Inactivation of the Luteinizing Hormone/Chorionic Gonadotropin Receptor by an Insertional Mutation in Leydig Cell Hypoplasia
Shao-Ming Wu,
Karen M. Hallermeier,
Louisa Laue1,
Caroline Brain2,
A. Caroline Berry,
David B. Grant1,
James E. Griffin,
Jean D. Wilson,
Gordon B. Cutler, Jr.3 and
Wai-Yee Chan
Departments of Pediatrics (S-M.W., L.L., W-Y.C.), Biology (K.M.H.),
and Cell Biology (W-Y.C.) Georgetown University Washington, DC
20007
Hospital for Sick Children (C.B., D.B.G.) and
Guys Hospital (A.C.B.) London, WC1N 3JH United Kingdom
Department of Internal Medicine (J.E.G., J.D.W.) University
of Texas Southwestern Medical Center Dallas, Texas 75235
Developmental Endocrinology Branch (G.B.C.) National
Institute of Child Health and Human Development National Institutes
of Health Bethesda, Maryland 20892
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ABSTRACT
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We previously identified a nonsense mutation
(Cys545Stop) in the paternal human LH/CG receptor (hLHR) allele in a
family with two 46,XY children afflicted with Leydig cell
hypoplasia. This mutation abolished the signal transduction
capability of the affected hLHR. We have now examined all coding exons
and the transcript of both alleles of the hLHR gene of the
affected children. A 33-bp in-frame insertion was found in the maternal
hLHR allele. This insertion occurred between nucleotide 54 and 55 and
might be the result of a partial gene duplication. Genomic DNA-PCR
showed that this defective maternal hLHR allele was inherited by the
two affected children. However, examination of the inheritance of the
935-A/G polymorphism of the hLHR by genomic- and RT-PCR
indicated that the maternal hLHR allele was not expressed in cultured
fibroblasts of the patients. The effect of the in-frame insertion on
the biological activity of the hLHR was examined by expressing the
mutated hLHR construct, generated by site-directed mutagenesis, in HEK
293 cells. The expression of the mRNA for the mutant hLHR in HEK 293
cells was not affected. Response of cells expressing the mutated hLHR
to hCG stimulation was impaired as demonstrated by reduced
intracellular cAMP biosynthesis. This change in signal transduction was
the result of a profound reduction in hormone binding at the cell
surface due to altered expression and processing of the mutated
receptor. We conclude that Leydig cell hypoplasia in this family is the
result of compound heterozygous loss-of-function mutations of the
hLHR gene.
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INTRODUCTION
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The human LH/CG receptor (hLHR) is a member of the G
protein-coupled receptor family. In the testis, it mediates the effects
of LH on testosterone biosynthesis by transducing the signal of agonist
binding via the interaction with Gs protein and
biosynthesis of cAMP (1). Two inherited diseases have been attributed
to mutations that alter the biological activities of the hLHR. Leydig
cell hypoplasia (LCH) is a disorder due to the failure of LH in
enhancing fetal testicular Leydig cell differentiation. It is a form of
male pseudohermaphroditism, characterized by female external genitalia,
absent Müllerian derivatives, cryptorchidism, occasional presence
of the vas deferens with epididymis, and 46,XY genotype. These patients
have elevated levels of LH but low basal and hCG-stimulated levels of
serum testosterone (2, 3, 4). Seven single-base substitutions leading to
either non-sense mutations (Cys545Stop and Arg554Stop) or missense
mutations (Cys131Arg, Glu354Lys, Ala593Pro, Ser616Tyr, and Ile625Lys)
were identified in eight unrelated kindreds (5, 6, 7, 8, 9, 10, 11, 12). The majority of
these mutations were found in affected homozygotes. A homozygous
deletion of six nucleotides (18221827,
Leu608-Val609) was also
observed in one kindred (13). Compound heterozygous mutations were
observed in two kindreds, one with a missense mutation and partial gene
deletion (8) and the other with a nonsense mutation in the paternal
hLHR allele, and the genetic status of the maternal allele was
undetermined (6). In vitro expression studies of the mutated
receptors demonstrated impaired or no ligand binding and cAMP
production in response to hCG stimulation, indicating the inactivation
of the mutated receptor (5, 6, 7, 8, 9, 10, 11, 12, 13).
A family with two children affected with LCH was previously examined by
us. The children were both 46,XY males with undescended gonads and
phenotypic female external genitalia. Fibroblasts from the labia majora
revealed normal 5
-reductase activity and androgen receptor binding.
Serum testosterone level was unresponsive to hCG stimulation. These
children inherited a paternal hLHR allele with the Cys545Stop nonsense
mutation (6). In this report, we show that the maternal hLHR allele in
this family carried a 33 bp in-frame insertion in exon 1 of the gene.
This mutation caused altered expression and processing of the mutated
hLHR, resulting in impairment of hormone binding at the cell surface
and subsequent failure of response to hCG stimulation. Thus, LCH in
this family was the result of compound heterozygous mutations of the
hLHR.
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RESULTS
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Genotype of Patients and Their Parents at the Nucleotide 935
Polymorphic Site
The primer strategy for amplification and sequencing of
hLHR exons is shown in Fig. 1
.
Examination of exons 210 of the hLHR gene revealed no
mutation (results not shown). The G/A polymorphism of the hLHR at
nucleotide 935 in exon 10 of the hLHR gene (14) was
informative in this family. The father was homozygous A, the mother was
homozygous G, and both children were heterozygous A/G at this
polymorphic site. This result suggested that the children inherited the
allele with A-935 from the father and the allele with G-935 from the
mother. Since the father was shown to give both children an hLHR allele
that had the nonsense mutation (6), the paternal hLHR allele with A-935
must carry the nonsense mutation.

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Figure 1. Receptor Map and Sequencing Strategy
The size of the exons and introns was not drawn to scale. Exons
represented by boxes are numbered from 1 to 11. Introns
are represented by thin lines. The positions of the
translation initiation (ATG) and termination (TGA) codons are
indicated. Arrows above the receptor represent
5'-primers, and arrows underneath the receptor represent
3'-primers. Arrow indicates 5' to 3' direction. a to v
denotes PCR primers. The exons and the primer sets used to amplify them
were: exon 1, a/b; exon 2, c/d; exon 3, e/f; exon 4, g/h; exon 5,
intron 5, and exon 6, i/j; exon 7, k/l; exon 8, m/n; exon 9, o/p; exon
10, q/r; and exon 11, s/t. Primers u/v were used in RT-PCR to amplify
the transcript containing exon 2 to exon 11. Primer sequences were as
described by Atger et al. (36 ) unless specified. a, 5'U3
(nucleotide -231 to -210) (34 ); b, GhLHR2; c, GhLHR3; d, GhLHR4; e,
GhLHR5; f, GhLHR6; g, GhLHR7; h, GhLHR8; i, GhLHR9; j, GhLHR12; k,
GhLHR13; l, GhLHR14; m, GhLHR15; n, GhLHR16; o, GhLHR17; p, GhLHR18; q,
GhLHR19; r, GhLHR20; s, GhLHR21; t, LL6 (nucleotide 21112135,
5'-AATAATGCAGTTACTGATGTAACAG-3'); u, 5'-exon 2 primer (nucleotide
162182, 5'-ATCACTTGCCTACCTCCCTGT-3'); v, 3'-exon 11 primer
(nucleotide 12831304, 5'-GTCTGCCAGTCTATGGCATGGT-3'); w, 5'-exon 9
primer (nucleotide 681704, 5'-GGATATTTCTTCCACCAAATTGCA-3'); x,
5'-exon 10 primer (nucleotide 867891,
5'-ACAGAATTTTTCACATTCCATTTCT-3').
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Examination of the 935 (A/G) Polymorphism at the Transcript
Level
RT-PCR using 5'-exon 2 primer (primer u) and 3'-exon 11 primer
(primer v) and fibroblast total cellular RNA as template gave an
amplified DNA band of 1132 bp in both children. No mutation was found
in the amplified cDNA. However, examination of the genotype at
nucleotide 935 showed that only the A allele was expressed. On the
other hand, the children were heterozygous A/G at this site when their
genomic PCR products were examined. Figure 2
compares the sequences of the
genomic-PCR and RT-PCR products of the children at this polymorphic
site. The results obtained from both children were the same. Only the
result of child I was presented in Fig. 2
.

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Figure 2. Comparison of the Genomic and the cDNA Sequences of
the hLHR of the Children at the G 935 A Polymorphic Site
Position of the polymorphic nucleotide is indicated by open
arrowhead. The sequences are indicated along the side of the
gel. Genomic DNA for the analysis was isolated from fibroblasts
obtained from both children and from whole blood obtained from the
parents. Exon 10 was amplified using primers q and r. For RT-PCR, total
cellular RNA was extracted from confluent cultures using the acid
guanidinium thiocyanate-phenol-chloroform procedure described by
Chomczynski and Sacchi (38 ). One microgram of total cellular RNA was
used for a 20-µl RT reaction using a random polyhexamer primer
(PdN6). The RT product was used as template for amplifying the sequence
encoded in exon 2 to exon 11 using primers u and v in a 28-cycle PCR.
Conditions of the PCR were the same as described previously (8 ).
Single-stranded DNA for sequence determination was generated by
asymmetrical PCR and was concentrated before sequencing by the dideoxy
chain termination method using Sequenase. Templates for the asymmetric
PCR were derived from two or more independent PCR products. Nucleotide
sequence analysis was performed on both strands derived from several
PCRs. The genomic sequence was A/G at the polymorphic site while the
cDNA sequence was A at this site.
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Examination of Exon 1 of the hLHR
Results of analysis of the PCR-amplified product of exon 1 of the
hLHR using genomic DNA as template are shown in Fig. 3A
. Normal control (N) had a single
PCR-amplified fragment of 453 bp. In the father (F), a broad band,
likely to be composed of two overlapping bands at 453 bp and at 459 bp,
respectively, was present. The mother (M) and the two children (CI and
CII) had two bands. The mother had a 486-bp band and a 453-bp band.
Both children had a 486-bp band and a 459-bp band. Apparently, the
larger band in the children was derived from the mother and the smaller
band was derived from the father. Nucleotide sequence determination
showed that the broad band in the father was caused by the presence of
the 6-bp CTGCAG insertion allele and the noninsertion allele of the
hLHR as reported recently (12). The mother had the noninsertion allele
(453-bp band) and an allele that had a 33-bp in-frame insertion after
nucleotide 54. The positions of the 6-bp polymorphism and the 33-bp
insertion are shown in Fig. 3B
. The children inherited the 6-bp
insertion allele from the father and the 33-bp in-frame insertion
allele from the mother.

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Figure 3. Amplification of Exon 1 of the hLHR
Gene
A, Amplification of exon 1 of the hLHR gene. S, Size
markers, X 174 digested with HaeIII. The size of the
marker bands is shown on the left; F, father; M, mother;
CI, child 1; CII, child II; N, normal. The size of the amplified
fragments is shown on the right. B, In-frame insertion
found in exon 1 of the maternal hLHR allele. Numbers
indicate the position of the amino acid residue from the N-terminal
methionine and the nucleotide base from the beginning of the coding
sequence of the hLHR. The inserted sequences are indicated by
arrows. The insertion above the
nucleotide sequence was a polymorphism. The insertion
beneath the sequence was the mutation observed in the
maternal allele. Exon 1 of the hLHR gene was amplified
by PCR using 5'-U3 primer (primer a) and 3'-GhLHR2 primer (primer b)
with genomic DNA as template. Conditions of the PCR were the same as
described previously (8 ) except that the buffer contained 20
mM Tris-HCl, pH 8.4, 5 mM KCl, 1.7
mM MgCl2, and 0.1 mg BSA/ml. The amplified DNA
fragments were separated by electrophoresis through a 3.8%
polyacrylamide gel.
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Expression of the Insertional Allele Construct in HEK 293 Cells
Construct for in vitro expression of the hLHR carrying
the 33-bp insertion was generated by site-directed mutagenesis.
Expression of the mutated hLHR in the transfected cells was examined by
RT-PCR using specific primers. Figure 4
shows the result of PAGE analysis of RT-PCR product of transfected
cells. The 261-bp band represented amplified glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) cDNA, and the 438-bp band was an amplified
fragment of the hLHR cDNA encoded by exon 10 and part of exon 11. After
28 cycles of PCR, no amplified hLHR cDNA was visible in the transfected
cells while G3PDH bands of equal intensity were observed in all three
cells (lanes 4 to 6), indicating that roughly equal quantities of RNA
were used for the RT-PCR. After another 21 cycles of nested PCR,
amplified hLHR cDNA bands became visible in cells transfected with the
pcDNA3hLHR-Ins construct (lane 2) and those transfected with
pcDNA3hLHR-WT construct (lane 3). No amplified hLHR cDNA was present in
untransfected cells (lane 1), indicating that HEK 293 cells did not
have endogenous LHR. Transfection with pcDNA3hLHR-Ins was more
efficient than with pcDNA3hLHR-WT, as indicated by the activity of the
ß-galactosidase, which was 0.73 mU/reaction for the pcDNA3hLHR-Ins
transfectants vs. 0.32 mU/reaction for the pcDNA3hLHR-WT
transfectants. This corresponded with the difference in the intensities
of the hLHR bands between lanes 2 and 3. Thus, the 33-bp insertion did
not prevent expression of the hLHR mRNA in HEK 293 cells.

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Figure 4. In vitro Expression of hLHR Carrying
the 33-bp in-Frame Insertion
Lanes 1 and 4, Untransfected 293 cells; lanes 2 and 5, cells
transfected with pcDNA3hLHR-ins construct; lanes 3 and 6, cells
transfected with pcDNA3hLHR-WT construct; lane N, minus reverse
transcriptase control. Size of the cDNAs in base pairs is indicated on
the side. HEK 293 cells were cultured in T-25 or T-75 flasks and were
transfected with 3.6 µg or 10.8 µg of DNA (19/20 pcDNA3 construct
plasmid DNA and 1/20 ß-galactosidase plasmid DNA), respectively,
using lipofectamine. Cells were cultured for 48 h after
transfection before being used for experiments. To assay for the
expression of the hLHR insert, cells in T-75 flasks were lysed, and
total cellular RNA was extracted and used for RT as described in Fig. 2 . Half of the RT product was used as template for amplifying hLHR
using nested PCR. The first 28 cycles were primed with 5'-exon 9 primer
(primer w) and the 3'-exon 11 primer (primer v). The PCR product was
then diluted 1:85 and subjected to another 21 cycles primed with a
primer internal to primer w (primer x complementary to the 5'-end of
exon 10) and the same 3'-exon 11 primer. Five microliters of the PCR
product were analyzed by PAGE (lanes 13, 438 bp, hLHR). Half of the
RT reaction was used as template for a 28-cycle PCR amplification of
G3PDH as described previously (39 ) (lanes 46, 261 bp, G3PDH).
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cAMP Studies
Cells transfected with the pcDNA3hLHR-WT construct responded to
hCG stimulation with a dose-dependent increase in intracellular cAMP.
Cells transfected with the pcDNA3hLHR-Ins construct responded weakly to
the stimulation by hCG. Means and SEs of the means of two
experiments are shown in Fig. 5
. The
results were normalized with ß-galactosidase activity. Basal levels
of intracellular cAMP among the three type of cells, i.e.
vector-transfected control cells (pcDNA3), pcDNA3hLHR-WT-transfected
cells (hLHR-WT), and pcDNA3hLHR-Ins-transfected cells (hLHR-Ins) were
similar. Treatment with hCG caused a dose-dependent increase in
intracellular cAMP in HEK 293 cells expressing hLHR-WT with an
ED50 = 3.38 ± 1.18 ng/ml (n = 4).
Vector-transfected control cells did not respond to hCG stimulation.
The response of the hLHR-Ins transfectants to hCG stimulation was much
less than the hLHR-WT transfectants and was only slightly above that of
the vector-transfected control cells (Fig. 5
). These results indicated
that the 33-bp insertion caused profound impairment of signal
transduction mediated by the mutated hLHR. Similar results were
obtained in four independent experiments.

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Figure 5. Dose-Dependent cAMP Production in Transfected 293
Cells
, hLHR-WT cells transfected with hLHR-WT construct DNA; ,
hLHR-Ins cells transfected with hLHR-Ins construct DNA (pcDNA3); ,
cells transfected with unmodified pcDNA3 expression vector. Means and
SEM of triplicates in two independent experiments were
presented. To examine the synthesis of cAMP by cells expressing the
mutant hLHR construct, HEK 293 cells were cultured in 24-well plates
(4 x 104 cells per well). Cells were transfected with
0.35 µg of DNA/well (95% pcDNA3 construct plasmid DNA and 5%
ß-galactosidase plasmid DNA) using lipofectamine. To assay for
the effect of hCG on intracellular cAMP production, cells were treated
with 0.11000 ng/ml hCG (CR127: 14,900 IU/mg, obtained through NHPP,
NIDDK, NICHHD, USDA) for 2 h at 37 C, with 5% CO2,
0.25 mM 3-isobutyl-1-methyl-xanthine, and 0.1% BSA. Cells
were treated with 3% perchloric acid and neutralized with potassium
hydroxide, and the amount of cAMP in the lysate was assayed using the
cAMP enzyme-immunoassay system. Results were normalized for
ß-galactosidase activity by the same cell preparation to correct for
transfection efficiency.
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Binding Studies
Cell surface binding of hCG was assessed by incubating transfected
cells with a fixed quantity of labeled hCG and increasing amounts of
unlabeled hormone. While cells expressing hLHR-WT continued to bind hCG
until saturation, those expressing hLHR-Ins and vector-transfected
cells did not bind any appreciable amount of hCG (Fig. 6A
). This difference persisted when hCG
binding was compared between detergent-solubilized lysate of
transfected cells expressing hLHR-WT and those expressing hLHR-Ins
(Fig. 6B
). The means and SEs of the means of three
experiments are shown in both panels. The results were normalized with
ß-galactosidase activity. Scatchard analysis of the data obtained
with detergent-solubilized cell lysate gave a dissociation constant
(Kd) of (2.1 ± 0.5) x 10-10
M (n = 4) for hLHR-WT, which was comparable to the
value obtained previously (8). The number of binding sites as
determined by the X intercept of the Scatchard plot was 45.0
pM. Binding of hCG by total lysate of transfected cells
expressing hLHR-Ins, similar to that of vector-transfected control
cells, was minimal even at a total hCG concentration of more than 100
ng/ml. The amount of hCG bound was too small to give a meaningful
Scatchard analysis of the data.

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Figure 6. hCG-Binding Studies
A, hCG binding by intact cells. B, hCG binding in detergent-solubilized
cell extract. , hLHR-WT cells transfected with hLHR-WT construct
DNA; hLHR-Ins cells transfected with hLHR-Ins construct DNA
(pcDNA3); , cells transfected with unmodified pcDNA3 expression
vector. Means and SEM were presented. To examine cell
surface binding of hCG, the procedure of Lloyd and Ascoli (41 ) was
followed with modifications. 8 x 104 HEK 293 cells
were transfected with 0.54 µg of DNA/well (95% pcDNA3 construct
plasmid DNA and 5% ß-galactosidase plasmid DNA) using
lipofectamine. Forty-eight hours after transfection, cells were
chilled in the cold room for 20 min before the addition of a fixed
amount of [I125]hCG and designated amounts of unlabeled
hCG in binding buffer (200 mM HEPES, pH 7.4, 2.5% BSA in
DMEM-F12). The cells were left at 4 C for 24 h. The
radioactive media were aspirated and the cells washed with washing
buffer (92.5% binding buffer, 7.5% horse serum). The aspirate and the
washing buffer were pooled and counted. Cells were collected with
solubilizing solution (0.2 N NaOH, 150 mM NaCl, 40 µg
salmon sperm DNA/ml) and counted. For binding study using
detergent-solubilized receptor, 2 x 106 HEK 293 cells
cultured in T-75 flasks were transfected with 10.5 µg DNA/flask).
Cells were harvested in ice-cold buffer A (150 mM NaCl, 10
mM sodium phosphate, pH 7.4) supplemented with protease
inhibitors (20 µM leupeptin, 1
mM phenylmethylsulfonyl fluoride, and 100 kallikrein
inhibiting units/ml aprotinin) and lysed for 45 min on ice with 1%
Triton X-100, 30% glycerol in buffer B (150 mM sodium
chloride, 20 mM HEPES, pH 7.4) supplemented with protease
inhibitors. The supernatants were collected after centrifugation.
Equilibrium binding was performed by incubating aliquots of the
extracts (diluted with buffer B containing 0.1% Triton X-100 and 20%
glycerol and supplemented with protease inhibitors) at 4 C for 24
h with a fixed amount of [I125]hCG in the presence of
designated amounts of unlabeled hCG. Separation of receptor-bound
[I125]hCG from unlabeled hormone was performed by
polyethyleneglycol precipitation as described previously (8 ). All
determinations were done in duplicate.
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DISCUSSION
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LCH in the family presented in this report is the result of
compound heterozygous inactivating mutations of the hLHR. One allele,
which both children inherited from the father, has a point mutation at
nucleotide 1635 that causes the replacement of Cys545 by a stop codon,
resulting in a prematurely truncated receptor (6). At the time when
this mutation was found, only exon 9 and exon 11 were sequenced because
all mutations of the hLHR known then were found in exon 11. In
subsequent studies, exons 2 to 11 of total cellular RNA derived from
cultured fibroblasts of the patients was examined. With PCR
conditions that were used to amplify exons 211 of the hLHR, we were
unable to amplify exon 1 after numerous attempts. An examination of the
base composition of exon 1 shows that it is GC rich. Of the first 100
bases starting from the initiation codon, 70% are G or C. There is
also a stretch of 10 CTG triplets interrupted with 2 CAG and 2 CCG
triplets (15). Similar to that observed in othergenes such as FMR-1,
myotonin protein kinase, etc. (16, 17), the high GC content and the
potential of forming stable secondary structures might have impaired
the efficiency of the PCR. This impairment of amplification of exon 1
was eventually overcome by altering the K+ and
Mg++ concentrations of the PCR buffer as described in
Materials and Methods.
The maternal hLHR allele in the patients carries an insertional
mutation in the N terminus of the receptor as described in this report.
This insertion apparently affects expression of the hLHR in the
cultured fibroblasts of the children. It is clear that the homozygosity
of the 935-A of the RT-PCR product sequence (Fig. 2
) is questionable,
since only one parent, the father, has the hLHR alleles with A at this
position. According to the results from their genomic DNA, the children
inherited another hLHR allele from their mother with a G at nucleotide
935. Therefore, the RT-PCR result suggests that although the children
inherited the maternal hLHR allele, the transcript of this allele is
not present in the total cellular RNA prepared from the cultured
fibroblasts. This would suggest that the mutation of the maternal
allele prevents its expression. Another possibility is that exon 10 of
the maternal allele, in which the 935 A/G polymorphism lies,
might have been spliced out in the cultured fibroblasts due to an
unknown mechanism, leaving only the transcript of the paternal allele
with exon 10. Alternatively, the expression of the hLHR might be
affected by parental origin. Even though this genetic mechanism is not
known for hLHR, parental effect on gene expression is not a rare
phenomenon and is well established for a number of genes, including
small nuclear ribonucleoprotein-associated polypeptide
(SNRPN), H19, insulin-like growth factor-II
(Igf2), Igf2 receptor (Igf2r), etc. (18). Whether
the expression of hLHR in cultured fibroblast is affected by parental
imprinting could be investigated using the
established polymorphisms of the hLHR gene (14) and
informative families. Nonetheless, due to the reduced response to
hormone stimulation of cells expressing the mutated maternal allele as
demonstrated by in vitro expression studies, the children
would either have no or reduced total hLHR activity, which would
explain the occurrence of LCH.
It is well established that the extracellular domain of the LHR is
responsible for high-affinity binding of the hormone, which initiates
signal transduction (19, 20). By comparing to the rat LHR model of
Bhowmick et al. (21), the extracellular domain of the hLHR
can be viewed as having the first 25 amino acid residues comprising the
signal peptide, followed by the 5'-flanking cysteine cluster of
residues 2650, and nine leucine-rich repeats encoded by nine exons
each containing 2126 amino acids. The 33-bp insertion occurs between
amino acid residues 18 and 19, immediately upstream of the signal
peptide cleavage site (22). If normal cleavage of the signal peptide
was not affected by the insertion, the insertion mutation will be
cleaved with the signal peptide, and the mature protein should behave
like the wild-type receptor. If the signal peptide is not cleaved, it
is likely that the protein would not exit the endoplasmic reticulum and
move to the cell membrane. It might also make the unprocessed protein
unstable or interfere with hormone binding. Since no cell surface
binding of hCG was demonstrated for transfectants expressing the
mutated hLHR, it suggests that the insertion affects normal processing
of the hLHR, and that the signal peptide was not cleaved.
Detergent-solubilized lysate of transfectants expressing the mutated
hLHR also failed to bind hCG. This observation implies that the
insertion, which affects processing of the mutated hLHR, also
interferes with hormone binding and/or stability of the receptor. A
similar small in-frame insertion has been shown to affect ligand
binding of the neuronal nicotinic acetylcholine receptor (23).
Therefore, although hormone binding is achieved primarily through
interaction of the leucine-rich repeats (24), an insertion within the
putative signal peptide reduces hormone-binding affinity, probably by
altering the conformation of the receptor protein.
It is notable that a 6-bp CTGCAG polymorphism occurs at the same site
as the insertion (14). As discussed earlier, both the polymorphism and
the insertion occur in a region with a high number of CTG repeats. A
number of human disorders, including Huntingtons disease, spinobulbar
muscular atrophy, dentatorubropallidoluysian atrophy, spinocerebellar
ataxia 1 (SCA1), and Machado-Joseph disease (SCA3), have been shown to
be caused by expansion of CTG/CAG triplet repeats in the coding region
of the gene (25, 26). The disease-causing mechanism of the repeat
expansions sometimes involves silencing of expression of the gene (26, 27). The possibility that a similar mechanism is responsible for the
failure of expression of the maternal allele in the gonadal fibroblasts
of the LCH patients cannot be excluded.
Comparison of the sequence of the insertion with that of the hLHR shows
that it is identical to the sequence immediately upstream, indicating
the possibility of partial gene duplication. Gene duplication has been
suggested to play a role in organism evolution as exemplified by the
multigene families in the genome of humans and other higher organisms
(28, 29, 30, 31). Gene duplication may also involve part of a gene resulting in
duplication of internal protein domain (32). Partial gene duplication
may be pathogenic, especially for rearrangements that duplicate only a
subset of exons or part of an exon (33). An example is the in-frame
duplication of 45 bp in exon 48 of the type II collagen gene, giving
rise to spondyloepiphyseal dysplasia (34), and internal duplication of
seven exons in the lysyl hydroxylase gene in type VI variant of
Ehlers-Danlos syndrome (35).
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MATERIALS AND METHODS
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Patients
Two children with LCH and their parents were included in the
study. Clinical features of the patients were as described previously
(6). This study was approved by the Institutional Review Board of
Georgetown University.
PCR Amplification and Sequencing of Genomic DNA
Genomic DNA was isolated from cultured fibroblasts obtained from
both children and from whole blood obtained from the parents. The
entire exon 11 of the hLHR gene, encoding amino acid
residues 319699, was amplified by PCR using genomic DNA as template
as described previously (8). The location of the primers used in the
various PCRs described is shown in Fig. 1
. Exon 1 was amplified
using 5'-U3 primer (primer a, nucleotide -231 to -210) and 3'-GhLHR2
primer (primer b), while the other exons were amplified using primers
described by Atger et al. (36) (Fig. 1
). The buffer used in
PCR for amplifying exon 1 contained 20 mM Tris-HCl, pH 8.4,
5 mM KCl, 1.7 mM MgCl2, and 0.1 mg
BSA/ml (Boehringer Mannheim Corp., Indianapolis, IN), while that used
in amplifying the other exons contained 20 mM Tris-HCl, pH
8.4, 50 mM KCl, 2.5 mM MgCl2, and
0.1 mg BSA/ml. Single-stranded DNA for sequence determination was
generated by asymmetrical PCR and was concentrated using a Centricon
microconcentrator (Amicon Inc., Beverly, MA) before sequencing by the
dideoxy chain termination method using Sequenase (United States
Biochemicals, Cleveland, OH) as described previously (8). If multiple
species of amplified DNA were generated by PCR, the PCR products were
resolved by PAGE and the DNA fragments were recovered as described
previously (37) before being used as template for asymmetric PCR.
Templates for the asymmetric PCR were derived from two or more
independent PCR products. PCR controls with no DNA and minus DNA
polymerase were always performed. Nucleotide sequence analysis was
performed on both strands derived from several PCRs.
RNA Isolation and Generation of cDNA by RT-PCR
Gonadal fibroblasts from patient 1 (CI) and skin fibroblasts of
the labia majora of patient 2 (CII) were cultured in DMEM with 10% FBS
and 1% antibiotic-antimycotic (Life Technologies, Inc., Gaithersburg,
MD). Total cellular RNA was extracted from confluent cultures using the
acid guanidinium thiocyanate-phenol-chloroform procedure described by
Chomczynski and Sacchi (38). One microgram of total cellular RNA was
used for a 20 µl RT reaction using a random polyhexamer primer (PdN6)
as described previously (39). The RT product was used as template for
amplifying the sequence encoded in exon 2 to exon 11 using a 5'-exon 2
primer (nucleotides 162182, 5'-ATCACTTGCCTACCTCCCTGT-3') (primer u)
and a 3'-primer located within exon 11 (nucleotides 12831304,
5'-GTCTGCCAGTCTATGGCATGGT-3') (primer v) in a 28-cycle PCR. The
conditions of the PCR were the same as described for genomic PCR.
Control experiments with HEK 293 cell RNA (irrelevant RNA) and minus
reverse transcriptase were always performed with the RT-PCR. Nucleotide
sequence of the transcript cDNA was determined using appropriate
primers.
Generation of hLHR-Ins Construct
The pcDNA3hLHR-WT cDNA recombinant plasmid was a generous gift
from Dr. Aaron J. W. Hsueh. The KpnI-Eco 47III fragment
in exon 1 of the hLHR-WT cDNA insert in pcDNA3hLHR-WT recombinant
plasmid was released by double digestion of the plasmid with
KpnI and Eco 47III. The released fragment was removed by
electrophoresis through 1% SeaPlaque gel (FMC BioProducts, Rockland,
ME). A KpnI-Eco 47III fragment containing the 33-bp
insertion was obtained by KpnI-Eco 47III double digest of
the amplified exon 1 of the maternal hLHR gene containing
the insertion. This fragment was purified by electrophoresis through
3.5% NuSieve gel (FMC BioProducts). After ligation, the pcDNA3hLHR-Ins
construct was used to transfect HEK 293 cells.
Examination of the Expression of the Mutated hLHR in HEK 293
Cells by PCR
Human embryonic kidney (HEK) 293 cells were cultured in DMEM/F12
supplemented with 5% FBS, 100 µg penicillin/streptomycin, and 2
mM glutamine as described previously (8). Cells in a T-75
flask were transfected with 10.8 µg of DNA (19/20 pcDNA3 construct
plasmid DNA and 1/20 ß-galactosidase plasmid DNA), and T-25 flasks
were transfected with 3.6 µg of DNA using lipofectamine (Life
Technologies, Inc., Gaithersburg, MD) as described by Hawley-Nelson
et al. (40). Cells were cultured for 48 h after
transfection before being used for experiments. To assay for the
expression of hLHR-Ins, cells in T-75 flasks were lysed and total
cellular RNA was isolated as described above. One microgram of total
cellular RNA was used for a 20-µl RT reaction. Half of the RT product
was used as template for amplifying hLHR with nested PCR. Twenty-eight
cycles of PCR primed by 5'-exon 9 primer (primer w) and 3'-exon 11
primer (primer v) were first done. The PCR product was then diluted
1:85 and subjected to another 21 cycles primed with a primer internal
to primer w (primer x complementary to the 5'-end of exon 10,
nucleotides 866891) and the same 3'-exon 11 primer. Five microliters
of the PCR product were analyzed by PAGE. Half of the RT reaction was
used as template for a 28-cycle PCR amplification of G3PDH as described
previously (39).
cAMP Studies
To examine the synthesis of cAMP by cells expressing the mutant
hLHR construct, HEK 293 cells were cultured in 24-well plates (4
x 104 cell per well). Cells were transfected with 0.35
µg of DNA/well (19/20 pcDNA3 construct plasmid DNA and 1/20
ß-galactosidase plasmid DNA) using lipofectamine. Cells were cultured
for 48 h after transfection before use in experiments. To control
for transfection efficiency, cells were harvested, and the activity of
ß-galactosidase in the cell lysate preparations was assayed as
described previously (8). To assay for the effect of hCG on
intracellular cAMP production, cells were treated with 0.11000 ng/ml
hCG [CR127: 14,900 IU/mg, obtained through National Hormone and
Pituitary Program (NHPP), National Institute of Diabetes and
Digestive and Kidney Diseases (NIDDK), National Institue of Child
Health and Human Development (NICHHD), US Drug Adminstration (USDA)]
for 2 h at 37 C, with 5% CO2, 0.25 mM
3-isobutyl-1-methyl-xanthine, and 0.1% BSA. Cells were treated with
3% perchloric acid and neutralized with potassium hydroxide, and the
amount of cAMP in the lysate was assayed using the cAMP enzyme
immunoassay system (Amersham Life Science, Arlington Heights,
IL). Results were normalized for ß-galactosidase activity by the same
cell preparation to correct for transfection efficiency.
Binding Studies
To examine cell surface binding of hCG by transfectants
expressing mutant LHR, the procedure of Lloyd and Ascoli (41) was
followed with modifications. Each well of a 12-well plate was seeded
with 8 x 104 HEK 293 cells. Cells were transfected
with 0.54 µg of DNA/well (19/20 pcDNA3 construct plasmid DNA and 1/20
ß-galactosidase plasmid DNA) using lipofectamine. Forty-eight hours
after transfection, cells were chilled in the cold room for 20 min
before the addition of a fixed amount of [I125]-hCG
(
8 x 105 cpm/10 ng hCG/well) (DuPont NEN, Boston,
MA) and designated amounts of unlabeled hCG in binding buffer [200
mM HEPES (Sigma Chemical Co., St. Louis, MO), pH 7.4, 2.5%
BSA (Fisher Scientific, Fairlawn, NJ) in DMEM-F12 (Biofluids, Inc.,
Rockville, MD). The cells were left at 4 C for 24 h. The
radioactive media were aspirated and the cells were washed with washing
buffer [92.5% binding buffer, 7.5% horse serum (Biofluids, Inc.,
Rockville, MD)]. The aspirate and the washing buffer were pooled and
counted. Cells were collected with solubilizing solution (0.2
N NaOH, 150 mM NaCl, 40 µg salmon sperm
DNA/ml) and counted in a Beckman
-4000 counter.
For binding study using detergent-solubilized receptor, 2 x
106 HEK 293 cells were cultured in T-75 flasks. Cells were
transfected with 10.5 µg DNA/flask (95% construct plasmid DNA and
5% ß-galactosidase plasmid DNA), using lipofectamine.
Forty-eight hours after transfection, cells were harvested in ice-cold
buffer A (150 mM NaCl, 10 mM sodium phosphate,
pH 7.4) supplemented with protease inhibitors (20 µM
leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 100
kallikrein inhibiting units/ml aprotinin, all from Sigma) as described
previously (42). Cells were lysed for 45 min on ice with 1% Triton
X-100, 30% glycerol in buffer B (150 mM sodium chloride,
20 mM HEPES, pH 7.4) supplemented with protease inhibitors.
The supernatants were collected after centrifugation and used for
binding assay (8). Equilibrium binding to the detergent-solubilized
receptors was performed by incubating aliquots of the extracts (diluted
with buffer B containing 0.1% Triton X-100 and 20% glycerol and
supplemented with protease inhibitors) at 4 C for 24 h with a
fixed amount of [I125]hCG (
4.7 x 105
cpm/5 ng hCG) in the presence of designated amounts of unlabeled hCG.
Separation of receptor-bound [I125]hCG from unlabeled
hormone was performed by polyethyleneglycol precipitation as described
previously (8). All determinations were done in duplicate.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Aaron J. W. Hsueh for the pcDNA3-hLHR-WT
construct and his most valuable help and advice.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Wai-Yee Chan, Department of Pediatrics, Georgetown University Childrens Medical Center, 3800 Reservoir Road, NW, Washington, DC 20007. E-mail:
wchan02{at}gumedlib.dml.georgetown.edu
This work was supported in part by NIH Grant HD-31553.
1 Deceased. 
2 Present address: St. Georges Hospital, Blackshaw Road, London SW17
0QT, UK. 
3 Present address: Lilly Research Laboratories, Eli Lilly and Company,
Lilly Corporate Center, Indianapolis, Indiana 46285. 
Received for publication February 6, 1998.
Revision received July 14, 1998.
Accepted for publication July 16, 1998.
 |
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