kosel, S. Burggraf, R. Fingerhut, H. G. Dorr, A. A. Roscher, and B. Olgemoller Rapid Second-Tier Molecular Genetic Analysis for Congenital Adrenal Hyperplasia Attributable to Steroid 21-Hydroxylase Deficiency Clin. Chem., February 1, 2005; 51(2): 298 - 304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pang, W. Wang, B. Rich, R. David, Y. T. Chang, G. Carbunaru, S. E. Myers, A. F. Howie, K. J. Smillie, and J. I. Mason A Novel Nonstop Mutation in the Stop Codon and a Novel Missense Mutation in the Type II 3{beta}-Hydroxysteroid Dehydrogenase (3{beta}-HSD) Gene Causing, Respectively, Nonclassic and Classic 3{beta}-HSD Deficiency Congenital Adrenal Hyperplasia J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2556 - 2563. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Lutfallah, W. Wang, J. I. Mason, Y. T. Chang, A. Haider, B. Rich, M. Castro-Magana, K. C. Copeland, R. David, and S. Pang Newly Proposed Hormonal Criteria via Genotypic Proof for Type II 3{beta}-Hydroxysteroid Dehydrogenase Deficiency J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2611 - 2622. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
