Variant LH Research Articles

The effects of luteinising hormone gene polymorphism on the outcomes of in vitro fertilisation and embryo transfer

Trp8Arg polymorphism of the LH beta gene has decreased bioactivity in vivo and previous studies showed conflicting data on the effect of LH beta gene polymorphism on the IVF outcome. In this study, 591 IVF patients were recruited. Patients with the variant allele(s) were the carrier group. In GnRH antagonist cycles, the clinical pregnancy rate was significantly lower in the carrier group (18.9%) than in the noncarrier group (37.1%). In long GnRH agonist cycles, the clinical pregnancy rate was comparable between both groups. To clarify the effect of COH protocols, IVF outcomes in the GnRH antagonist and long GnRH agonist protocol groups in carriers were analysed. Among carriers, the clinical pregnancy rate was significantly lower in the GnRH antagonist protocol group (18.9%) than in the long GnRH agonist protocol group (45.2%). Single nucleotide polymorphism analysis may contribute to the individualisation of COH protocols for each patient in the future. Impact Statement What is already known on this subject? Trp8Arg polymorphism of the LH beta gene is known to have decreased bioactivity in vivo. Previous studies have demonstrated hypo-sensitivity in the patients with the variant LH beta protein, while other study showed similar carrier frequency between the poor and the normal response group. What the results of this study add? The variant LH beta gene was associated with a lower clinical pregnancy rate in GnRH antagonist cycles but not in long GnRH agonist cycles. What the implications are of these findings for clinical practice and/or further research? Single nucleotide polymorphism analysis may contribute to the individualisation of COH protocols for each patient in the future.

A common polymorphic allele of the LH beta-subunit gene is associated with higher exogenous FSH consumption during controlled ovarian stimulation for assisted reproductive technology

BackgroundV-betaLH is a common genetic variant of LH caused by two polymorphic base changes in the beta subunit gene, altering the amino acid sequence (Trp8Arg and Ile15Thr). In a previous-preliminary trial performed in women undergoing IVF, it was demonstrated that carriers of v-betaLH show sub-optimal ovarian response to a standard long GnRH-agonist down -regulation protocol when stimulated with pure recombinant FSH (r-hFSH). The aim of this study was to confirm the hypothesis that women with v-betaLH display hypo-sensitivity to exogenous FSH in a larger IVF population and to explore the frequency of this variant in a Danish female population.MethodsIn the present study, the effect of v-betaLH was retrospectively investigated in a larger series of women undergoing controlled ovarian stimulation (COS) and, for the first time, in a Danish IVF population. A total of 220 normogonadotrophic women following a long GnRH-agonist down-regulation protocol received an individualized dose of r-hFSH (100 IU and 375 IU s.c. daily) according to antral follicle count, baseline FSH, body mass index and age. The LH genotype was assessed in all patients by immunofluorometric assay.ResultsV-betaLH was present in 11% of patients, whereas the allelic frequency was 12%. The study population was divided into two groups according to their LH genotype. Group A consisted of 196 wt/wt women. Group B included 24 individuals with v-betaLH (21 heterozygous and 3 homozygous). No statistically significant differences in the mean number of oocytes retrieved, fertilization rate and pregnancy rate per cycle were observed between groups. However, Group B received a significantly higher cumulative-dose of r-hFSH than Group A (2435.86 +/− 932.8 IU versus 1959.8 +/− 736.45 p = 0.048). When one-way ANOVA in a within design was applied, the LH genotype had a statistically significant effect (p < 0.01) on the cumulative dose of r-hFSH, showing a progressive increase from wt/wt (1959.8 +/− 736.45 IU) to v-betaLH hetero- (2267.5 +/− 824.3) and homozygotic women (3558.3 +/− 970.9).ConclusionsThese results confirm that carriers exhibit hypo-sensitivity to exogenous FSH during COS, documenting that the frequency of v-betaLH in Denmark is similar to a number of European countries.

The Common Genetic Variant of Luteinizing Hormone Has a Longer Serum Half-Life than the Wild Type in Heterozygous Women

The common genetic variant of human LH has two mutations and an extra N-linked oligosaccharide chain, a modification expected to affect the half-life in the circulation. Our objectives were to determine the half-lives of variant and wild-type forms of LH during GnRH receptor blockade in heterozygous women and to determine the time-related changes in isoform composition. Serum samples were obtained from three healthy women heterozygous for variant LH before and up to 20 h after administration of the NAL-GLU GnRH antagonist. The half-lives were estimated by monoexponential decay. The number of sialic acid and sulfonated N-acetylgalactosamine residues per wild-type and variant LH molecule and the distribution of molecules with zero, one, two, or three sulfonated residues were measured. The variant LH had a half-life that was approximately 40% longer than the corresponding forms of wild-type LH (148 vs. 108 min; P < 0.001). Variant LH had more sialic acid residues per molecule than wild type (3.6 vs. 2.4; P < 0.05), whereas the number of sulfonated residues was similar (1.0 vs. 0.98). The decline in the variant LH during GnRH receptor blockade was associated with a decrease in sulfonated and an increase in sialic acid residues similar to that for in wild-type LH. Isoforms of either variant or wild-type LH with two to three sulfonate groups per molecule had the shortest half-life. Variant LH remains longer in circulation than wild type during GnRH receptor blockade in heterozygous women, in accord with its higher content of sialic acid.

Comparison and Problems of Measured Values of LH, FSH, and PRL among Measurement Systems

Several measurement systems are used for LH, FSH, and PRL in Japan, but the comparison of the measured values is difficult in some cases. In this study, we compared the measured values of 3 types of measurement systems widely used in Japan, SPAC-S, ARCHITECT, and Centaur, in females with a normal menstrual cycle and with various ovulatory disorders. Variant LH was discriminated by the criterion, SPAC-S LH/ARCHITECT LH ratio <0.5. Excluding the variant LH, the correlation of the measured LH values among the measurement systems was high, 0.94-0.99, and the value measured by ARCHITECT LH and Centaur LH was slightly higher than that measured by SPAC-S LH. As for FSH, the correlation was also high, 0.94-1.00, and the value measured by ARCHITECT FSH and Centaur FSH was slightly lower than that measured by SPAC-S FSH. The important judgment criterion of polycystic ovary syndrome (PCOS), a high level of LH relative to FSH, has been set to 1.0 or higher LH/FSH using SPAC-S. When the regression line of the LH/FSH ratio among the measurement systems was investigated, it can be said that 1.25 or higher by ARCHITECT and 1.40 or higher by Centaur are appropriate for the judgment criteria of a high level of LH relative to FSH. As for PRL, the correlation was high, 0.96-0.99, but the value showed large differences among the systems. For the diagnosis of hyperprolactinemia, the higher limit of the normal range may be 15 ng/mL for SPAC-S, 30 ng/mL for ARCHITECT, and 24 ng/mL for Centaur.

Preparation of specifically activatable endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications

Clostridium botulinum neurotoxins are potently toxic proteins of 150 kDa with specific endopeptidase activity for SNARE proteins involved in vesicle docking and release. Following treatment with trypsin, a fragment of botulinum neurotoxin serotype A that lacks the C-terminal domain responsible for neuronal cell binding, but retains full catalytic activity, can be obtained. Known as the LH N fragment, we report the development of a recombinant expression and purification scheme for the isolation of comparable fragments of neurotoxin serotypes B and C. Expressed as maltose-binding protein fusions, both have specific proteolytic sites present between the fusion tag and the light chain to facilitate removal of the fusion, and between the light chain endopeptidase and the H N translocation domains to facilitate activation of the single polypeptide. We have also used this approach to prepare a new variant of LH N/A with a specific activation site that avoids the need to use trypsin. All three LH Ns are enzymatically active and are of low toxicity. The production of specifically activatable LH N/A, LH N/B, and LH N/C extends the opportunities for exploitation of neurotoxin fragments. The potential utility of these fragments is discussed.

Association of Common LH Variant with Hyperfunctional Promoter in a Japanese Infertile Woman

A common LH variant (V-LH) with Trp8Arg and Ile15Thr is often associated with ovarian dysfunction primarily in the Japanese population, and the LHB gene encoding V-LH is linked with a hyperfunctional promoter that could partly compensate for the somewhat weak biological effect of the V-LH in the Finnish and other several populations. We analyzed the promoter region in a Japanese infertile woman homozygous for the V-LH, to examine whether the hyperfunctional promoter is present or absent in the Japanese V-LH carriers with ovarian dysfunction. Direct sequencing was performed for a 661 bp promoter region from -8 to -668 bp of LHB, revealing homozygosity for eight nucleotide substitutions (-238A>G, -276G>A, -489C>A, -490T>A, -504T>A, -506T>C, -525T>G, and -552C>T) that are identical to those found in the hyperfunctional promoter. The results suggest that ovarian dysfunction frequently observed in the Japanese V-LH carriers would be due to some population-specific genetic and/or environmental factor(s) rather than to the lack of the hyperfunctional promoter and the resultant low biological effect of the V-LH. In addition, the tight linkage between the two missense substitutions in the coding region and the eight nucleotide substitutions in the promoter region of LHB appears to be common to various ethnic groups.

Altered Kinetics of Pituitary Response to Gonadotropin-Releasing Hormone in Women with Variant Luteinizing Hormone: Correlation with Ovulatory Disorders

Objective: The LH response of pituitary gland to gonadotropin-releasing hormone (GnRH) stimulation is not well defined in patients with mutant β-subunit (Trp⁸ to Arg⁸ and Ile¹⁵ to Thr¹⁵). Here we compared the relative activities and dynamics of LH secretion in patients with wild-type and variant LH following injection of GnRH. Methods: A GnRH stimulation test was performed in 33 patients with ovulatory disorders (patient group) and 29 women with normal ovulatory cycles (control group) heterozygous for the variant LHβ allele. Blood samples were obtained up to 120 min after GnRH injection. Serum LH response was determined by comparing the results of LH immunoassays using a monoclonal antibody that recognizes wild-type LH only with those of another assay using a polyclonal antibody that recognizes equally both variant and wild-type LH (total LH). The ratio of variant LH to total LH (LH ratio) was used to determine the serum LH status. Results: The LH ratio in the control group showed the peak 15 min after GnRH injection, while that in the patient group showed the peaks 30–60 min after injection. The LH ratio in the patient group at 120 min after injection was significantly lower than that in the control group. The percent increases in LH ratio in both groups showed the peak 15 min after injection. The patient group had significantly lower changes of LH ratio at 15, 60, 90 and 120 min after GnRH injection compared with that in the control group. Conclusion: Differences in circulatory kinetics of the two types of LH may explain the differences in LH function between patients with ovulatory disorders and women with normal ovulatory cycles.

A novel Ala(-3)Thr mutation in the signal peptide of human luteinizing hormone beta-subunit: potentiation of the inositol phosphate signalling pathway and attenuation of the adenylate cyclase pathway by recombinant variant hormone.

Upon screening for polymorphisms in the human luteinizing hormone beta-subunit (LH beta) gene, we discovered a novel mutation in the LH beta signal peptide with functional consequences for signal transduction in mouse Leydig tumour cells (mLTC-1). This G(52)A point mutation in exon 2 of the LH beta gene, detected in heterozygous form in several normal DNA samples, caused an Ala(-3)Thr amino acid substitution. Recombinant forms of wild-type (WT) and Ala(-3)Thr variant (V) LH were produced in human embryonic kidney (HEK) 293 cells and purified. The immunoreactivities of the recombinant LH were determined by immunofluorometric assays and in-vitro bioactivities in mLTC-1 cells were assessed by using cAMP, progesterone and inositol trisphosphate (IP(3)), and activation of mitogen-activated protein kinase (MAPK) as end-points. Whereas both LH forms stimulated progesterone production and MAPK in similar fashion, WT-LH was more potent in stimulating cAMP, and V-LH was more potent in stimulating IP(3) generation. Both LH forms bound to LH receptors with similar affinities. No evidence was found for influence of the signal peptide mutation on efficacy of alpha- and beta-subunit dimerization. Sequencing of the recombinant V-LH beta protein also revealed that the mutation did not interfere with signal peptide cleavage. In summary, the present findings indicate that the Ala(-3)Thr mutation in the LH beta-subunit signal peptide has functional consequences, in the form of dissociation of stimulatory potency for different signal transduction pathways in vitro.

Disparate response of wild-type and variant forms of LH to GnRH stimulation in individuals heterozygous for the LHbeta variant allele.

There is a common genetic variant of LH due to two amino acid changes in the LHbeta subunit, Trp(8)Arg and Ile(15)Thr. In order to compare the relative activities of wild type (wt-LH) and variant LHbeta (v-LHbeta) genes in LH production and secretion, we performed gonadotrophin-releasing hormone (GnRH) stimulation tests for healthy females (n = 7) and males (n = 10) heterozygous for the v-LHbeta allele. Blood samples were drawn up to 180 min after injection of GnRH. The serum samples were subjected to two immunofluorometric assays, one detecting wt hormone, the other detecting equally both LH types. The wt/total ratio increased significantly (P < or = 0.016) after GnRH injection in males. This indicates that the proportion of wt-LH increases in the circulation in men but not in women, and that women consequently secrete relatively more v-LH. An in-vitro bioassay was performed on 0 and 60 min samples, and the bio/immunoreactivity (B/I) ratio decreased in both sexes (P = 0.010-0.012). This supports the previously reported lower B/I ratio of wt. than v-LH, since wt-LH is expected to accumulate in circulation because of its longer half-life. In conclusion, these findings demonstrate that wt.- and v-LH respond differently to GnRH stimulation in men and women heterozygous for v-LHbeta. These results are in agreement with previously documented differences of the two forms in circulation, as well as with different promoter activities of the two LHbeta alleles.

Increased prevalence of luteinizing hormone β-subunit variant in patients with premature ovarian failure

Objective: To evaluate the significance of an LH variant with a mutant β-subunit (Trp 8 to Arg 8 and Ile 15 to Thr 15) in gynecologic disease, including infertility. Design: Clinical study. Setting: Department of Obstetrics and Gynecology, Shimane Medical University Hospital, Izumo, Japan. Patient(s): Two hundred forty-five Japanese women with endocrine disorders and/or gynecologic disease and 153 healthy, nonpregnant, fertile Japanese women. Intervention(s): A blood sample was collected. Main Outcome Measure(s): The ratio of LH values from the SPAC-S and Immulyze assays (LH ratio: SPAC-S LH/Immulyze LH) was used to determine variant (≤0.5) or wild-type (>0.5) LH status according to a demonstrated relation between the ratio and the sequence of the LH β-subunit gene. Result(s): The LH ratio was lower (0.80 ± 0.31) in the 245 patients than in the controls (1.00 ± 0.38), and the variant was more frequent in the patients (18.4%) than in the controls (8.5%). We found no difference in the frequency of the variant between infertile and fertile patients. The prevalence of infertility did not differ between patients with variant LH and patients with normal LH. Ovulatory disorders, hyperprolactinemia, premature ovarian failure, menstrual disorders, and luteal insufficiency were significantly more frequent in patients with the variant. Conclusion(s): Variant LH may contribute to female reproductive disorders, including infertility and premature ovarian failure.

Luteinizing hormone and different genetic variants, as indicators of frailty in healthy elderly men.

We investigated the possible clinical correlates between the serum LH concentration and characteristics of frailty and determined the presence and concentration of a genetic LH variant in an independently living population of elderly men. After exclusion of subjects with severe mobility problems and signs of dementia, 403 healthy men (aged 73-94 yr) were randomly selected from a population-based sample. Total testosterone (T), sex hormone-binding globulin (SHBG), and leptin were determined by RIA. Non-SHBG-bound T was calculated. LH and the presence of the genetic LH variant were measured using immunofluorometric assays. The characteristics of frailty were leg extensor strength using dynamometry, bone mineral density of total body and proximal femur, and body composition, including lean mass and fat mass, measured by dual energy x-ray absorptiometry. Disability was further assessed by the Modified Health Assessment Questionnaire and by a measure of physical performance. LH significantly increased with age and inversely correlated with T and non-SHBG-bound T. LH was inversely related to muscle strength and lean mass, and both relations were independent of T. LH was positively related to self-reported disability (Modified Health Assessment Questionnaire); 12.5% of the study population was heterozygous for the LH variant allele. T levels and the degree of frailty were not different in the wild-type LH group compared with the heterozygote LH variant group. A significant positive relation between LH and fat mass as well as leptin was only present in the heterozygote LH variant group. In conclusion, serum LH levels increases with age in independently living elderly men and correlates inversely with a variety of indicators of frailty. The observed relation between LH and frailty, independent of T, suggests that LH reflects serum androgen activity in a different way than T, possibly reflecting more closely the combined feedback effect of estrogen and androgen. A difference in biological response between the two LH forms is suggested, as a difference exists in the relation between LH and fat mass, respectively, and leptin in the heterozygote LH variant subjects vs. the wild-type LH subjects.

Determination of a common genetic variant of luteinizing hormone using DNA hybridization and immunoassays.

An immunologically anomalous form of LH, due to two point mutations in codons 8 and 15 of the LH beta gene, has previously been described. LH status, i.e. the discrimination between wild-type (WT) and variant (V) LH, is usually determined by immunoassays, which can be unreliable at low serum concentrations of LH. A DNA hybridization assay was therefore developed to score the LH genotype in all subjects, independent of their serum LH concentrations. To evaluate the performance of the hybridization method, and to expand our observations of the worldwide occurrence of the V-LH, we determined its frequency in additional populations. To confirm the connection between the anomalous immunoreactivity and the V-LH beta gene, we also sequenced the LH beta subunit gene of a homozygous person. According to the ratio of two immunoassays, one detecting only WT-LH and the other detecting equally WT and V-LH, individuals can be classified as homozygotes for the V-LH beta allele, heterozygotes or WT. DNA samples from persons with known LH status, according to the immunoassays, were used for the development and evaluation of a new allele-specific DNA hybridization assay. This assay, and PCR and restriction fragment length polymorphism analysis, were used to determine the frequency of the V-LH beta allele in DNA samples obtained from eight populations. Ambulatory adult men and women, apparently healthy and with no endocrine disorders. The LH genotyping by immunoassays and by the new hybridization method gave identical results with all samples analysed (n = 25). The V-LH beta subunit was observed to always have the two point mutations, and to be identical with the ones previously reported. The V-LH beta carrier frequency in the DNA samples collected from various populations varied between 0 and 53.5%. The immunoassay technique and the hybridization assay can be used as alternatives to determine the LH status. A great variation in carrier frequency of the V-LH beta allele is observed in different populations.

Mutations and polymorphisms in the gonadotrophin genes; clinical relevance.

Recent studies on molecular pathogenesis of reproductive disorders have demonstrated an important genetic component in subfertility and infertility. The current methods of molecular biology, enabling detailed identification of mutations and polymorphisms, have revealed numerous mutations in the key genes involved in regulation of the reproductive functions. A good example are the common microdeletions that are detectable in the Y chromosome of azoospermic and oligozoospermic men (see, e.g. Najmabadi et al., 1996; Foresta et al., 1997; Kremer et al., 1997). When genes involved in hormone action are mutated, the phenotypic consequences are determined by the nature of the mutation. In a gain-of-function mutation, heterozygosity is sufficient to produce a phenotype, whereas in loss-of-function mutations usually only the homozygotes are affected. Both types of mutations are known with the receptor genes, but those of the ligands, i.e. hormones, are almost always of the loss-of-function type. When the genes involved in reproductive functions are affected, it is possible, but still rarely proven, that a given mutation affects the fertility of only one sex. Although the mutations impairing fertility should be self-limiting, they stay in the genetic pool due to their variable mode of inheritance and penetrance, and are in fact more common than assumed. Concerning gonadotrophins, the key hormones of the hypothalamic-pituitary-gonadal axis, microheterogeneity, due to variability in their carbohydrate structures, has been known for a long time. This phenomenon has been found to alter the intrinsic bioactivity of gonadotrophins, and in some cases to result in pathologies of gonadal function (Dufau & Veldhuis, 1987; Beitins & Pabmanabhan, 1991; Tsatsoulis et al., 1991). The variability of gonadotrophin glycosylation has usually been considered to be hormonally regulated, and its eventual genetic basis remains totally unknown. Our recent studies on bioactivity/immunoreactivity ratios of gonadotrophins (especially of LH) in various physiological and pathophysiological conditions have shown that this parameter varies much less than previously demonstrated, and probably has less clinical value than proposed (Jaakkola et al., 1990; Huhtaniemi et al., 1992, 1996). With regard to genuine mutations of the gonadotrophin genes, surprisingly few reports still exist. Since such mutations would severely compromise fertility, they are apparently sporadic and will be rapidly eliminated from the genetic pool. In contrast, a surprisingly common genetic variant of LH (v-LH) was recently described. It alters the functional characteristics of LH, and is likely to be an important contributing factor in the individual physiological variation of LH action and in some pathologies of the pituitary-gonadal function. The purpose of this review is to summarize our current knowledge on mutations of gonadotrophin genes and the common genetic variant of LH. In addition, we review the data obtained from experimental studies on targeted disruption of the gonadotrophin subunit genes in mice, since these genetically modified animal models are very informative as regards the role of gonadotrophins in reproductive functions.

Misleading Results with Opus® Magnum hLH Assay

We recently found unsatisfactory agreement of luteinizing hormone (hLH) results of the hLH Spec assay (Wallac) and Opus Magnum hLH assay (Behring Diagnostics) (Fig. 1⇓ A). Particularly disturbing were the occasional marked discrepancies. Figure 1. Comparison of results for samples from 35 fertile women: (A) Opus Magnum LH assay vs AutoDELFIA LH Spec assay ( y = −0.985 + 0.778 x , S b = 0.093, S y / x = 6.7); (B) Opus Magnum LH assay vs I3/A2 intact LH assay ( y = −0.257 + 0.772 x , S b = 0.016, S y / x =1.4); (C) ratio between I3/A2 intact LH assay and AutoDELFIA LH Spec assay results in relation to serum LH concentration determined by AutoDELFIA LH Spec; (D) ratio between Opus Magnum LH assay and AutoDELFIA LH Spec assay results in relation to serum LH concentration determined by AutoDELFIA LH Spec assay. In C and D , the population is distributed into three categories: (○) homozygotes, (□) heterozygotes, and (×) normal type for the variant LH allele. S b = standard error of slope, S y / x = standard error of estimates. According to the manufacturers, both assays are standardized against the WHO 2nd International Standard for pituitary hLH for immunoassay (code 80/552). Both assays are claimed to be highly …

Worldwide frequency of a common genetic variant of luteinizing hormone: an international collaborative research

Objective: To determine the worldwide frequency of a common immunological LH variant because of two point mutations in the LH β-subunit gene (Trp8Arg and Ile15Thr). Design: Cross-sectional study on LH status (variant and wild-type) in serum (or DNA) samples from Finland (Finns and Lapps), Estonia, Poland, Sweden, The Netherlands, United Kingdom, Italy, South Africa (blacks), Thailand, China, Japan, and the United States (Hispanics and blacks). Setting: Academic research environment. Patient(s): Ambulatory adult men and women (n = 2,936) with minor illnesses and no known endocrinological disorders. Intervention: A single blood sample was collected from each subject. Main Outcome Measure(s): The LH status was determined by two immunofluorometric assays using monoclonal antibodies. One (assay 1) only recognizes the wild-type LH, the other (assay 2) recognizes equally variant and wild-type LH. The ratio of assay 1 to assay 2 indicates the LH status: wild-type, >0.9; heterozygote, 0.2 to 0.9; and homozygote, < 0.15. One population (Lapps) was studied by DNA analysis using polymerase chain reaction and allele-specific oligonucleotide hybridization. Result(s): The carrier frequency of the variant LHβ allele varied from 7.1% in U.S. Hispanics to 41.9% in Lapps of northern Finland. The variant LHβ allele tended to be more common in populations from Northern Europe as compared with those from Asia. Conclusion(s): The high frequency of the LHβ variant worldwide makes it an important confounding factor when obtaining disproportionately low LH levels with some immunometric assays. The LH variant may contribute to some pathologies of the pituitary-gonadal function.

Inherited disorders of the gonadotropin hormones

Inherited disorders of the pituitary gonadotropins, LH and FSH, are rare. No mutations of the common α-subunit gene have been described. A single case of an FSHβ gene mutation has been reported. This mutation consisted of a two nucleotide deletion that caused a frameshift of codons 61–86 followed by premature termination. A homozygous patient with this mutation presented with primary amenorrhea and infertility. Serum FSH levels were low and LH levels were elevated. A postmenopausal heterozygous relative had subnormal FSH and LH and it was postulated that the mutant FSHβ subunit may have impaired gonadotrope function. Only a single example of an LHβ gene mutation has been described. This case was reported in a male who failed to undergo puberty, had elevated immunoreactive LH, but low bioactive LH and low testosterone. The LHβ gene is a member of the CGβ LHβ gene cluster that resides on chromosome 19q. No rearrangements or deletions were observed and there was a homozygous substitution of Gln 54 with Arg. The substituted Gln residue is conserved in each of the glycoprotein hormone β-subunits. Recombinant mutant LH was expressed in CHO cells and was shown to be immunologically active, but it did not bind to the LH receptor, explaining the absence of bioactivity. This finding suggests that Gln 54 is either a contact site for the receptor or that the mutation alters the conformation of LH to prevent binding to the receptor. The serum LH bio/immuno (B/I) ratio in heterozygotes was 50% of control samples, consisent with normal production and stability of the mutant hormone in vivo. Male heterozygotes exhibited slightly reduced testosterone and only one of four was fertile. Female heterozygotes had regular menses and were fertile. A polymorphic variant of LH has been reproted. The variant is prevalent in Finland (24% heterozygotes) and several cases have been reported in Japan. The LH variant consists of two amino acid substitutions (W8R; I15T) that correspond to residues normally found in CGβ. The I15T substitution may introduce a glycosylation site. The variant LH has increased bioactivity, but a reduced serum half-life. It is unclear whether the LH variant is of clinical significance aside from altering immunoactivity in some assays. In addition to gonadotropin mutations, defects in gonadotrope viability (SF-1; DAX-1 mutations) and in GnRH secretion (Kallmann syndrome: SF-1; DAX-1) can also lead to hypogonadotropic hypogonadism (Fig. 1). As noted in other talks, the LH-R and FSH-R are also targets for mutations. Thus, genetic defects have now been identified at each level of the H-P-G axis.

Polymorphism of gonadotropin action; molecular mechanisms and clinical implications.

Various structural alterations of gonadotropins and their receptors (R) contribute to the polymorphism of gonadotropin action. One reason is the microheterogeneity of gonadotropins due to variations in the degree of their glycosylation. This alters the intrinsic bioactivity of gonadotropins, as reflected by changes in their bioactivity to immunoreactivity ratios in various physiological and clinical conditions. We have reassesses 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 demonstrated. The second part of this chapter deals with a common polymorphism that was recently discovered in the gene of the LH beta-subunit. The variant LH beta allele contains two point mutations, both altering the amino acid sequence (Trp8Arg and Ile 15Thr), the latter one in addition introduces a new glycosylation signal to the LH beta peptide. The variant seems to represent an evolutionary early form of LH, being structurally closer to hCG than wild-type LH. The LH variant is common world-wide, with the carrier frequency varying from 28% in Finland to 7.5% in North American Hispanics. The LH variant differs functionally from wild-type LH, and it seems to predispose the carriers to mild aberrations of reproductive function. The third section summarizes our findings on the first mutation of the FSHR gene. This inactivating missense mutation is located in the gene sequence encoding the extracellular domain of the FSHR (Ala 189Val). The mutated receptor protein is apparently incorrectly folded and devoid of biological activity. The mutation explains about 50% of the hereditary form of hypergonadotropic ovarian dysgenesis in the Finnish population.

The role of luteinizing hormone-beta gene polymorphism in the onset and progression of puberty in healthy boys.

An immunologically anomalous LH with two point mutations in its beta-subunit gene (Trp8Arg and Ile15Thr) has recently been described. This polymorphism is common in Finland; 28% of the population are homo- or heterozygous for the variant allele. To assess the effect of the LH variant on LH action, we correlated its presence in a group of 49 healthy boys with the onset and progression of puberty. This group was followed-up longitudinally from a mean age of 11.7 +/- 0.1 yr for 3 yr at 3-month intervals. In addition, we studied the prevalence of the variant LH in boys with constitutional pubertal delay (testicular volume < or = 4 mL after 13.5 yr of age). The LH beta gene status of each subject in this study was judged from a single venous blood sample using two immunofluorometric LH assays with different combinations of monoclonal antibodies: one detecting both the variant and wild-type LH, and the other detecting only wild-type hormone. Of the boys with pubertal onset at a normal age, 36 (74%) were homozygous for the wild-type LH beta allele, 12 (24%) were heterozygous, and 1 (2%) was homozygous for the variant LH beta allele. Clear differences in pubertal parameters were found between the boys with normal and mutated (homo- or heterozygous) LH genotypes. During the follow-up, the boys with the mutated genotype had smaller testicular volumes (P < 0.03), were shorter (P < 0.02), had slower growth rates (P < 0.04), and had lower serum insulin-like growth factor I-binding protein-3 levels (P < 0.03) than the boys with the normal LH genotype. In the boys with delayed onset of puberty, the frequency of the variant LH beta allele did not differ from that in the reference population, indicating that the variant LH is not associated with conditions due to disturbed control of the reactivation of GnRH secretion. We conclude that during the progression of puberty, the variant LH may be less active in stimulating testicular growth than wild-type LH. Thus, the gene may affect tempo, contributing to the wide normal variation in pubertal progression in healthy boys. Our results also suggest that the variant LH not only affects the course of puberty, but is already involved in the regulation of the GH-insulin-like growth factor I axis during childhood.

Prevalence of an immunological LH β‐subunit variant in a UK population of healthy women and women with polycystic ovary syndrome

An immunological LH beta-subunit variant has been described, which is undetectable using monoclonal antibodies directed to the intact LH molecule alone. Subjects have been found homozygous or heterozygous for nucleotide mutations within codons 8 and 15 in the LH beta-subunit gene. The prevalence of the variant LH beta-subunit has been estimated in a healthy UK population of women of reproductive age and in women with polycystic ovary syndrome (PCOS). The relationship of the variant molecule to the clinical and hormonal parameters of the subjects has been evaluated. The control and PCOS subjects were screened for the presence of the mutation by using a ratio of two immunofluorometric assays using monoclonal antibodies (Mab). One assay, not detecting the LH variant, uses a Mab directed to the intact LH molecule and a beta-specific Mab. The other assay, detecting both the variant and wild-type LH, uses two beta-subunit specific Mabs. The mutations in the LH beta-subunit gene were confirmed by restriction fragment length polymorphism. The relationship of the presence of the variant to the clinical and hormonal parameters was assessed by ANOVA. Two hundred and twelve normal ovulatory women, of whom 66 (31%) were obese (body mass index > 25) and 146 (69%) non-obese, and 153 women with PCOS, 115 (75%) obese and 38 (25%) non-obese participated in the study. The variant LH was detected in 31 (15%) controls and 32 (21%) PCOS subjects (P = 0.124) using specific Mab. Obese PCOS had a higher incidence of the heterozygous LH variant compared to obese controls (odds ratio 2.5, P = 0.03), and compared to non-obese PCOS (odds ratio 6.3, P = 0.01). The previously described two mutations in codon 8 and codon 15 were present in all subjects detected to be mutant hetero of homo-zygous by RFLP. There was no relationship between the presence of the variant LH and the clinical and hormonal parameter in the PCOS subjects; however, in the controls the presence of the variant LH was associated with a higher serum total testosterone (P = 0.046), oestradiol (P = 0.03) and SHBG (P = 0.002). The results of this study show that the variant LH beta-subunit is a common polymorphism occurring in 15% of a healthy UK population of women. The prevalence was not higher in women with PCOS, though it was over represented in obese women with PCOS. The presence of the variant did not alter the clinical or hormonal expression of the disorder in women with PCOS. Its presence in the controls was however associated with higher serum oestradiol and probably secondary elevation of SHBG and testosterone, suggesting that the variant form of LH may be associated with subtle changes in the function of the hypothalamic-pituitary-gonadal axis.

Occurrence and biological properties of a common genetic variant of luteinizing hormone.

We have characterized the frequency and selected biological properties of a variant form of LH caused by two point mutations in the gene of the LH beta-subunit. Detection of the LH variant (or polymorphism) is based on aberrant immunoreactivity; it is not detected by a monoclonal antibody (Mab) recognizing a specific epitope in the LH alpha/beta-dimer (assay 1), but an assay using two LH beta-specific Mab recognizes this LH form normally (assay 2). Hence, the ratio of LH measured by assays 1 and 2 is 1.18-2.10 (range of mean +/- 2 SD) in wild-type subjects, 0.54-0.98 in heterozygotes, and below 0.15 in homozygotes with regard to the mutant LH beta allele. Analysis of sera from 249 healthy male and female subjects of Finnish origin revealed a frequency of 24.1% heterozygotes and 3.6% homozygotes for the mutation, with similar proportions in each sex. The ratio of in vitro bioactivity to immunoreactivity (assay 2) of the variant LH was significantly (P < 0.01) increased (2.9 +/- 0.1; n = 11) compared to that of wild-type LH (2.2 +/- 0.1; n = 13). No difference was observed in LH pulsatility, measured from blood samples collected at 5-min intervals for 5 h, between three male and three female subjects homozygous for the LH variant and three matched male and three female controls with wild-type LH. Likewise, the responses of LH immunoreactivity (assay 2) to GnRH stimulation were similar with both types of LH. The half-time of the variant LH in rat circulation from both sexes was significantly shorter than that of LH from control subjects (males, 25.5 +/- 3.8 vs. 48.3 +/- 2.7 min, respectively; P < 0.01; n = 3). Upon isoelectric focusing of peripheral serum samples, the isoform distribution of the variant LH was similar to that of wild-type LH. In conclusion, the LH variant discovered by us appears to occur with high frequency in the Finnish population (28% homo- or heterozygotes). It has increased in vitro bioactivity and a decreased half-time in vivo. These differences are compatible with a putative extra carbohydrate chain in the LH beta-chain, as one of the two mutations introduces an extra glycosylation signal. The subjects homozygous for the LH polymorphism are apparently healthy. However, the altered bioactivity and in vivo kinetics of the LH variant may induce subtle changes in LH action, either predisposing the affected individuals to or protecting them from disease conditions related to LH action.