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Polymorphism
of gonadotropin action: clinical implications
Ilpo
T. Huhtaniemi
Department
of Physiology, University of Turku, Kiinamyllynkatu
10, 20520 Turku,
Finland
Department of Obstetrics and Gynaecology, University of Aberdeen, Aberdeen
AB 24 2ZD, Scotland, UK
Asian J Androl 2000 Dec; 2: 241-246
Keywords:
LH;
FSH; bioassay; immunometric assay; mutation; polymorphism; glycosylation
Abstract
It has recently became apparent that the structural heterogeneity of gonadotropin molecules can contribute to variations of their action in different physiological and pathophysiological conditions. One reason for the structural variations of circulating gonadotropin molecules is the microheterogeneity caused by the variability of glycosylation of individual gonadotropin molecules. The carbohydrate moieties of gonadotropins are important for their intrinsic bioactivity, as reflected by measurement of their bioactivity to immunoreactivity (B/I) ratios. We have reassessed this phenomenon by improved in vitro bioassay and immunoassay methods, and it appears that the intrinsic bioactivity of gonadotropins, in particular of LH, is more constant than previously assumed. Many of the previously documented differences, some even considered diagnostic for certain clinical conditions, have turned out to be methodological artifacts. The first part of this review summarizes our recent findings on the B/I ratios of LH, with special reference to the male.
The second part of this review describes a common polymorphism that was recently discovered in the gene of the LH -subunit. The variant LH allele contains two point mutations, which introduce to LH two amino acid changes and an extra glycosylation site. The LH variant is common world-wide, with carrier frequency varying from 0 to 52% in various ethnic groups. The LH variant differs functionally from wild-type LH, and it seems to predispose its carriers, both men and women, to mild aberrations of reproductive function. It is important for the clinician to be aware of this variant LH form, not detected by all immunoassays, because it may explain some aberrant results of LH measurements in patient samples.
1 Introduction
The
two pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating
hormone (FSH), together with chorionic gonadotropin (CG), belong to the
family of glycoprotein hormones.
The fourth member of this family of hormones is thyroid-stimulating
hormone (TSH). They
all are composed of the common -subunit (C) and a hormone-specific
-subunit, which are coupled by noncovalent interactions[1].
In addition, there are N-linked carbohydrate side chains
in the gonadotropin subunits, two in C, one in LH, two in FSH, and
two in GC, and there are four O-linked carbohydrate side chains in the
24-amino acid C-terminal extension of the CG chain.
Each gonadotropin dimer has a molecular weight of about 30,000.
While
the -subunits determine the functional specificity of gonadotropins,
their intrinsic bioactivity is to a great extent determined by their degree
of glycosylation. Weakly
glycosylated forms of the hormones have short circulatory half-time, and
although totally deglycosylated gonadotropins are able to interact with
their cognate receptor, they are unable to evoke generation of the second
messenger signal[2].
Since gonadotropins are secreted from the pituitary as a mixture
of differently glycosylated isoforms, with composition varying according
to the physiological state, it is reasonable to hypothesize that the intrinsic
bioactivity of gonadotropins is one variable determining their overall
function.
The
physiologic functions of gonadotropins are relatively well-known today.
FSH stimulates in the ovary follicular maturation and granulosa
cell estrogen production,
whereas in the testis it plays a role in stimulating Sertoli cell functions,
thereby furthering indirectly spermatogenesis.
LH stimulates in the ovary theca cell androgen production, triggers
ovulation, and maintains progesterone production of corpus luteum.
In the testis, LH stimulates Leydig cell androgen production.
Hence, the two gonadotropins are essential for normal gonadal function
and fertility. It is therefore
understandable that mutations in gonadotropin genes are extremely rare,
since they severely affect fertility.
However, sporadic cases of mutations have been detected in all
gonadotropin subunit genes[3].
Despite their rarity, these cases have appeared very elucidating,
by demonstrating the specific functions of the two gonadotropins in conditions
where one of them
is selectively eliminated.
2 Novel findings on bioactivity to immunoreactivity (B/I) ratios of LH
Besides
immunoassays (RIA, IRMA, ELISA etc.), in vitro bioassays, also
sensitive and specific, can be used for measurement of gonadotropins in
clinical serum samples[4-8].
The newest forms of these assays use cell lines expressing recombinant
human gonadotropin receptors, providing greatly improved interassay variability.
The immunoassays mainly monitor the amount of immunoreactive hormone molecules
in the sample, without taking into account the biological activity of
the hormone measured. The
bioassays measure this very feature of the hormone, but do not always
correlate with the number of hormone molecules.
This discrepancy has been attributed to the microheterogeneity
of gonadotropin molecules, in particular to variations in their glycosylation.
The differently glycosylated gonadotropin molecules have variable
intrinsic bioactivities. Therefore,
the concentrations of gonadotropins measured by the two principally different
assay systems do not always agree.
The bioactive (B) to immunoreactive (I) ratio of LH and FSH in
a serum sample, i.e., the bio/immuno (B/I) ratio, has been widely used
as an indicator of the quality of gonadotropins.
It reflects the average bioactivity of a gonadotropin molecule
in the sample, and it has been shown to vary in predictable manner in
various physiological and pathophysiological conditions.
The
current in vitro bioassay methods for LH are technically easier
and more reliable than those for FSH, and still the majority of information
on the B/I ratio measurements
of gonadotropins are on LH[4-8].
The B/I ratios of LH have been documented to increase in males
during endogenous and GnRH-stimulated LH pulses[9], during
pubertal maturation[10-12] and after orchidectomy[13].
Low or decreased
B/I ratios of LH have been measured in men in hypogonadotropic
hypogonadism[14], during treatment with GnRH agonists[15],
estrogen[16] and androgen[17], during renal failure[18],
in idiopathic infertility[19] and in impotence[20].
However, although alterations in the degree of glycosylation of
the circulating LH molecules have been discovered, the biochemical basis
of the altered B/I ratio changes has so far remained elusive in this large
number of studies.
With
the advent of more sensitive and specific immunometric assay methods for
gonadotropins, e.g. the immunofluorometric assay, IFMA[21],
it has become apparent that many, though not all, of the previously detected
changes in the B/I ratio of LH are due to biased immunoreactivity measurements
by conventional radioimmunoassays (RIA), which often overestimate low
hormone concentrations[22-25]. The
B/I ratios are therefore low at low hormone concentrations and systematically
increase when the hormone levels increase.
A higher fold-increase occurs in B-LH
resulting in artifactual elevation of the B/I ratio.
We have previously demonstrated this bias during pulsatile LH secretion[25]
and after GnRH stimulation[24] in healthy men, after gonadotropin
suppression during GnRH agonist treatment
of prostatic cancer patients[24], and in boys with hypogonadotropic
hypogonadism[23].
One widely cited finding on increased B/I ratios of LH is that during pubertal maturation[10-12]. This information, also cited in textbooks of endocrinology, states that a crucial event during puberty is, besides reactivation of gonadotropin secretion, an increase in the intrinsic bioactivity of LH. Since the earlier studies reporting this finding used conventional RIA for I-LH measurement, we found it important to reassess this finding by eliminating the bias of RIA with a novel sensitive and specific IFMA[26]. Since also the B-LH levels are low in peripubertal serum samples, we sensitized the in vitro bioassay, as originally described by Debertin & Pomerantz[27], for measurement on these samples. On the average, a 10-fold increase in the sensitivity of the bioassay was achieved, down to 0.05 IU/L.
Figure 1 shows the levels of B-LH, I-LH and the B/I ratios at different stages of puberty in healthy boys. I-LH increased between pubertal stages I and IV (according to Tanner) from 0.420.13 to 2.240.34 IU/L (P<0.01), and B-LH from 1.350.49 to 5.040.78 IU/L (P<0.01). In contrast, no significant change was observed during the same period in the B/I ratios of LH which varied between 2.58-2.84. In conclusion, if sensitive IFMA and in vitro bioassay methods are used, no alteration can be demonstrated in the intrinsic bioactivity of LH, as reflected by the B/I ratio, during the pubertal maturation of healthy boys.
Figure
1. The mean (meanSEM) level of B-LH (top panel), I-LH (middle panel),
and the B/I ratio
(bottom panel) in 14 perimenopausal boys studied according to their stage
of puberty (I-IV). The
number of subjects analyzed at each pubertal stage is presented in brackets
in the bottom panel. When
different letters are above the bars these results differ significantly
from each other (A vs B, P<0.01)[26].
The above study showed yet another physiological condition where a previously demonstrated changes in the quality of LH could not be confirmed with more sensitive and specific assay methods. It is therefore apparent that the clinical value of in vitro bioassays of LH is not as great as previously assumed. The improved immunoassays monitor reliably the LH levels and correlate well with the bioactivity of the hormone. The clinical use of in vitro bioassays of LH may therefore be limited to cases where a discrepancy is found between the I-LH level of the patient and the clinical picture. There may be conditions where the immunoassays using monoclonal antibodies are too specific and do not detect all LH isoforms, or the hormone of the patient may represent a structural variant. An example of such a condition is described in the next paragraph.
3 A common genetic variant of luteinizing hormone
When
we tested the suitability of various monoclonal antibody (Mab) combinations
for LH measurement by the immunofluorimetric assay (IFMA), we identified
a healthy woman with two children, whose LH was not at all detected by
a specific Mab combination[28].
The antibody that did not recognize her LH was directed against
an epitope in the intact LH / dimer
(assay 1). Interestingly,
all other Mab combinations test (, and / specific) measured normal
LH concentrations in her serum.
Likewise, her LH bioactivity and the B/I ratio of LH (using a subunit-specific
IFMA for I-LH measurement, assay 2) were within the normal range, and
in accordance with her normal clinical status, including normal fertility.
Since the FSH and TSH levels of the subject were also normal, we
concluded that her LH subunit had to be structurally abnormal, probably
due to altered gene structure.
The LH levels of her family members were also analyzed, and it
turned out that her mother had similar nondetectable LH by assay 1, but
her father and children
displayed LH levels by assay 1 that were about 50% of the levels measured
by the reference assay 2, hence the ratio of LH with assay 1/assay 2 was about
0.5. This further strengthened
the assumption that the aberrant LH form was due to a mutation with Mendelian
fashion of inheritance. The
subject and her mother were apparent homozygotes, and the father and children
heterozygotes with respect to a putative mutated LH allele.
A scheme of the molecular alteration of the variant LH and its
influence on reactivity of the different epitopes with the Mab's test
is presented in Figure 2.
Figure
2. Schematic
presentation of the reason of aberrant immunoreactivity in individuals
with the variant form of LH in two-site immunometric assays.
The changes in the amino acid sequence and/or an extra carbohydrate
chain in the LH -chain block or eliminate the antigenic epitope present
in the / dimer. Combination
of monoclonal antibody (MAB) / and MAB- was
used in assay 1, and MAB- and MAB- in assay 2. #=carbohydrate chain.
When
a larger number (n=249) of serum samples from healthy Finnish volunteers
were analyzed (Figure 3), and
the ratio of LH by assay 1/assay 2 was measured, the results fell clearly
into three categories:
(1) 1.0-2.0, i.e., normal ratio individuals, (2)
0.5-0.75, i.e., low ratio individuals, and (3) the ratio near 0,
i.e., zero ratio individuals.
The combined frequency of the low (heterozygotes) and zero (homozygotes)
ratio LH types in the Finnish population was 28%, and the
distribution was in the Hardy-Weinberg equilibrium.
Figure
3. The distribution of 249 normal Finnish subjects in the normal (squares),
low (circles), and zero (triangles) ratio groups according to the results
of the ratios of LH measured by assays 1 and 2.
The LH level measured by assay 2 is shown on the abscissa.
As no sex differences were detected, the male and female data
are compiled[31].
We
then sequenced the LH subunit gene of our original subject with the
zero ratio LH[29].
Two point mutations, both resulting in amino acid change, Trp8Arg8
(TGGCGG) and Ile15Thr15 (ATCACC), were detected
(Figure 4).
The latter mutation introduces a new glycosylation signal (Asn-X-Thr)
into the LH chain,
which results in
oligosaccharide chain attachment into Asn13[30].
The same tripeptide glycosylation signal is present in the hCG
-chain where Asn13
Figure
4. Sequences of the first 20 amino terminal amino acids in the the
-chains of wild-type
LH (LH-), variant LH (LH-VAR-) and hCG
(hCG). The nucleotide
changes between wild-type LH and the other -chains are
marked above amino acids 8 and 13.
In addition, amino acids 3 and 10 in hCG- are surrounded by boxes
to indicate their difference from the other two LH -chains.
The
homozygote men and women for the LH variant allele that have been detected
in Finland are apparently healthy, with no reported infertility.
In this respect our findings differ from reports from Japan[34-36],
where the same LH variant was related to infertility.
We have observed some functional differences between the LH variant
as compared to wild-type hormone.
Its B/I ratio
is higher, but the half-time in circulation shorter[31].
Since the pulse-frequency of the variant LH was normal, the overall
action of the variant hormone, although more potent
at the receptor site, is shorter in duration in vivo.
Such a change in
the half-time of LH may be of physiological importance.
We are gradually accumulating data that this is indeed the case.
In some cohorts, men heterozygous for the LH variant have slightly
but significantly lower serum concentration of testosterone than men with
wild-type LH (our unpublished observation).
Women with at least one variant LH allele have higher levels of
serum testosterone, estradiol and sex hormone-binding globulin, and the
frequency of the variant alleleis altered in certain subtypes of polycystic
ovarian disease[32,37].
Boys with variant LH have normal age of onset, but slower progression
of puberty[38]. During the final stages of puberty their height,
testis weight, plasma IGF-1 binding protein-3 concentration and blood
hemoglobin are significantly lower than in matched control boys with normal
ratio LH.
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Correspondence
to: Ilpo
Huhtaniemi, M.D., Ph.D. Professor of Reproductive Biology,
Department of Obstetrics and Gynaecology,
University of Aberdeen, Aberdeen AB24 3ZD, Scotland, UK.
Tel: +44-1224-559 483 Fax: +44-1224684 880
e-mail: ilpo.huhtaniemi@utu.fi
Received
2000-07-26 Accepted 2000-08-28
