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Original Studies |
IRIBHN (P.R., F.C., S.C., M.T., L.D., C.G., G.V.) and Service de Génétique Médicale (G.V.), Faculté de Médecine, Université Libre de Bruxelles, B-1070 Brussels, Belgium; Department of Obstetrics and Gynecology (T.M.), Gumna University Japan
Address correspondence and requests for reprints to: Patrice Rodien, Faculte de Medecine, Campus Hospital Erasme, Route de Linnik 808, Bruxelles 1070 Belgium.
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Abstract |
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LQ) allele are 0.26 and 0.74 respectively. In
contrast, the LQ allele is virtually absent from the Japanese
population. Functional characterization of both alleles by transient
expression in COS-7 cells did not reveal any difference between the two
receptors, neither for cell surface expression nor for cAMP production
and sensitivity to hCG/LH. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the frame of our studies aiming at understanding the mechanisms of glycoprotein hormone receptor activation, we had been using systematically the shorter LH/CG complementary DNA lacking the Leu-Gln insertion. We were thus concerned that this receptor would constitute a variant, not representative of the more prevalent "wild-type" sequence. This led us to explore the structure of exon 1 of the human LH/CGr in samples of the Caucasian and Japanese populations. Whereas Atger’s sequence with the Leu-Gln insertion constituted a frequent polymorphic variant in the Caucasian population, it was virtually absent in the Japanese. While this study was in process, Tsai Morris et al. (6) described the isolation of both sequences from a genomic library, which led them to propose the existence in the haploid human genome of at least two human LH/CGr genes, located on the same chromosome. Contrary to this conclusion, our study indicates the existence of a single hLH/CGr locus with two different alleles displaying identical functional characteristics.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genomic DNA was studied from 102 unrelated Caucasian subjects, provided by the CEPH, and from 110 unrelated Japanese patients exhibiting thyroid nodules.
PCR amplification of exon 1 of the human LH/CGr was conducted according to Atger et al. (4) with the following primers: hLH/CGr1: CACTCAGAGGCCGTCCAAG and hLH/CGr2: GGAGGGAAGGTGGCATAGAG. Amplicons were then submitted to electrophoresis on a 2.2% agarose gel following standard procedures, either directly or after digestion with PVUII. (Gibco BRL Life Technology).
Sequencing reactions were performed on both strands with the Taq Dye Deoxy Terminator Cycle sequencing kit (Applied Biosystems), and sequencing products were run and analyzed on an automated sequencer (Applied Biosystems 373).
Construction of the LQ variant To introduce the LQ, insertion amplification of the first exon of the hLHr was realized on genomic DNA of a Caucasian subject homozygote for the presence of the insertion with hLH/CGr2 and a direct primer introducing a XhoI site and a consensus sequence (GGAAAA): TGGCCTCGAGGAAAAATGAAGCAGCGGTTCTCGGC. A HxoI–NarI fragment was ligated in orthotopic position in the hLHr encoding plasmid previously described (7). Sequence of the constructs was verified by sequencing both strands up to the ligation sites.
Functional characterization of receptors Transient transfection in COS-7 cells, cAMP measurement, and binding experiments were essentially performed as previously described (8), except that binding incubations lasted overnight at 18C. Cell surface expression was assessed by FACS analysis using a mouse polyclonal serum generated by DNA immunization (9, and manuscript in preparation). Products Taq polymerase and restriction enzymes were purchased from Gibco BRL Life Technology. 125I hCG (Specific activity: 84mCi/mG) was purchased from Dupont de Nemours, NEN Research Products, Belgium. Cold hCG was purchased from Sigma-Aldrich, Belgium.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PCR amplification of the first exon of the hLH/CGr generated a
segment migrating in 2.2% agarose gel as a 290-bp band. In some
Caucasian samples a two-band pattern was observed, suggestive of a
heterozygous state (Fig. 1a
). Direct DNA
sequencing (Fig. 2) of such a sample
confirmed heterozygosity with an inframe insertion of 6 bp, encoding
Leu-Gln at nucleotide positions 55–60 (aminoacid residues 19–20).
However, the difference in size between the two alleles could not
account for the apparent size differences of the two bands on agarose
gel. The slow migrating band resulted from the aberrant migration of
heteroduplexes in heterozygous samples: mixing of two samples
homozygous for each of the alleles generated a two-band pattern only
when allowing for denaturation and rehybridization before
electrophoresis.
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LQ) were 0.26 and 0.74,
respectively. The genotype frequencies of the two types of homozygotes
and of heterozygotes were 0.088 (LQ/LQ), 0.558 (LQ/LQ), and 0.354
(LQ/LQ), respectively. This is in close agreement with the expected
frequencies, assuming Hardy-Weinberg equilibrium (0.07, 0.55, and
0.38). In contrast, in a population of Japanese subjects (220
chromosomes investigated), the LQ allele was never found. Comparison of functional characteristics of the two alleles
Constructs encoding the LQ or LQ LHr were transfected in COS-7
cells. The same level of expression was achieved at the cell surface
for both receptors, as assessed by flow immunocytometry using a
polyclonal antibody generated in mice by DNA immunization (Fig. 3). Basal as well as hCG-stimulated cAMP
production were similar for the LQ and LQ receptors (Fig. 4a). Similarly, binding experiment did
not reveal any significant difference between the two constructs, with
apparent Kd of 299 ± 25 mUI/mL and 395 ± 33 mUI/mL for LQ
and LQ receptors, respectively (Fig. 4b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LQ)
from a human placental genomic DNA library. Sequencing of the 5'
untranslated and promoter regions revealed additional divergences
between the two sequences. They concluded to the existence of two
LH/CGr genes with different tissue-specific transcription patterns.
We consider that the present data, together with previous studies
reporting direct sequencing of LH/CG receptor mutants (10), indicate
that the two sequences are allelic. Indeed, if more than one LH/CG
receptor locus existed, homozygosity for point mutations would not be
displayed by direct sequencing, as demonstrated in patients with
aminoacid substitutions leading to hypergonadotrophic hypogonadism
(10). In addition, our finding that both kinds of homozygous
individuals (LQ/LQ and LQ/LQ) do exist in Caucasians at the
frequency expected from the Hardy-Weinberg equilibrium, makes a
duplication of human LH/CGr gene extremely unlikely. The gene dosage
experiments, as performed by Tsai Morris et al. (6), are
reportedly very difficult and should be interpreted with extreme
caution (11). From our population study, the shorter form of the
LH/CGr, the major form of ovarian LH/CGr according to Tsai Morris
et al. would simply be missing in around 9% of Caucasians
who are homozygous for the LQ allele. We suggest that the isolation of
the two sequences by Tsai Morris et al. most probably
reflects the heterozygosity of the genomic DNA library they used as
their starting material. Interestingly, whereas the LQ allele is
present at a relatively high frequency in a normal Caucasian
population, it is completely absent in the Japanese group we have
studied here.
When comparing the hLH/CGr aminoacid sequence with the recently cloned
monkey sequence (12), it appears that a Leu-Leu insertion also exists
in the same position in the simian sequence. This indicates that the
allelic LQ "variant" would actually be closer to the ancestral
primate gene than the LQ allele described originally. It suggests
also that the absence of the LQ allele in Japanese results from a
founder effect.
No functional difference could be documented between the short and long receptors, which, in agreement with the high frequency of both alleles, even in the homozygous state, seems to rule out any direct involvement of this polymorphism in pathological conditions. However, it must be acknowledged that subtle differences in maturation of the protein, due to the proximity of the insertion with the signal peptide cleavage site, could have been overlooked in our experimental eucaryotic overexpression system. It is conceivable that such undetected differences, in association with other factors like LH variants (13), could have functional significance. Finally, this LH/CGr polymorphism constitutes a convenient marker in screening families segregating testotoxicosis or hypogonadism due to LH/CGr inactivating mutations.
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Acknowledgments |
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Footnotes |
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2 Present addresses: Clinique des maladies endocriniennes
et metaboliques, hopital Cochin, Paris, France.
3 Present address: Istituto di Endocrinologia,
Universita di Pisa, Italy.
Received July 16, 1998.
Accepted August 31, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or hLHrLQ (
) in response to hCG
stimulation. Panel B: 125I hCG binding on COS 7 cells
transiently expressing hLHr (