Genomics and Genetics of Gonadotropin Beta-Subunit Genes: Unique FSHB and Duplicated LHB/CGB Loci

Molecular and Cellular Endocrinology 329 (2010) 4–16

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Molecular and Cellular Endocrinology

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Review Genomics and genetics of gonadotropin beta-subunit genes: Unique FSHB and duplicated LHB/CGB loci

Liina Nagirnaja a, Kristiina Rull a,b,c, Liis Uusküla a, Pille Hallast a, Marina Grigorova a,c, Maris Laan a,∗ a Institute of Molecular and Cell Biology, University of Tartu, Riia St. 23, 51010 Tartu, Estonia b Department of Obstetrics and Gynecology, University of Tartu, Puusepa 8 G2, 51014 Tartu, Estonia c Estonian Biocentre, Riia St. 23b, 51010 Tartu, Estonia article info abstract

Article history: The follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (HCG) play Received 5 January 2010 a critical role in human reproduction. Despite the common evolutionary ancestry and functional related- Received in revised form 13 April 2010 ness of the gonadotropin hormone beta (GtHB) genes, the single-copy FSHB (at 11p13) and the multi-copy Accepted 26 April 2010 LHB/CGB genes (at 19q13.32) exhibit locus-specific differences regarding their genomic context, evolution, genetic variation and expressional profile. FSHB represents a conservative vertebrate gene with a Keywords: unique function and it is located in a structurally stable gene-poor region. In contrast, the primate-specific Gonadotropin hormones LHB/CGB gene cluster is located in a gene-rich genomic context and demonstrates an example of evolu- FSHB LHB tionary young and unstable genomic region. The gene cluster is shaped by a constant balance between HCG beta selection that acts on specific functions of the loci and frequent gene conversion events among dupli- Gene duplications cons. As the transcription of the GtHB genes is rate-limiting in the assembly of respective hormones, the Gene conversion genomic and genetic context of the FSHB and the LHB/CGB genes largely affects the profile of the hormone Selection production. Mutations © 2010 Elsevier Ireland Ltd. All rights reserved. Polymorphisms Gene expression

Contents

1. Introduction ...... 5 2. Comparative genomic context of gonadotropin beta genes ...... 5 2.1. FSHB genomic region ...... 5 2.2. LHB/CGB genomic region ...... 5 2.2.1. LHB/CGB genes in human ...... 5 2.2.2. LHB/CGB genes in primates ...... 6 3. Forces affecting diversity and evolution of gonadotropin beta genes...... 8 3.1. Diversity patterns of gonadotropin beta genes ...... 8 3.2. Role of gene conversion ...... 9 3.3. Role of selective pressures ...... 9 3.3.1. FSHB gene ...... 9 3.3.2. LHB/CGB genes ...... 9 4. Genetic variation of human gonadotropin beta genes ...... 10 4.1. Gonadotropin beta gene mutations with known functional effects ...... 10 4.2. Polymorphisms associated with phenotypic traits ...... 10 4.2.1. FSHB gene ...... 10 4.2.2. LHB gene ...... 10 4.2.3. CGB genes ...... 12 4.3. Non-synonymous amino acid substitutions with unknown phenotypic consequence ...... 12

∗ Corresponding author. Tel.: +372 7375008; fax: +372 7420286. E-mail addresses: [email protected], [email protected] (M. Laan).

5. Comparative expressional profile of human gonadotropin beta genes ...... 12 5.1. Profile of FSHB and LHB gene expression ...... 12 5.2. Profile of HCG beta gene expression ...... 13 5.3. Tissues of minor expression of gonadotropin beta genes...... 13 6. Conclusive remarks ...... 14 Acknowledgements ...... 14 Appendix A. Supplementary data...... 14 References ...... 14

1. Introduction monkey and great ape lineages additional gene duplication events have occurred within the LHB/CGB gene cluster, further diversify- The follicle stimulating hormone (FSH), luteinizing hormone ing this genomic region among species (Maston and Ruvolo, 2002; (LH) and chorionic gonadotropin (CG; HCG in human) are function- Fortna et al., 2004; Dumas et al., 2007). ally and evolutionarily related hormones regulating reproductive function (Pierce and Parsons, 1981). FSH and LH are produced in 2. Comparative genomic context of gonadotropin beta the anterior lobe of pituitary gland in a pulsatile manner and their genes function in gonads is mediated over distinct FSH and LH receptors, respectively (Table 1)(Dalkin et al., 2001; Ascoli et al., 2002; Dias et The evolution of single-copy FSHB and multi-copy LHB/CGB loci al., 2002). FSH is required for follicle maturation and stimulation of represent contrasting scenarios of functionally related GtHB gene ovarian estrogen production in women. In males, FSH promotes family members, which are physically separated in the genome. Sertoli cell proliferation and indirectly spermatogenesis (McGee The evolution, divergence and diversity patterns of the GtHB genes and Hsueh, 2000; Plant and Marshall, 2001). LH stimulates female have been determined by the sequence properties and dynamics of progesterone synthesis and ovulation and also theca cell androgen the respective genomic regions. production. In males, LH stimulates testosterone production in Ley- dig cells (Moyle and Campbell, 1996). Apart from FSH and LH, the 2.1. FSHB genomic region evolutionarily young primate-specific CG is synthesised in placenta (Table 1). Although CG and LH bind to the same receptor LH/CGR, The beta-subunit of the human FSH (111 aa mature protein) is their properties are different as CG is more stable (hormone half-life coded by the FSHB gene (4262 bp) at chromosome 11p13. FSHB is for LH <1 h compared to ∼24 h for HCG), the production of hormone a single-copy gene located in a G/C-nucleotide deficient and gene- is continuous and pregnancy-specific (Wehmann and Nisula, 1981; poor genomic region. The G/C content in FSHB exonic regions is Moyle and Campbell, 1996). The main function of CG is to delay 43–52% and 30–33% in introns. Only one more gene (C11org46; apoptosis of the corpus luteum gravidium, prepare endometrium ∼100 kb downstream from FSHB) has been mapped within the for the implantation of the fetus in early pregnancy and promote flanking ±100 kb region (Fig. 1). The closest gene upstream of FSHB, fetal testicular testosterone production. KCNA4, is located ∼200 kb away. Interestingly, another member FSH, LH and CG are all hetero-dimeric proteins that share of the same gene family, KCNA7, is located 11.7 kb downstream a common alpha-subunit, but have a hormone-specific beta- of the LHB/CGB gene cluster at chromosome 19 (Fig. 1). This subunit that mediates binding to the respective receptors (Morgan is consistent with earlier studies showing the common origin et al., 1975; Moyle et al., 1975; Rathnam and Saxena, 1975). of the gonadotropin beta genes (Li and Ford, 1998; Querat et The unique CGA gene coding for the alpha-subunit (116 aa) is al., 2000; Querat et al., 2001; Querat et al., 2004). The repeat- located at chromosome 6q12–q21. It has a conservative nature content of the flanking region of the human FSHB gene (∼44%) in forming a functional hormone with all beta-subunits of falls within the average range estimated for the human genome gonadotropins as well as thyroid-stimulating hormone, and also (40–50%) (Lander et al., 2001). The FSHB gene and its immedi- contributes to the binding of the three receptors (FSHR, LHR, ate flanking regions (±100 kb) are characterized by low genome TSHR) (Pierce et al., 1971). So far no human patients have been dynamics and high structural conservation since it has preserved described carrying CGA variants, although four non-synonymous very similar features not only in primates but also among mammals changes have been predicted to exon 2 (dbSNP, build 131; (Fig. 1). http://www.ncbi.nlm.nih.gov/projects/SNP/). The genes coding for FSH, LH and CG beta-subunits have a common evolutionary ances- 2.2. LHB/CGB genomic region try and belong to gonadotropin hormone beta-subunit gene family (GtHB). Loci for FSH beta (denoted FSHB in primates, Fshb in mam- 2.2.1. LHB/CGB genes in human mals and GTH-I in fishes) and LH beta (denoted LHB in primates, The human genes coding for the beta-subunits of LH (121 aa Lhb in mammals and GTH-II in fishes) are conserved among verte- mature protein) and HCG (145 aa mature protein) are located brates and functional genes have been cloned in fishes, amphibians, in tandem in a shared genomic region (45,165 bp) at 19q13.32 reptiles, birds and mammals (Gharib et al., 1989; Guzman et al., (Fig. 1; Table 1)(Policastro et al., 1986). The LHB/CGB gene clus- 1991; Kumar et al., 1995; Rosenfeld et al., 2001; Shen and Yu, 2002; ter consists of one LHB gene (1111 bp), four HCG beta coding genes Kawasaki et al., 2003; Watanabe et al., 2007). The beta-subunit of (CGB, CGB5, GGB8 and CGB7; 1467 bp) and two gene copies with CG (CGB) probably emerged from a duplicate LHB gene copy approx- unknown function (CGB1 and CGB2; 1366 bp) (Policastro et al., imately 55–35 million years ago before the divergence of New 1983, 1986; Fiddes and Talmadge, 1984). In contrast to the gene- World and Old World monkeys (Talmadge et al., 1984; Policastro poor genomic context of the FSHB locus, the LHB/CGB region is et al., 1986; Bailey et al., 1991; Maston and Ruvolo, 2002; reviewed flanked by several genes. In total, 18 annotated genes are mapped in Henke and Gromoll, 2008; Hallast and Laan, 2009). Only a few within the ±100 kb proximate region of this gene cluster (Fig. 1). sequence changes led to the novel function and different expres- The LHB/CGB region has high G/C-nucleotide content (57% com- sion pattern of the derived CGB gene compared to the ancestral LHB pared to 41% for the human genome average) and is rich in (Fiddes and Goodman, 1980; Talmadge et al., 1984). In Old World Alu-repeats (23.2% compared to 13.1%) (Lander et al., 2001; Hallast 6 L. Nagirnaja et al. / Molecular and Cellular Endocrinology 329 (2010) 4–16

Table 1 Characteristic features of gonadotropin speciﬁc beta-subunits.

FSH LH HCG

Protein Mature ␤-subunit protein 111 amino acids 121 amino acids 145 amino acid Time of production Postnatal Postnatal Prenatal Production pattern Pulsatile Pulsatile Continuous Biologic half-life 3–4 h 20–30 min ∼24 h Main function Stimulates and regulates Stimulates steroidogenesis in Promotes implantation and spermatogenesis in men and testicular Leydig cells in men placentation, and production of follicular maturation in women and promotes ovulation in progesterone during ovarian luteal cells in women pregnancy, stimulates sexual differentiation of the male fetus Receptor FSHR LH/CGR LH/CGR

Gene Speciﬁc ␤-subunit coding gene FSHB LHB CGB, CGB5, CGB8, CGB7 Chromosomal localisation 11p13 19q13.32 19q13.32 Gene length 4262 bp 1111 bp 1467 bp Major site of expression Anterior lobe of pituitary gland Anterior lobe of pituitary gland Syncytiotrophoblastic cells in placenta Alternative splice forms Yes No No et al., 2005, 2008). Alu-repeats represent primate-speciﬁc repeat the 132 aa protein predicted for CGB1 and CGB2 has no similarity to elements, which have extensively spread by ‘copy-paste’ mech- HCG beta-subunit or to any other known protein due to an alterna- anism during the past 35–40 million years. These elements may tive open reading frame (Supplementary Figure S2)(Bo and Boime, trigger various genomic rearrangements and have been associ- 1992; Hollenberg et al., 1994; Dirnhofer et al., 1996). Additionally, ated with recent abundant duplication events in primate genomes CGB1 and CGB2 have been shown to undergo extensive alternative (Bailey et al., 2003). splicing that potentially encode distinct protein isoforms (Rull et The structure and sequence content of the human LHB/CGB al., 2008a,b; Bo and Boime, 1992). gene cluster represents a typical young genomic region evolved by duplication events. There is a high DNA identity between the 2.2.2. LHB/CGB genes in primates genes (85–99%) as well as between the inter-genic regions (81–97%) Functionally divergent CGB probably emerged from a duplicate (Hallast et al., 2005). The amino acid sequence identity between the LHB gene copy in the common ancestor of anthropoid primates HCG beta-subunit is 98–100% and to LH beta 85% (Bo and Boime, 55–35 million years ago and it demonstrates an excellent example 1992; Hollenberg et al., 1994). The DNA sequence identity between of the power of evolution by gene duplication (Ohno, 1970). After the HCG beta-subunit non-coding genes CGB1 and CGB2 and the the initial gene duplication event, the evolution of the LHB/CGB four HCG beta genes is 85% (Supplementary Figure S1). However, gene cluster in New World monkeys (Platyrrhini) compared to Old

Fig. 1. Schematic representation of the genomic context of the FSHB and the LHB/CGB genes (±100 kb). The ﬁgure was drawn based on Ensembl database (http://www.ensembl.org/; Release 54). Boxes denote the genes and triangles above or below them point to the direction of transcription. The black boxes and arrow- heads indicate CGB, white LHB and grey neighbouring genes. The CGB genes of rhesus macaque (Macaca mulatta) and common marmoset (Callithrix jacchus) are indicated as CGB A–C, since their ancestral status relative to the human CGB genes is unknown (reviewed in Henke and Gromoll, 2008; Hallast and Laan, 2009). L. Nagirnaja et al. / Molecular and Cellular Endocrinology 329 (2010) 4–16 7

Fig. 2. Density of polymorphisms in the human FSHB and LHB/CGB genes. The graph was drawn based on the resequencing data obtained from Hallast et al. (2005) and Grigorova et al. (2007). Bars represent the number of DNA variants calculated per 1000 bp of the re-sequenced region. The length of the analysed region for LHB, CGB, CGB2 and CGB1 genes is 1.5 kb, CGB5 and CGB8 1.6 kb, CGB7 2.2 kb and for FSHB 2.9 kb. The density fraction of common variants (minor allele frequency, MAF >10%) and rare variants (MAF <10%) is shown by the height of black and grey bars, respectively. Each gene, except for CGB8, was analysed in three populations: Est – Estonians (n = 47), Han – Chinese Han (n = 25), Man – African Mandenkalu (n = 23). CGB8 has been studied only in Estonian samples (n = 95; Rull et al., 2008a,b).

World monkeys, apes and humans (Catarrhini) have followed dif- tions of LH beta and CG beta (Fig. 1)(Simula et al., 1995; Maston ferent scenarios (reviewed in detail in Henke and Gromoll, 2008; and Ruvolo, 2002; Scammell et al., 2008). In Old World monkeys Hallast and Laan, 2009). All the New World monkey species stud- and apes additional duplication events have lead to variable num- ied so far (e.g. common marmoset Callithrix jacchus) harbour one bers of gene copies among the species. The number of mapped CGB pseudogenized LHB gene and one CGB gene coding for dual func- genes ranges from three in rhesus macaque (Macaca mulatta)tosix

Fig. 3. Distribution of known DNA variants across the human FSHB and LHB/CGB genes. The intron/exon structure of full genic regions is drawn to scale; for FSHB only the re-sequenced region is shown. Black, white and grey boxes represent exons, introns and untranslated regions, respectively. Vertical lines mark the positions of detected polymorphisms (Grigorova et al., 2007; Rull et al., 2008a,b; Hallast et al., 2005; Tables 2–4). Non-synonymous mutations causing clinical consequences (from Table 2) are indicated with underlined font and polymorphisms associated with a distinct phenotype (from Table 3) are marked with a star above the positions. S - singleton polymorphism detected for only one individual. 8 L. Nagirnaja et al. / Molecular and Cellular Endocrinology 329 (2010) 4–16

in humans and potentially even up to ∼50 copies in gorilla (Gorilla

). gorilla)(Crawford et al., 1986; Maston and Ruvolo, 2002; Fortna et al., 2004; Dumas et al., 2007). The active ongoing genome dynamics in the primate LHB/CGB Layman et ,

Lofrano-Porto locus was further proved by the ﬁnding that the CGB1-like gene , copies are speciﬁc only to African great apes (Hallast et al., 2007). Berger et al.

Phillip et al. (1998) Most intriguingly, the upstream region distinct to CGB1-like genes and harbours a novel small gene snaR (small NF90-associated RNA) and transcribed from an antisense strand predominantly in testis and Supplementary Figure S1 ( weakly in placenta and brain (Parrott and Mathews, 2007). As

CGB7 CGB1-like genes, snaR orthologs are only present in hominoids, and Valdes-Socin et al. (2004) Lindstedt et al. (1998) Kottler et al. (2010) et al. (2008) (2005) Layman et al. (2002) Weiss et al. (1992) Layman et al. (1997) Matthews et al. (1993) al. (1997) Reference Lofrano-Porto et al. (2007) / were shown to descend from Alu-transcripts (Parrott and Mathews, CGB8

/ 2009). The CGB1-speciﬁc insert disrupting the ORF of HCG beta orig- inates from a cluster of snaR genes mapped to chromosomes 19 in CGB5 / human.

CGB Additionally, it was determined that even human and chim- / panzee harbour a discordant number of CGB genes (six and ﬁve LHB copies, respectively) (Fig. 1)(Hallast et al., 2008). The structural differences of the LHB/CGB region in two sister-species were best explained by the scenario of independent lineage-speciﬁc duplication events. 000019.8 for ,NC Absent Aberrant tertiary structure of the protein, loss of bioactivity Loss of bioactivity, truncated protein Loss of bioactivity, truncated protein Absent, no binding to receptor Aberrant tertiary structure of the protein, loss of bioactivity Loss of bioactivity, truncated protein level Absent, abnormal splicing of mRNA

FSHB 3. Forces affecting diversity and evolution of gonadotropin beta genes

Intra- and interspecies diversiﬁcation of a genomic segment is 000011.8 for inﬂuenced by various forces such as selective pressures, mutation rate and DNA sequence rearrangements, but also by demographic history of the species (is not addressed in this review). ) c

3.1. Diversity patterns of gonadotropin beta genes pubertal development (one case) Primary amenorrhea, partial breast development (two cases) retarded sexual development (one case) Primary amenorrhea, infertility, retarded sexual development (two cases) Secondary amenorrhea, infertility (one female FSHB has been a subject to evolutionary constraints due to its unique function essential for mammalian reproduction (Wallis, 2001). The FSHB gene is characterized by low genetic variation and excess of polymorphisms with intermediate allele frequencies (Lamminen et al., 2005; Grigorova et al., 2007). Resequencing study in three human populations (European Estonians, Chinese Han and African Mandenkalu) identiﬁed a density of polymorphisms of 3 SNPs/1 kb (Fig. 2)(Grigorova et al., 2007). Majority of these were common polymorphisms located in non-coding regions and were shared by three human populations (Fig. 3).

) In contrast to FSHB, the LHB/CGB genes are among the top diverse c genes in the human genome (Hallast et al., 2005). The highest den- males hypoplastic Leydig cells (one case) low FSH (one case) gynecomastia (one case) low testosterone level (one case) low FSH (one case) Male phenotype Female phenotype Bioactivity/effect at protein sity of polymorphisms was detected within the genes located at the edges of the gene cluster (12.6 SNPs/kb for LHB, 14.6 SNPs/kb for CGB and 15.6 SNPs/kb for CGB7)(Figs. 1–3). For the central genes b CGB5 (8.1 SNPs/kb) and CGB8 (7.5 SNPs/kb) the density of polymorphic positions was lower, but still more than twofold higher as detected for the FSHB. Compared to FSHB, the LHB/CGB genes har- 2bp/87X Azoospermia, hypogonadism, very 1bp/108Xbour anotably Primary amenorrhea, impaired higher fraction of population-speciﬁc variants. For all LHB/CGB genes the most variable region was intron 1, whereas

Amino acid change the polymorphisms in exons have been less tolerated due to their apparent functional consequences (Fig. 3; Table 2). The variation of two HCG beta non-coding genes, CGB2 (9.8 SNPs/kb) and CGB1

a (6.7 SNPs/kb), is not as high as reported for the rest of the gene copies. On the one hand, this could be explained by relatively shorter period for accumulation of variants due to their evolutionary younger age compared to LHB and HCG beta genes (Hallast et al., 2007). On the other hand, CGB1 and CGB2 are located in the cen-

mutations tral part of the LHB/CGB region, which has been shown to be less mutations 545G>C intron 2 Azoospermia, hypogonadism (two 528G>A exon 2 Gly36Asp Impaired spermatogenesis, 2693T>C exon 3 Cys82Arg Azoospermia, hypogonadism, very 2684G>del exon 3 Ala79 2631TG>del exon 3 Val61 2600T>G exon 3 Cys51Gly Primary amenorrhea, infertility, 2677C>A exon 3 Tyr76X Azoospermia, hypogonadism, mild 818A>G exon 3 Gln54Arg Absence of spontaneous puberty, Nucleotide positions are deﬁned relative toAmino the acid transcription positions start are site deﬁned on based theConsanguineous on genomic siblings. the DNA sequence sequence; of GenBank a references: mature NC protein. active as a gene conversion acceptor and thus mostly unaffected by c a LHB Nucleotide substitution FSHB b its diversifying effect (see following section). Table 2 Mutations in the human gonadotropin beta-subunit genes with a clinical phenotype. L. Nagirnaja et al. / Molecular and Cellular Endocrinology 329 (2010) 4–16 9

tional gene conversion: the donor loci are located in the centre and the acceptor genes at the edges of the cluster (CGB and CGB7). Con- sistently, the highest density of polymorphisms is mapped for the most active gene conversion acceptors, the CGB and CGB7 genes (Hallast et al., 2005). However, in addition to increasing diversity of entire LHB/CGB gene cluster, gene conversion could also be the source of mutations affecting the function of the acceptor gene (reviewed in Chen et al., 2007). The process may have a functional consequence if a DNA segment is transferred from HCG beta genes to the unique LHB gene or from more divergent CGB1-like genes to the protein-coding genes in the cluster (see examples in Section 4).

3.3. Role of selective pressures

3.3.1. FSHB gene The major factor shaping the genomic context of FSHB is a strin- gent selective pressure. No mutations, which severely affect the function of this locus, are passed through to the next generation as the carriers fail to reproduce an offspring. A closer look at the diversity patterns across the FSHB gene reveals a strong allelic association between all common polymorphisms that form two major gene variants called haplotypes (Fig. 4B) (Grigorova et al., 2007). A haplotype is defined as a combination of allelic variants of sequen- tial polymorphisms, which are often inherited together. The two major FSHB haplotypes spread worldwide (58% and 26% of studied individuals, respectively) could be denoted as “yin-yang” gene Fig. 4. Schematic description of gene conversion and definition of haplotype vari- variants since they have a contrasting composition of alleles in all ants. (A) During gene conversion the genetic material is transferred between highly common polymorphic positions (Fig. 4B). Minority of the individu- identical gene copies or alleles. One locus acts as a donor of a gene conversion tract als (in total 16% of subjects in three studied populations) carry rare and the other as an acceptor. In case the two gene copies differ in some sequence positions within the converted tract, the variant specific to the acceptor is replaced haplotypes with distinguishing mutated positions (Fig. 5), thus the by copying the donor-specific variant. The donor DNA sequence remains unchanged. two major FSHB gene variants must have had a selective advan- In the figure, the donor locus is displayed as a black line with circles indicating tage in humans contributing to the reproductive success. Functional the gene-specific positions. The acceptor locus is drawn as a grey dashed line and effects and biological consequence of the two major FSHB haplo- the gene-specific positions as triangles. (B) Definition of gene haplotypes using the types are still to be addressed. two major FSHB gene variants as an example (Grigorova et al., 2007). Haplotypes are drawn schematically and the average frequency across European Estonians, Chinese Han and African Mandenkalu is given in brackets. The allelic composition of the two FSHB gene variants differs in every polymorphic position shown as 3.3.2. LHB/CGB genes black circles. The alternative alleles of the FSHB common polymorphisms are shown Compared to FSHB, the LHB/CGB genes are evolving by a more for haplotype 1 and haplotype 2. The locations of the polymorphisms within the complex scenario. The maintenance of duplicated, highly homolo- FSHB gene are indicated. Rs-numbers correspond to NCBI human genome build 36 gous LHB and CGB genes must occur through the balance between (http://www.ncbi.nlm.nih.gov/). inter-locus gene conversion activity and selection targeted to the specific functions of the loci. The unique functions of LH beta- subunit are guaranteed mainly by the preservation of LHB-specific 3.2. Role of gene conversion promoter and nucleotide positions that mediate the LH beta speci- ficity (Henke and Gromoll, 2008). As the gene and its function are Gene conversion is a mechanism often found in duplicated conserved in the entire vertebrate lineage, the LHB gene in Old genomic regions whereby DNA sequence information is transferred world monkeys and apes must evolve under constant competition between a pair of highly identical sequences (Fig. 4A). On the between gene conversion events and strong selective pressures one hand, it preserves the sequence similarity between duplicated (Hallast et al., 2008). In human and chimpanzee, there is evidence gene copies of multigene families leading to concerted evolution for Darwinian selection acting on LHB and the major HCG beta of duplicons (Walsh, 1985; Ohta, 1991). On the other hand, it also coding genes, CGB5 and CGB8 (Hallast et al., 2008). These genes spreads polymorphisms between homologous genes (Ohta, 1991; provide 18–39% and 27–64% of the total HCG beta transcript pool, Huber et al., 1993; reviewed in Chen et al., 2007). Thus, when a poly- respectively (Bo and Boime, 1992; Miller-Lindholm et al., 1997; Rull morphism occurs in one gene copy, it can be transferred to another and Laan, 2005). Still, as HCG beta coding genes are represented duplicated locus. in the human genome by four copies, the selective pressure act- The high sequence similarity among the genes (up to 99%) as ing on these genes is more relaxed and facilitates accumulation of well as inter-genic regions (up to 97%) of the human LHB/CGB gene polymorphisms either by de novo substitutions or gene conversion cluster provides an ideal surface for the gene conversion activity. events. In three populations representing Africa, Asia and Europe, Due to the homogenizing effect of gene conversion events, the the CGB5 and LHB genes comprised of one (61% of individuals) or intra-specific LHB/CGB genes have become more closely related two (67% of individuals) major haplotypes, respectively (Fig. 5). to each other than to their counterparts in closely related species The two major haplotypes in LHB differ from each other in seven (Maston and Ruvolo, 2002; Hallast and Laan, 2009). The observed out of 16 identified polymorphic positions. In contrast, FSHB has transferred sequence tracts between the LHB/CGB genes have been two completely different major haplotypes with no shared alleles. determined to range from a few bp to maximum 796 bp (Hallast et For all HCG beta genes 30–61% of studied individuals were carrying al., 2005). The entire gene cluster revealed the tendency of direc- various minor gene variants. 10 L. Nagirnaja et al. / Molecular and Cellular Endocrinology 329 (2010) 4–16

Fig. 5. Haplotype networks for the human CGB5, LHB and FSHB genes. The networks were derived based on resequencing data (Hallast et al., 2005; Grigorova et al., 2007) and using the Median-Joining network algorithm implemented in NETWORK 4.201 software (http://www.ﬂuxus-technology.com). Singleton polymorphisms were excluded from the analysis. Each node represents a single combination of SNP alleles within a gene. The size of a node is proportional to the haplotype frequency in the total dataset and the length of a line connecting two nodes correlates with the number of sequence differences between two haplotypes. The frequency of prevalent haplotypes across studied populations is given next to the major nodes.

4. Genetic variation of human gonadotropin beta genes been demonstrated to be more frequent among obese patients with polycystic ovary syndrome (PCOS) than in healthy females (Table 3; A few mutations that affect hormonal function and metabolism Fig. 3; Figure S1)(Tong et al., 2000). have been characterised in the single-copy LHB and FSHB genes. A substitution G>T located in FSHB promoter region at posi- As an analysis of the multi-copy CGB genes is technically challeng- tion −211 from mRNA transcription start site has originally been ing and the phenotypic effect of mutations is probably weaker due described in a male patient with azoospermia and isolated FSH to redundancy, the mutational spectrum of these genes is not as deficiency (Mantovani et al., 2003). This SNP is located within a extensively studied. In addition to mutations, a list of polymor- progesterone response element (PRE) conserved among numerous phisms has been found to be associated with specific phenotypes. placental mammals and capable of enhancing the gene transcription up to 9-fold (Webster et al., 1995). A cohort of young European 4.1. Gonadotropin beta gene mutations with known functional men (n = 554) showed an association between the minor allele T effects carrier status and reduced serum FSH level (Grigorova et al., 2008). Compared to the wild-type homozygotes (GG), the heterozygotes In total five mutations in FSHB have been reported in three male (GT) and the homozygotes (TT) for the minor allele had on average and six female patients, all having a severely impaired sexual devel- 15.7% and 40% lower levels of FSH in their bloodstream, respec- opment and infertility (Table 2; Fig. 3; Figure S1). Three mutations, tively. The association with lower male serum FSH level was further Val612 bp/87X, Tyr76X and Ala791 bp/108X in exon 3 lead to confirmed in a cohort of men diagnosed with infertility (n = 1029) a premature stop codon and truncation of the FSH beta protein (Grigorova et al., 2010). Moreover, the minor allele of this polymor- (Matthews et al., 1993; Layman et al., 1997; Phillip et al., 1998; phism was shown to be overrepresented among infertility patients. Kottler et al., 2010). Two other identified mutations in exon 3, FSHB −211G/T is the first described polymorphism shown to sig- Cys51Gly and Cys82Arg, alter a cystein knot structure of the FSH nificantly affect the male serum FSH levels. beta peptide. The cystein knot is crucial for hormone dimerization and bioactivity (Layman et al., 1997; Lindstedt et al., 1998). Three mutations have been described in the LHB gene: Gly36Asp, 4.2.2. LHB gene Gln54Arg and a substitution G to C at position 545 (from tran- The most studied variation in LHB gene is a combination of two scription start) in intron 2 (Table 2; Fig. 3)(Weiss et al., 1992; amino acid changes that are always found together as a haplotype Valdes-Socin et al., 2004; Lofrano-Porto et al., 2007). The latter on one chromosome: Trp8Arg/Ile15Thr (Table 3; Fig. 3; Figure S1) mutation disrupts splicing of intron 2 and results in an insertion (Furui et al., 1994; Okuda et al., 1994). Trp8Arg substitution is of 236 bp into LHB mRNA sequence and a frameshift in exon 3 mainly responsible for an altered immunoreactivity of the hor- (Lofrano-Porto et al., 2007). As LH is not necessary for sexual dif- mone and Ile15Thr substitution introduces an extra glycosylation ferentiation before birth, all patients had a normal phenotype at site into the altered LH beta peptide. The carrier frequency of the birth, including descended testes in males (Hughes, 2001). Clinical variant allele (V-LH beta) differs widely between ethnic groups signs caused by the lack of bioactive LH appear after pubertal age: (0–50%) (Nilsson et al., 1997; Lamminen and Huhtaniemi, 2001). delayed puberty and arrested spermatogenesis in males, secondary Compared to normal LH, the hormone formed by the V-LH beta amenorrhoa and infertility in a female patient (Table 2). possesses an increased in vitro biopotency and altered half-life in circulation, although the published data on the length of the half- 4.2. Polymorphisms associated with phenotypic traits life is contradictory (Manna et al., 2002; Wide et al., 2010). There are numerous published studies addressing the carrier status of the 4.2.1. FSHB gene V-LH beta variant in relation to various clinical conditions (Table 3). Among the three common synonymous changes described in The variant LH form has been suggested to suppress gonadal func- the coding region of FSHB, the 2623T>C (Tyr58Tyr) in exon 3 has tion and to be involved in the development of subfertility (Furui et L. Nagirnaja et al. / Molecular and Cellular Endocrinology 329 (2010) 4–16 11

Table 3 Variants in the human gonadotropin beta-subunit genes associated with phenotypic traits.

Nucleotide substitutiona Amino acid Assessed phenotype Number of carriers/total Association Reference changeb number of studied individuals

Variants in FSHB −211G>T 5upstream Azoospermia, FSH One male patient with na Mantovani et al. (2003) deﬁciency normal virilization Serum FSH concentration 131/554 cohort of young Yes – minor allele Grigorova et al. (2008) men carriers had lower FSH level 283/1029 male partners of Grigorova et al. (2010) infertile couples 2623T>C exon 3 Tyr58Tyr Polycystic ovary 89/135 female patients Yes Tong et al. (2000) syndrome (PCOS) 54/105 normal control females Variants in LHB 443T>C/465T>C exon 2 Trp8Arg/Ile15Thr PCOS 32/153 female patients No Rajkhowa et al. (1995) (v-LH) 31/212 healthy females PCOS 1/30 female patients No Elter et al. (1999) 5/30 healthy females PCOS 46/328 female patients No Tapanainen et al. (1999) 169/881 control females Subfertility/infertility 3/3 female patients na Furui et al. (1994) 0/2 fertile females Infertility 16/97 female patients Yes Takahashi et al. (1998) 14/169 fertile females Infertility 12/145 male patients No Ramanujam et al. (2000) 15/200 fertile males Delayed tempo of 13/49 healthy boys Yes - v-LH carriers Raivio et al. (1996) pubertal progression had smaller testicular volume, slower growth rate Endocrine disorder 45/245 female patients Yes Takahashi et al. (1999) and/or gynecologic disease 13/153 fertile females Menstrual disorders 21/176 female patients No Ramanujam et al. (1999) 20/200 normally ovulating females Endometriosis 16/85 female patients No Gazvani et al. (2002) 29/145 females with unknown fertility Male eunucoid syndrome One fertile male patient na Shiraishi and Naito (2003) Non-obstructive male 12/95 male patients No Lee et al. (2003) infertility 29/200 fertile males 961G>A exon 3c Gly102Ser Infertility, endometriosis, 2/52 female patients No Liao et al. (1998) PCOS 0/212 healthy females Menstrual disorders 7/176 female patients Yes Ramanujam et al. (1999) 0/200 normally ovulating females Infertility 5/145 male patients Yes Ramanujam et al. (2000) 0/200 fertile males

Variants in CGB5 −155G>C/−147G>del/ Recurrent miscarriage 26–27/184 RM couples Yes Rull et al. (2008b) (RM) −144T>C/−142T>A 5upstream 45–48/195 fertile females 1038C>T intron 2 RM 30/184 RM couples Yes Rull et al. (2008b) 51/195 fertile females 1178G>C exon 3 Val56Leu RM 1/184 RM couples na Rull et al. (2008b) 0/195 fertile females

Variants in CGB8 806C>T exon 2 Arg8Trp RM 1/184 RM couples na Rull et al. (2008b) 0/195 fertile females 1045C>T intron 2 RM 1/184 RM couples Yes Rull et al. (2008b) 7/195 fertile females 1237C>G exon 3 Pro73Arg RM 1/184 RM couples na Rull et al. (2008b) 0/195 fertile females

a Nucleotide positions are deﬁned according to the location of the transcription start site in the genomic DNA sequence; GenBank references: NC 000011.8 for FSHB, NC 000019.8 for LHB/CGB/CGB5/CGB8/CGB7 (Supplementary material, Figure S1). b Amino acid positions are deﬁned based on the sequence of a mature protein. c Originally described as 1502G>A; na – statistical tests were not applicable. 12 L. Nagirnaja et al. / Molecular and Cellular Endocrinology 329 (2010) 4–16

Table 4 Non-synonymous amino acid substitutions with unknown phenotypic consequence.

Gene Position relative to mRNAa Position relative to ATGb Amino acid changec Reference

FSHB 946G>T Ser2Ile Cargill et al. (1999)

LHB 406G>A 397 Met-6Ile Hallast et al. (2005) 413G>A 404 Ala–3Thr Jiang et al. (2002) Hallast et al. (2005) 450A>G 441 His10Arg Hallast et al. (2005)

CGB 1363A>C 998 Asp117Ala Hallast et al. (2005)

CGB5 794G>A 429 Arg6Gln Hallast et al. (2005) 1247G>A Val79Met Miller-Lindholm et al. (1997) 1362A>C 997 Asp117Ala Hallast et al. (2005) CGB8 869G>A Val29Ile Rull et al. (2008b)

CGB7 782G>A 417 Arg2Lys Hallast et al. (2005) 787/788AT>CC 422/423 Met4Pro 1162G>A 797 Ala51Thr 1232C>T 867 Arg74Cys 1363C>A 997 Ala117Asp

a Nucleotide positions are defined according to the location of the transcription start site in the genomic DNA sequence; GenBank references: NC 000011.8 for FSHB, NC 000019.8 for LHB/CGB/CGB5/CGB8/CGB7 (Supplementary material, Figure S1). b Translation start site as in original reports; GenBank references: NC 000011.8 for FSHB,NC000019.8 for LHB/CGB/CGB5/CGB8/CGB7 (Supplementary material, Figure S1). c Amino acid positions are defined based on the sequence of a mature protein. al., 1994; Haavisto et al., 1995; Lamminen and Huhtaniemi, 2001). vents desensitisation of LH/HCG receptor and therefore supports The V-LH beta variant could have originated by an ancient gene con- the continuous function of corpus luteum during gestation (Cameo version event where one of the CGB genes has acted as a donor and et al., 2004). The polymorphism leading to Val79Met substitution LHB as an acceptor (see Section 3.2; Hallast et al., 2005). The con- in the exon 3 of CGB5 was shown in in vitro experiments to code certed substitutions (443T>C and 465T>C) in V-LH exon 2 leading to for the altered protein unable to fold correctly and assemble with Trp8Arg/Ile15Thr changes correspond to the nucleotide sequence alpha-subunit (Miller-Lindholm et al., 1999). Individuals carrying in the respective positions of all six CGB genes. this polymorphism could possibly have a subtle deficiency of bioac- Additionally, Gly102Ser substitution in exon 3 has been found tive HCG but its association with an impaired reproductive outcome to be associated with reproductive disorders in some populations has not been established. (Ramanujam et al., 2000). 5. Comparative expressional profile of human 4.2.3. CGB genes gonadotropin beta genes A resequencing study of the CGB5 and CGB8 genes detected three rare non-synonymous substitutions (Val56Leu in CGB5 and Pituitary FSH and LH are produced in a pulsatile manner with Arg8Trp, Pro73Arg in CGB8) only among recurrent miscarriage differential frequencies and amplitude regulated by gonadotropin patients as possible variants increasing the risk of recurrent preg- releasing hormone (GnRH) (reviewed in Marshall et al., 1993; Miller nancy loss (Table 3; Fig. 3; Figure S1)(Rull et al., 2008b). Similarly, et al., 2002). On the other hand, CGB genes are expressed and a rare T>A mutation in CGB8 located adjacent to an initiator ele- HCG produced continuously in placenta over two alternative signal ment for HCG beta transcription (4 bp before transcription start transduction pathways in order to avoid receptor desensitisation site) was identified as a potential risk variant in developing a (Cameo et al., 2004). Rate-limiting for the respective hormone pro- recurrent miscarriage (Rull et al., 2008b). A significant protec- duction is the transcription of FSHB, LHB and HCG beta coding genes. tive effect toward recurrent miscarriage was associated with two Thus, the genetic and genomic context of the FSHB, LHB and HCG SNPs located at identical positions in intron 2 in both, CGB5 beta genes affects the profile of the hormone production. (1038C>T) and CGB8 (1045C>T), and with four CGB5 promoter For the FSHB gene at least four alternative transcripts have been variants (−155G>C/−147G>del/−144T>C/−142T>A) (Table 3)(Rull described that encode an identical unmodified protein. Different et al., 2008b). These variants reduced the risk of recurrent mis- transcripts arise due to combinations of alternate splicing of the carriages up to 1.8-fold. The haplotype-forming alternative CGB5 first non-coding exon and at least two potential polyadenylation promoter variant may have arisen by the transfer of a gene con- signals (Jameson et al., 1988). In case of LHB and HCG beta coding version tract derived from the CGB8 promoter sequence as a genes, no splice variants have been identified. Although alterna- donor. tive transcripts have been reported for the HCG beta non-coding genes CGB1 and CGB2, their functional effect is yet to be deter- 4.3. Non-synonymous amino acid substitutions with unknown mined (Bo and Boime, 1992; Berger et al., 1994; Rull and Laan, phenotypic consequence 2005).

Several additional variants have been reported in the human 5.1. Proﬁle of FSHB and LHB gene expression FSHB, LHB and HCG beta coding genes that cause a non-synonymous substitution, but the data about the phenotypic consequences in Pituitary LH and FSH have been preserved during evolution as the study participants is unavailable (Table 4; Figure S1)(Jiang et functionally critical hormones that guarantee successful reproduc- al., 2002; Hallast et al., 2005). The Ala-3Thr change in the signal tion and survival of a species. Thus, large-scale ﬂuctuations in the peptide of LH beta has been shown to cause different in vitro signal transcriptional activity of the FSHB and the LHB genes might be transduction properties compared to a wild-type preprotein pre- fatal for the reproductive success. An optimal concentration of sumably due to altered conformation of secreted LH (Jiang et al., LHB and FSHB transcripts and the coded protein is kept strictly 2002). The alternative signal transduction pathway apparently pre- in narrow ranges in men as well as in women during follicular L. Nagirnaja et al. / Molecular and Cellular Endocrinology 329 (2010) 4–16 13

Fig. 6. Normal ranges of (A) serum FSH and LH concentrations in men and women; (B) serum HCG dynamics during pregnancy and (C) levels of HCG beta mRNA transcripts in placental tissue. The graphs (A) and (B) are drawn based on reference values of United Laboratory of University of Tartu Clinics, Estonia. The graph C was drawn based on the data from Rull et al. (2008a,b). The boxes represent the 25th and 75th percentile and median is denoted as the line that bisects the boxes. and luteal phases, but undergoes a rise during ovulation and post- Although wide ranges in HCG beta transcriptional activity are menopausal period (Fig. 6A) (Owen, 1975; Sherman and Korenman, tolerated during the pregnancy, biparental expression of CGB genes 1975; Zumoff et al., 1982; Baird, 1983). Overexpression of FSHB is required in order to provide a necessary amount of HCG beta- could cause polycystic ovary syndrome as in female FSH overex- subunits and avoid complications in early pregnancy (Uusküla et pressing mice, whereas severe reduction in FSHB transcription and al., unpublished data). Too low mRNA expression could lead to mis- FSH production could cause infertility in females or poor sperm carriage and excessively high amount of HCG beta transcripts could quality in males (Kumar et al., 1997, 1999; Plant and Marshall, be a marker for ectopic or molar pregnancy (Rull and Laan, 2005; 2001). Large fluctuations in LHB expression could lead to hypog- Rull et al., 2008a). onadism, infertility and several other endocrine disorders in both sexes as can be seen in patients with various mutations in the 5.3. Tissues of minor expression of gonadotropin beta genes LHB gene (Tables 2 and 3) (reviewed in Huhtaniemi and Themmen, 2005). In addition to major sites of expression – pituitary and pla- Serum FSH levels depend on the selective regulation of FSHB centa – gonadotropin hormone production in minor concentrations mRNA transcription by the action of pituitary ja gonadal factors has been reported in other tissues. An ectopic production of such as activin and inhibin, while the expression of glycoprotein pituitary gonadotropins LH and FSH in normal non-malignant tis- alpha-subunit (CGA) mRNA remains unaffected (Carroll et al., 1989; sues is limited; minimal amount of LHB transcripts have been Farnworth, 1995; Lin and Ge, 2009). The critical role of the FSHB detected in testis and placenta (Berger et al., 1994; Giovangrandi transcription in determining FSH levels was also confirmed by map- et al., 2001). Both hormones have been detected in tumours that − ping of a polymorphism in a regulatory region 211 bp upstream of arise from gonadotroph cells of the pituitary gland. Gonadotroph FSHB. The alternative allele of this polymorphism reduces the FSHB adenomas account for approximately 40% of all clinically recog- transcription by 46–58% and leads to lower FSH production in men nized macroadenomas and approximately 80% of surgically excised (Table 3)(Hoogendoorn et al., 2003; Grigorova et al., 2008, 2010). clinically non-functioning adenomas (Valimaki et al., 1999). In In case of LHB, several variants in the promoter region of V-LH beta these adenomas the glycoprotein hormone beta-subunits may be have been identified, which increase expressional activity and alter detected by molecular, immunological or immunohistochemical the response to hormonal stimulation (Jiang et al., 1999). staining. The clinical manifestation is delayed due to inefficient secretion of the hormone and its subunits (Jaffe, 2006; Valimaki 5.2. Profile of HCG beta gene expression et al., 1999). HCG beta genes have been reported to be expressed in mini- In contrast to pituitary gonadotropins, placental HCG beta mal amount in normal non-trophoblastic tissues, mostly in testis, mRNA is expressed by four duplicate genes and larger inter- prostate, thymus, skeletal muscle and pituitary gland (Bellet et al., individual differences in the total amount of produced mRNA and 1997; Reimer et al., 2000). The role of HCG in normal tissues may in hormone levels are tolerated (Miller-Lindholm et al., 1997; Rull be related to autoregulatory mechanisms, beta-subunit of the hor- and Laan, 2005; Rull et al., 2008a). The highest variability in hormone has been shown to exert growth-promoting activity (Butler mone levels is seen during the I trimester when HCG concentration and Iles, 2004). HCG probably acts in an auto-/paracrine way, a doubles every 48–72 h and causes up to ∼11-fold inter-individual transcrine pathway has been suggested for the testis, prostate and differences (Fig. 6B) (Hay, 1988). Large fluctuations in gene expres- uterus (Berger et al., 2007). sion probably reflect high genetic variation in the HCG beta genes Several non-trophoblastic tumors, like bladder, renal, prostate, (see Section 3.1). Transcriptional activity of the HCG beta genes lung, gastrointestinal, neuroendocrine, breast and gynecological changes through the course of a pregnancy and in correlation with cancers, have been shown to produce HCG beta-subunit and to a the dynamics of the HCG hormone (Fig. 6B and C). lesser extent intact HCG (Stenman et al., 2004). The role of HCG 14 L. 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