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 Guy’s 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (1822–1827, {Delta}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{alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go. Examination of exons 2–10 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 2111–2135, 5'-AATAATGCAGTTACTGATGTAACAG-3'); u, 5'-exon 2 primer (nucleotide 162–182, 5'-ATCACTTGCCTACCTCCCTGT-3'); v, 3'-exon 11 primer (nucleotide 1283–1304, 5'-GTCTGCCAGTCTATGGCATGGT-3'); w, 5'-exon 9 primer (nucleotide 681–704, 5'-GGATATTTCTTCCACCAAATTGCA-3'); x, 5'-exon 10 primer (nucleotide 867–891, 5'-ACAGAATTTTTCACATTCCATTTCT-3').

 
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 2Go 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. 2Go.



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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.

 
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. 3AGo. 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. 3BGo. 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, {phi}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.

 
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 4Go 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. 2Go. 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 1–3, 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 4–6, 261 bp, G3PDH).

 
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. 5Go. 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. 5Go). 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

{circ}, hLHR-WT cells transfected with hLHR-WT construct DNA; •, hLHR-Ins cells transfected with hLHR-Ins construct DNA (pcDNA3); {triangleup}, 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 0.1–1000 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.

 
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. 6AGo). This difference persisted when hCG binding was compared between detergent-solubilized lysate of transfected cells expressing hLHR-WT and those expressing hLHR-Ins (Fig. 6BGo). 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. {circ}, hLHR-WT cells transfected with hLHR-WT construct DNA; • hLHR-Ins cells transfected with hLHR-Ins construct DNA (pcDNA3); {triangleup}, 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 2–11 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. 2Go) 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 26–50, and nine leucine-rich repeats encoded by nine exons each containing 21–26 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 Huntington’s 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).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 319–699, 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. 1Go. 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. 1Go). 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 162–182, 5'-ATCACTTGCCTACCTCCCTGT-3') (primer u) and a 3'-primer located within exon 11 (nucleotides 1283–1304, 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 866–891) 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 0.1–1000 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 {gamma}-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 Children’s 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. Back

2 Present address: St. George’s Hospital, Blackshaw Road, London SW17 0QT, UK. Back

3 Present address: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285. Back

Received for publication February 6, 1998. Revision received July 14, 1998. Accepted for publication July 16, 1998.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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