Variation of 52 new Y-STR loci in the Y Chromosome Consortium worldwide panel of 76 diverse individuals

We have established 16 small multiplex reactions of two–four loci to amplify 52 recently described single-copy simple Y-STRs and typed these loci in a worldwide panel of 74 diverse men and two women. Two Y-STRs were found to be commonly multicopy in this sample set and were excluded from the study. Of the remaining 50, four (DYS481, DYS570, DYS576 and DYS643) showed higher diversities than the commonly used loci and can potentially provide increased haplotype discrimination in both forensic and anthropological work. Ten loci showed occasional missing alleles, duplicated peaks or intermediate-sized alleles.
Introduction
Y-STRs have key roles in the fields of forensic genetics, anthropological genetics and genealogy because of their ability to discriminate between male lineages and provide information about the relationships between them [1, 2]. The Y chromosome haplotype reference database [3] provides a widely used compilation of haplotype information constructed from a “minimal haplotype” of nine loci or a “minHt + SWGDAM core set” of 11 loci (http://www.yhrd.org/index.html). Some applications, however, require more Y-STRs. For example, a study of ∼1,000 men from east Asia found that almost 3% (27/1,003) shared the same 16-STR haplotype [4] and thus would not be distinguished by standard analyses. Most of the STRs on the Y chromosome have now been identified [5], and a set of 52 was highlighted that seemed particularly useful because their unit size was ≥3, they were single-copy, had a simple structure and showed variation in a set of eight diverse men. These additional loci proved to be useful in the east Asian study where 46 of them allowed a male lineage characteristic of the Qing Dynasty to be defined [4], but they clearly varied considerably in their diversity [4, 5] and may vary in other properties that affect their usefulness as well. In addition, it may often be impractical or impossible to type such a large number of markers. Further studies of these loci are therefore needed to identify the most useful subset. US population data for 16 of them have been presented [6], but data from other loci and populations are lacking. We have therefore established multiplex typing procedures for all of them and examined their variation in the Y Chromosome Consortium (YCC) worldwide panel of men [7].
Materials and methods
The YCC panel consists of 74 male and two female DNAs; the men may be broken down into 26 from Africa, 26 from Asia and the Americas and 22 from Europe or the Middle East. In addition, the haplogroup R individual previously typed with all of the new markers [5] was included in this study to facilitate consistent allele calling. DNA was amplified before use with the GenomiPhi whole genome amplification kit (Amersham Biosciences, Amersham, UK) according to the manufacturer’s recommendations.
A total of 52 polymorphic simple single-copy Y-STRs [5] were included in the present study. The published primers had been designed to operate under a common set of conditions and were therefore used in this study, except that a G was added to the 5’ end of the unlabelled primer if it was not already present to facilitate non-templated addition of an A to the labelled product strand [8]. Loci were tested in silico for potential interactions between primers using the AutoDimer software [9], and suitable sets were assembled into small multiplexes for experimental assessment resulting in 16 multiplexes each consisting of 2–4 loci (Table S1).
Polymerase chain reactions (PCRs) were set up in 20 μl volumes containing 1× PCR buffer (Invitrogen, Paisley, UK), 1.75 mM MgCl2, 200 μM deoxynucleotide triphosphates (dNTPs; Amersham Biosciences), 1.0 unit of Platinum Taq DNA polymerase (5 U/μl, Invitrogen) with 10 pg–2 ng whole-genome-amplified DNA and primer pairs at the concentrations shown in Table S1. Thermal cycling was carried out in an MJ Research (Genetic Research Instrumentation, Braintree, UK) DNA Engine Tetrad™ 2 starting with denaturation at 95°C for 15 min, followed by 20 cycles of touchdown PCR: 94°C for 30 s, 70°C for 45 s, 72°C for 1 min, with a 1°C decrease in annealing temperature every cycle and then 15 cycles of standard PCR (94°C for 30 s, 50°C for 45 s, 72°C for 1 min) and finishing with extension at 60°C for 45 min and storage at 4°C.
Products were analysed by mixing 1 μl of PCR product with 15 μl Hi–Di formamide and 0.2 μl size marker (CXR 60–400 bases, Promega UK, Southampton, UK) and running on 36 cm × 50 μm capillaries containing POP-4 polymer (Applied Biosystems) on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Warrington, UK). Electrophoresis was carried out at 3 kV for 3 s followed by 15 kV for 45 min with a run temperature of 60°C. Allele sizes were measured using GeneMapper v3.0 (Applied Biosystems). Most loci were sequenced because of the lack of previous sequence data, to confirm previous results or to investigate the structure of intermediate-sized sizes. Such alleles were amplified using unlabelled primers and sequenced by the Wellcome Trust Sanger Institute small-scale sequencing facility using standard methods.
Results
The 52 Y-STRs were examined in the 76 YCC samples and haplogroup R control individual, but the analyses presented in this paper (Tables S2, Tables S3) are based only on the YCC data to facilitate comparisons with other YCC results [10]. As expected, no specific products were obtained from the two female YCC samples in the size range examined, and single peaks were seen in all males for 40 of the STRs. The other 12 loci showed more complex patterns (Table 1). Products from four loci were missing in one (DYS525, DYS589, DYS636) or two (DYS556) individuals. These findings were reproducible and occurred in multiplex reactions that successfully amplified other loci, so that they may represent null alleles, but their structural basis remains to be determined, and they were treated conservatively as missing data in our analyses.
Table 1Loci showing multiple peaks, missing peaks or intermediate allelesLocusIntermediate alleleaCommentsDYF386S1Two peaks in many individuals, excluded from analysisDYF390S1Two peaks in many individuals, excluded from analysisDYS448Two peaks in two individualsDYS52210 (U2Ains)Intermediate-sized allele. This insertion converts the reference sequence, which can be written ATAG ATG (ATAG)10 into ATAG AT A G (ATAG)10, which therefore has 12 copies of the ATAG repeat, but differs from the regular allele 12DYS525No product in one individual, two peaks in one individualDYS53111 (D6Tins)Intermediate-sized alleleDYS549Two peaks in one individualDYS556No product in two individualsDYS567Two peaks in two individualsDYS576Two peaks in two individualsDYS589No product in one individualDYS636No product in one individualU upstream, D downstream, ins insertionaNomenclature based on the standard recommendations [1]
Two peaks were observed in many individuals for DYF390S1 and DYF386S1, and we interpreted these as duplicated loci that happened to have the same sized alleles in the small number of individuals examined before [5]; these two STRs were excluded from subsequent analyses. Five loci also showed two peaks of similar height in one (DYS525, DYS549) or two (DYS488, DYS567, DYS576) individuals, which may reflect rare duplications or somatic mutations in the YCC cell lines. In addition, two loci showed fragment sizes that did not fall into the expected size classes: DYS522 in one individual and DYS531 in 11 individuals corresponding precisely to haplogroup Q [7] and thus representing a variant characteristic of this haplogroup. The structural basis of these variants was determined by sequencing and found to arise from insertion events in the flanking sequences between the STRs and the primers (Table 1). Null alleles, occasional duplications and intermediate alleles have been found in the standard Y-STRs [1], and so we concluded that 50 of the 52 new Y-STRs merited further consideration as loci for wider use.
We next examined the variation of these 50 STRs. The number of alleles ranged from two to 11, the diversity from 0.05 to 0.90 and the variance from 0.04 to 7.89 (Table 2). All of these characteristics were correlated, probably because of their common dependence on the repeat count. To interpret the values obtained, we have compared them with published data on the standard single-copy loci in the YCC panel [10]. Of the new loci, four (DYS481, DYS570, DYS576 and DYS643) showed higher diversity than the most variable standard locus DYS390 (diversity = 0.79) and 15 showed higher diversity than DYS393 (diversity = 0.66; Table 2). The discrimination of haplotypes that are not distinguished by the commonly used markers is a particularly useful property. As reported [10], eight pairs of YCC individuals carry haplotypes that are identical when the standard minimal set of Y-STRs is used. Two of these are from different populations (Mbuti Pygmy/Bantu speaker; English/German) and these were distinguished by seven and nine of the new loci, respectively. The other six pairs are from isolated populations, and these were distinguished by 2, 1, 1, 0, 0 and 0, respectively, of the new markers (Table S4). Although a total of 15 loci contribute to this increased discrimination, all of the five distinguishable haplotypes could be separated using just two of the most variable loci, DYS570 and DYS576.
Table 2Variation of 50 new Y-STR loci in the YCC panelLocusMean repeat countNumber of allelesDiversityVarianceDYS48123.3110.907.87DYS57017.6110.863.89DYS57617.370.822.16DYS64311.190.822.33DYS48515.970.781.92DYF406S110.670.751.26DYS52211.260.741.03DYS58912.360.731.07DYS53311.260.721.03DYS54912.150.720.92DYS50512.160.711.00DYS50811.480.711.44DYS5259.970.710.95DYS53111.040.690.39DYS55611.760.671.03DYS57210.350.640.61DYS56511.560.640.71DYS5949.570.630.87DYS54011.440.620.52DYS48713.260.621.38DYS51110.950.620.73DYS5739.940.620.73DYS61712.450.621.06DYS49515.350.610.76DYS56710.460.610.72DYS49714.240.600.51DYS48813.460.580.91DYS49211.650.560.45DYS49012.970.562.43DYS56811.170.560.92DYS63611.350.560.49DYS53710.940.530.43DYS61811.840.510.37DYS63810.940.490.34DYS49112.150.460.40DYS5788.140.410.40DYS64011.230.390.39DYS47611.240.360.29DYS4949.040.350.28DYS6419.640.330.93DYS5549.050.310.36DYS57510.040.250.22DYS5838.030.220.12DYS5907.930.220.23DYS4807.930.220.16DYS56911.030.180.11DYS5309.120.170.09DYS5809.140.110.19DYS4728.020.080.04DYS5799.020.050.89Loci are ordered according to their diversity.
Discussion
We have investigated the properties of 52 new Y-STRs in a diverse worldwide set of males. We found that two of the Y-STRs were multicopy and thus not well suited to some applications and that the remaining 50 loci differed substantially in their properties. Our measurements of allele numbers, diversity and variance were overall consistent with the previous report [5]; correlation coefficients (R2 values) were 0.47, 0.58 and 0.67, respectively, but differed for some individual loci. The most variable Y-STR, in all respects, was DYS481, and this was not previously considered in detail because sequence data were not available before. Several other loci (e.g., DYS570, DYS576 and DYS643) may be particularly useful for increasing discrimination in forensic work, and the simple structure and mutational properties of this set make them the markers of choice for many population genetic studies. This is illustrated by considering the correlation between mean repeat count and variance in repeat number of the 50 simple loci: it was far higher (R2 = 0.67) than the value reported for complex Y-STRs (R2 = 0.34, [5]), suggesting that the simple STRs have simpler mutational mechanisms and may lead to more precise dates of lineages. The data in Table 2 and Table S3 now provide a basis for choosing the best simple loci and assembling them into a high-level multiplex reaction for more extensive population screening.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Table S1
Multiplex organization and primer concentrations (DOC 102 kb)
Table S2
PCR product size range and allele range (DOC 92 kb)
Table S3
Haplotypes of the YCC DNAs (XLS 58 kb)
(Note: this table is provided as an Excel file and is the table mentioned in CE7. We transformed it into text because some journals insist on this, but the text version is difficult to interpret as CE5 highlights. The loci are ordered according to their positions in multiplexes 1–16 in
Table S1
and
Table S2
, and it seems most consistent to keep the same order for
Table S3
).
Table S4
Subdivision of minimal haplotypes by new Y-STRs (DOC 87 kb)


Structural and functional evolution of the P2Y12-like receptor group

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y12-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y12, have been deorphanized. The P2Y12-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y12 in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y12-like receptor members.
Introduction
Most cellular effects of nucleotides are mediated through two main families of specific cell surface receptors, the ionotropic P2X and metabotropic P2Y nucleotide receptors. The metabotropic receptors belong to the superfamily of G protein-coupled receptors (GPCR). The GPCR superfamily comprises at least five structurally distinct families/subfamilies (GRAFS classification) which share little sequence homology among each other [1]. The rhodopsin-like receptors (also called family A or 1) form the largest family in vertebrates. Eight different mammalian P2Y (P2Y1, 2, 4, 6, 11–14) receptor subtypes, all belonging to the rhodopsin-like receptors (family A), have been identified and proven to function as receptors for extracellular nucleotides so far [2]. Based on their preferential agonists, P2Y receptors have been subclassified into adenine nucleotide-activated (P2Y1, P2Y11, P2Y12, P2Y13), pyrimidine nucleotide-activated (P2Y4, P2Y6), ATP/UTP-activated (P2Y2), and UDP-sugar-activated (P2Y14) receptors. Most structural and functional studies were performed with the classic P2Y receptors P2Y1 and P2Y2 [3]. However, in comparison to other GPCR, large-scale structure/function data are rare, mainly due to difficulties in finding suitable P2Y-free heterologous expression systems [4]. Pharmacological characterization on both native and recombinant P2Y receptors is further hampered by the release of endogenous nucleotides and the hydrolysis or conversion of exogenous and endogenous nucleotides by ectoenzymes (for review see [5]). ATP and AMP are metabolized by cell surface enzymes, the ecto-NTPDase 1 (nucleoside triphosphate diphosphohydrolase 1, ectoapyrase, CD39, metabolizes ATP to AMP) and the 5′-ectonucleotidase (CD73, metabolizes AMP to adenosine). In addition, a nucleoside diphosphokinase catalyzes the transfer of the gamma phosphate of nucleoside triphosphates to nucleoside diphosphates. For example, in the presence of ATP, nucleoside diphosphokinase catalyzes the conversion of UDP to UTP. These specific problems may have contributed to many false and controversial declarations of P2Y receptors being activated by nucleotides [6–9]. Recent success in expressing GPCR in yeast, which is discussed below, may circumvent these problems and may engage structure-function-relation studies.
Although in-depth phylogenetic analysis of GPCR groups indicates clustering of P2Y receptors, when compared with other GPCR of family A [1, 10], the adenine nucleotide-activated P2Y1 and P2Y12, for example, display only minor structural relation at the protein level. Phylogenetic analysis even revealed that nucleotide specificity e.g. for ADP evolved independently in P2Y1 and P2Y12. So we suggest that grouping of P2Y receptors should be performed on phylogenetic aspects rather than on ligand specificity.
Based on their amino acid sequences P2Y receptors can be subdivided into at least two groups (Fig. 1). One comprises P2Y1, 2, 4, 6, 11, and the second group contains P2Y12–14. Additionally, several other structurally related GPCR, such as CysLT1R, GPR91, GPR99, and GPR34, cluster into both groups (Fig. 1). These receptors are activated by structurally different ligands such as leukotrienes, organic acids, and phospholipids. Probably because of their structural relation several P2Y receptors display agonist promiscuity. It was shown that not only ADP but also leukotriene E4 (LTE4) activates P2Y12 at nanomolar concentrations [11]. Cysteinyl leukotriene receptors (CysLT1R and CysLT2R) have been described to be activated by UDP in addition to activation by cysteinyl leukotrienes [12, 13]. Further, there is evidence that CysLT1R antagonists, such as pranlukast and montelukast, inhibit activation of P2Y1 and P2Y6 by 2MeS-ADP (2-methylthio-ADP) and UDP, respectively [14]. Although P2Y12 and cysteinyl leukotriene receptors are clinically used targets for prevention of thrombocyte aggregation (clopidogrel) and treatment of asthma (montelukast), respectively, the structural basis and (patho)physiological relevance of dual/multiple agonist specificity is obscure. Close structural relations between P2Y and other non-nucleotide receptors as well as the mentioned examples of ligand promiscuity indicate that nucleotides are probably only one of many natural agonists on the genuine P2Y receptors.
Fig. 1Phylogenetic relation of human P2Y receptors. To evaluate the structural relation of P2Y receptors and related GPCR, amino acid sequences of human orthologs were aligned and phylogenetic relations were estimated using CLUSTAL W. The derived tree was essentially identical to former analysis [2, 10]. The P2Y12-like receptors cluster into a group (framed) that is distinct from other nucleotide receptors such as P2Y1 and P2Y2
Structural evolution of P2Y12-like GPCR
Discovery and deorphanization of group members
The ADP-(P2Y12)-like receptor group is the most recently identified P2Y receptor group and includes P2Y12, P2Y13, and P2Y14 (see Fig. 1, Table 1). Based on high structural similarity, the lyso-phosphatidylserine receptor (GPR34) and several orphan GPCR (GPR34-like, GPR82, GPR87, GPR171) are considered members of the structural P2Y12-like receptor group (3,11). Most members of this group were deorphanized by the so-called reverse pharmacology approach. The orphan receptors predicted from sequence data are now often used to ascertain the ligands by testing them on tissue extracts and subsequent fractionation or on huge libraries of bioactive compounds, whereas, traditionally, the bioactive ligand was used to identify the receptor (classic approach).
Table 1Members of the P2Y12-like receptor familyReceptorG protein couplingNatural agonistTissue expressionP2Y12GiADP, CysLT-E4, phosphoribosyl pyrophosphatePlatelets, brain (glia) [19]P2Y13GiADP, diadenosine triphosphateSpleen and adult brain, lower expression in placenta, lung, liver, spinal cord, thymus, spleen, small intestine, uterus, stomach, testis, fetal brain, adrenal gland, lymph node, bone marrow, peripheral blood mononuclear cells, leukocytes [22, 45]P2Y14GiUDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-N-acetylglucosamineBrain (astrocytes, glia), placenta, adipose tissue, stomach, intestine, spleen, lung (epithelium), heart [15, 16, 71]GPR87Gin.kPlacenta, thymus, small intestine, colon, prostate, uterus, testis, peripheral blood leukocytes [28, 29]GPR171n.kn.kn.kGPR34Gilyso-phosphatidylserineBrain, glia cells, mast cells, spleen, heart, kidney, liver [27, 31, 34, 72]GPR82n.kn.kTestes, epididymis (unpublished own results)Based on phylogenetic analyses and structural similarities the seven GPCR clusters in a group which is distinct from other P2Y receptors (see Fig. 1). Although they share structural features it appears from current studies that not all members are receptors for nucleotides. The table lists the currently known receptor agonists, the receptor’s G protein specificity and expression patternn.k not known
As the first member of the P2Y12-like receptor group P2Y14 (synonyms KIAA0001, GPR105, VTR 15–20) was identified [15, 16] and then deorphanized in the year 2000 [17]. UDP-glucose was found to activate P2Y14 by screening multiple receptors, each expressed in individual yeast strains, against a large library of over 700 known and putative natural GPCR agonists.
In 2001, P2Y12 (initially named SP1999) was discovered and identified as the platelet clopidogrel-sensitive ADP receptor by a number of groups [18–21]. The P2Y12 was deorphanized with both the classic and the reverse pharmacology approaches. Hollopeter et al. used subfractionated transcripts from platelets and isolated the cRNA (complementary RNA, encoding e.g. the P2Y12 receptor) which mediated ADP-induced increases in K+ current after injection into Xenopus oocytes [19]. In contrast, fractionated rat spinal cord extracts were assayed for Ca2+ mobilization in cells transiently transfected with P2Y12 and chimeric Gα subunit [21]. Almost in parallel, ADP was shown to be an agonist for P2Y13 (former GPR86), a very close structural neighbor of P2Y12 [22–24]. 2MeS-ADP is the most potent agonist at P2Y12 followed by ADP, adenosine 5′-O-2-thiodiphosphate, and ATP. (N)-methanocarba-2-methylthio-ADP, a highly potent agonist at P2Y1, exhibited no and only minor agonist activity at P2Y12 and P2Y13, respectively [25].
Based on structural relations GPR34 [26, 27], GPR82 [28], GPR87 [29], and GPR171 (synonym H963) [30] also belong to the P2Y12-like group (see Fig. 1). Except for GPR34, which was recently identified as a receptor for lyso-phosphatidylserine [31] by a reverse pharmacology approach, all the other P2Y12-like receptors are still orphan GPCR.
The evolutionary origins
Based on structural similarities P2Y12-like receptors cluster into a group distinct from other P2Y receptors (see Figs. 1 and 2). This P2Y12-like receptor group can be subdivided into two subgroups. One subgroup encompasses P2Y12, P2Y13, P2Y14, GPR87, and GPR171, the other subgroup GPR34, GPR34-like, and GPR82 (Fig. 2). This may suggest that both subgroups evolved from gene duplications starting from two related members. The grouping is further supported by shared genomic localization. Chromosomal clustering in the human genome is found for P2Y12, P2Y13, P2Y14, GPR87, and GPR171 at 3q24-3q25 as well as for GPR34 and GPR82 at Xp11.4. This clustering is evolutionary well preserved. In the zebrafish genome P2Y12, P2Y14/GPR87, and GPR171 cluster at chromosome 15 and GPR34 type 2 and GPR82 are tandemly arranged at chromosome 9. Gene clustering is not only found for vertebrate P2Y12-like receptors but also for several other GPCR genes. Multiple copies of related GPCR genes, such as the human protease-activated receptors on 5q13, trace amine-associated receptors (TAAR, human 6q23.2), and CC-chemokine receptors (human 3p21.3), are the result of intrachromosomal gene duplications. The most impressive example is found in odorant receptors, where in humans, chromosome 11 contains nearly half of the odorant receptor repertoire, including a single cluster of more than 100 odorant receptors [32]. Gene amplification can be viewed as a dynamic and reversible regulatory mechanism that facilitates adaptation to variable environments. Clustered genes may confer selective benefits via their ability to be co-regulated and co-amplified. Indeed, even transcripts encoding a fusion protein of GPR34 and GPR82 have been observed (own unpublished results). However, the biological relevance of P2Y12-like receptor clustering in respect to transcriptional activity at their respective genomic loci, co-regulation, and transcript diversity need to be determined in the future.
Fig. 2Evolutionary origin of P2Y12-like receptors. To subclassify and evaluate the phylogenetic relations between the P2Y12-like receptors, the amino acid sequences of orthologs from one species of each vertebrate class (if available) were aligned using CLUSTAL W (identity matrices) and a phylogenetic tree was constructed (1,000 iterations). Bootstrap values >600 were considered as significant to support a new branch
Since P2Y12, P2Y14/GPR87, GPR34, and GPR34-like are all found in sharks (Mustelus manazo, Carcharodon carcharias) and chimeras (Callorhinchus milii) one can assume that P2Y12-like receptors arose more than 450 million years (Myr) ago, before cartilaginous and bony fish split. Our own polymerase chain reaction (PCR) amplification attempts as well as the ongoing lamprey (Petromyzon marina) and sea urchin (Strongylocentrotus purpuratus) genome projects revealed no P2Y12-like sequences yet, suggesting their origin in the very early Gnathostomata or, less likely, a loss in agnate and all non-vertebrates.
Gene duplication can be a primary source of the genetic material from which genes with new functions evolve. One copy of a duplicated gene may become mutated and acquire unique functionality without risking the fitness of the organism ensured by the homolog. On the other hand, if not advantageous, continuous accumulation of mutations (neutral drift) will eliminate one of the genes, a process named pseudogenization. In fish genomes, there is only one respective ortholog with relation to P2Y12/P2Y13 and to GPR87/P2Y14. In the case of P2Y12/P2Y13 the fish ortholog is more closely related to P2Y12 implicating that P2Y13 evolved later in tetrapod evolution probably by P2Y12 duplication. However, one cannot rule out that the gene was eliminated in the common ancestor of sharks and bony fishes. The evolutionary mechanism of P2Y14 and GPR87 evolvement remains unsolved since their structural relation to the fish orthologs is similar. The absence of P2Y14 in the African craw frog (Xenopus) genome but the presence of ortholog sequences in all other sequenced tetrapod genomes does not necessarily implicate that P2Y14 derived from GPR87 in early tetrapod evolution. Again, P2Y14 gene loss in amphibians must be considered.
Orthologs of GPR34, GPR87/P2Y14, and P2Y12 are found in all tetrapods and bony and cartilaginous fishes investigated so far. However, not all members of the P2Y12-like group appear to be present in all vertebrate classes. GPR82 has an ortholog in zebrafish but is not yet found in the genomes of pufferfishes and the lizard (Anolis carolinensis) genome. Although GPR171 is found in reptiles, birds, mammals, zebrafish, and chimera it appears to be absent in African craw frog, stickleback, and pufferfishes. This may implicate specific functions of some P2Y12-like receptors which are less important in some species or are compensated by other receptors and mechanisms.
Signatures of gene inactivation (pseudogenization) are rarely found for members of the P2Y12-like group. Following gene duplication in the common ancestor of evolutionary basic fishes, like eels and carps [33], pseudogenization of one GPR34 subtype was identified in a salmon species (Keta salmon) (own unpublished results). The GPR34-like receptor is an evolutionary old P2Y12-like receptor being present in cartilaginous and bony fishes, amphibians, and birds but not in mammals (Fig. 2). Sequences with residual relation to the GPR34-like receptor are found in the platypus genome but it is most likely a pseudogene.
Genomic organization of P2Y12-like receptors
Comparison of transcript and genomic sequences provides information on intron/exon structure of a GPCR gene. Since the gain or loss of spliceosomal introns are unique events in evolution, they can serve as markers for phylogenetic analysis. Further, such analyses may reveal splice variants and may be informative about the promoter structure and gene regulation. Introns are the basis of alternative splicing, exon skipping, and RNA editing events and, therefore, can contribute to receptor diversity at a supragenomic level.
Most coding regions of human P2Y12-like receptors do not contain introns (Table 2). One exception is GPR87 where the genomic sequence encoding the receptor’s N terminus is interrupted by an intron in mammalian and avian orthologs. Another intron which is rarely removed (cryptic intron) has been identified in the N terminus-encoding sequence of GPR34 [34].
Table 2Genomic organization of P2Y12-like GPCR. n.a. not analyzed because of lack of mRNA informationMemberNumber of introns in the 5′ non-coding regionNumber of introns in the coding regionApprox. size of genomic regionaP2Y12 Human1–2−47 kbp (2 different transcript starts) Mouse4−46.5 kbp Chickenn.a.−bn.a. (>1 kbp) African clawed frog−1 (N terminus)4 kbp Zebrafishn.a.−bn.a. (>1 kbp) Stickleback11 (TMD2)2 kbp Pufferfishn.a.1 (TMD2)>2 kbpP2Y13 Human−1 (rare transcript, N terminus, NM_176894) 3.2 kp (2 different transcript starts) Mouse1−3 kbp Chickenn.a.−bn.a. (>1 kbp) African clawed frog−1 (N terminus)14–19 kbpP2Y14 Human2−66 kbp (2 different transcript starts) Mouse1–2−16 kbp Chickenn.a.−bn.a. (>1 kbp)GPR87 Human11 (N terminus)22.7 kbp Mouse11 (N terminus)16 kbp Chicken21 (N terminus)9.5 kbp African clawed frogn.a.−bn.a. (>1 kbp)P2Y14/GPR87 Zebrafishn.a.−bn.a. (>1 kbp) Pufferfishn.a.−bn.a. (>1 kbp)GPR171 Human2−5 kbp Mouse1−4.5 kbp Chicken1−2.3 kbp Zebrafishn.a.−bn.a. (>1 kbp)GPR82 Human2−4 kbp Mouse2–3−6 kbp Chickenn.a.−bn.a. (>1 kbp) African clawed frogn.a.−bn.a. (>1 kbp) Zebrafishn.a.−bn.a. (>1 kbp)GPR34 Human3–41 cryptic (N terminus)8.2 kbp Mouse3–4−9.1 kbp Chicken1−2.5 kbp African clawed frog2−5.2 kbp Zebrafish  Type 11−7.3 kbp  Type 2−−1.2 kbp Pufferfishn.a.−bn.a. (>1 kbp)Except for GPR87, human P2Y12-like receptor members contain no intron in the coding region. However, this intronless gene structure is not well preserved in all vertebrates. The table summarizes the number of introns within the 5′ and coding region of selected vertebrate genes and estimates the size of the geneaBased on the 5′ longest transcriptbComplete open reading frame within the genomic sequence but intron within the very N terminus cannot be ruled out because of lack of mRNA information
The genomic organization of the individual P2Y12-like receptor genes is not well conserved during vertebrate evolution (see Table 2). For example, the P2Y12 gene gained an intron in bony fish evolution disrupting the open reading frame of the transmembrane domain 2 (TMD2)-encoding part. Similarly, one of the two GPR34 paralog genes acquired an intron in the more recent bony fish evolution [33]. The two long-standing alternative explanations for the origin of introns, the intron-early theory and intron-late theory, remain a matter of continuous debate not only for GPCR [35]. The intron-early theory suggests that introns are extremely ancient characteristics of genes and that early genes were created through the intron-mediated shuffling of exons. However, numerous gene and genome comparison studies provided evidence that at least some introns are more recently acquired (intron-late theory). The P2Y12-like receptor group is, therefore, a nice example where introns were acquired in the coding region in some species during more recent evolution.
In contrast to the coding region, the 5′ region of most members of the P2Y12-like receptor group displays a distinct intron/exon organization with sometimes multiple transcription starts. Also, the gene sizes strongly differ between the individual and even closely related receptors. Introns in the 3′-untranslated region (UTR) of P2Y12-like receptors have not been found yet. Large and complex organized 5′ non-coding regions of a gene may provide the basis for multiple promoter regions, cis-acting elements, and a variable 5′ UTR of the mRNA. Alternative structures of the 5′ UTR can contribute to expression regulation and alternative translation start points. For example, 5′ UTRs often contain small open reading frames (ORF) which can be translated via leaky scanning at the ribosome. Such leaky scanning can reduce translation of the downstream main ORF as shown for several genes including GPCR [36, 37].
It is of interest to note that both genes, GPR34 and GPR82, are located in antisense orientation within a large intron of the CASK gene. This position is conserved during vertebrate evolution. The CASK gene encodes a calcium/calmodulin-dependent serine protein kinase that is a member of the membrane-associated guanylate kinase (MAGUK) protein family. Since GPR34 and GPR82 transcripts are antisense orientated to CASK one can speculate that transcripts may regulate expression of CASK or vice versa. Such hypotheses will be addressed in receptor-deficient mouse models (see below) in the future.
Key residues defining the individual member
Many attempts have been made to identify structural signatures which are helpful in annotation and grouping of P2Y receptors. On the basis of available sequence data for validated P2Y receptors, key residues were extracted to define P2Y12-like and P2Y1-like receptor groups [11, 38, 39]. In recent studies, we have shown for several GPCR that a significant number of orthologs is required for identification of functional motifs and key residues [33, 40–42]. By mining public databases and by amplifying and sequencing P2Y12-like orthologs we have acquired large sets of sequences of P2Y12 (74 orthologs), P2Y13 (31 orthologs), P2Y14 (38 orthologs), GPR87 (51 orthologs), GPR171 (41 orthologs), GPR82 (34 orthologs), and GPR34 (133 orthologs) to determine structural conservation of the members and to identify amino acid sequence motifs and key residues that are unique for the P2Y12-like receptor group and the individual members (Fig. 3). These data sets were obtained from vertebrate species ranging from evolutionary old bony and cartilaginous fishes to the more modern mammals representing 450 Myr of evolution. The overall identity between P2Y12-like members is rather low ranging from 19% amino acid identity (human GPR82 vs human GPR87) to 47% (human P2Y12 and human P2Y13). Between the respective fish and mammal receptor orthologs P2Y12 (~49%) shows the highest identity followed by GPR171 (~48%), GPR34 (~40%), and GPR82 (~35%). Since not all P2Y12-like GPCR have orthologs in all vertebrate classes, we analyzed the structural conservation within this receptor group by comparing Ka/Ks values (ratio of the number of nonsynonymous substitutions per nonsynonymous site and the number of synonymous—or silent—substitutions per synonymous—or silent—site) of the P2Y12-like receptor ortholog set from species containing all group members (Table 3). Although all P2Y12-like receptors display a purifying selection mode of evolution (Ka/Ks <<1 indicates high conservation and elimination of deleterious mutations), there are significant differences between the members of this receptor group. P2Y12 and GPR87 were kept most conserved during evolution in birds and mammals. By contrast, GPR82 appears to be less constrained as already indicated by a relatively low conservation at the amino acid level (see above).
Fig. 3Conserved residues in P2Y12-like receptors. To identify conserved group and member-specific positions the amino acid sequences of P2Y12 (74 orthologs), P2Y13 (31 orthologs), P2Y14 (38 orthologs), GPR87 (51 orthologs), GPR171 (41 orthologs), GPR82 (34 orthologs), and GPR34 (133 orthologs) were aligned using CLUSTAL W. Residues that are 100% conserved among the respective orthologs were boxed. Only a few positions are almost fully conserved among all members of the P2Y12-like group (position number refers to the relative numbering system by Ballesteros and Weinstein [73]): TMD1: Phe/Tyr1.39, Phe1.57; TMD2: Leu2.43, Asn/Asp2.50, Pro/Ala2.58; TMD3: Tyr/His3.33, Arg/Gln3.50; TMD4: Trp4.50; TMD6: Cys/Ser6.47; Pro6.50; TMD7: Asp7.49, Pro7.50, and the two Cys residues bridging extracellular loops 1 and 2. The approximate positions of the seven transmembrane domains (TMD) are given above the sequencesTable 3Sequence conservation of P2Y12-like GPCRReceptorKa/KsPi(mean ± SD)(mean ± SD)P2Y120.049 ± 0.0180.145 ± 0.022P2Y130.141 ± 0.0700.187 ± 0.023P2Y140.103 ± 0.0360.190 ± 0.024GPR870.046 ± 0.0270.134 ± 0.018GPR1710.078 ± 0.0120.158 ± 0.021GPR340.082 ± 0.0370.137 ± 0.026GPR820.202 ± 0.0490.167 ± 0.034To compare the sequence conservation of P2Y12-like receptors, ortholog sequences [relative positions 1.48 (in TMD1) to 7.68 (in C-term)] from 18 species were aligned and DNA polymorphism analyses were performed using DnaSP (version 4.1). The Ka/Ks ratio is calculated from the number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks) for any pair of sequences. The nucleotide diversity (Pi) is the average number of nucleotide differences per site between two sequences. The orthologs of the following species were analyzed because their genome contained all members of the P2Y12-like group: Bos taurus, Equus caballus, Canis familiaris, Pteropus vampyrus, Ornithorhynchus anatinus, Monodelphis domestica, Callithrix jacchus, Pan troglodytes, Homo sapiens, Macaca mulatta, Microcebus murinus, Pongo pygmaeus, Tarsius syrichta, Cavia porcellus, Mus musculus, Rattus norvegicus, Tursiops truncates, Gallus gallus
As shown in Fig. 3, members of the P2Y12-like group share only eight fully conserved residues (Phe1.57, Leu2.43, Trp4.50, Pro6.50, Asp7.49, Pro7.50, and both Cys residues bridging extracellular loops 1 and 2) when sequences of more than 400 receptors of this group are compared. Although the ADP receptors P2Y12 and P2Y13 share more than 50 fully conserved residues none of these conserved residues is exclusively found in P2Y12 and P2Y13 but rather present also in other P2Y12-like receptors (see Fig. 3). Further, only a few residues are member specifically conserved (GPR171: Gln3.53 and Asn5.59; P2Y13: Met7.48; GPR82: Leu6.44 and Asp7.57; GPR34: Met7.52) and have not been found in other P2Y12-like receptors so far. These facts suggest that ligand and signaling specificity is determined by a combination of many, more or less conserved determinants. It has been proposed that His6.52/Arg6.55 and Lys7.35/Glu7.36/Leu7.39 within TMD6 and the ECL3, respectively, may present such motifs required for nucleotide binding [38]. However, such residue combination is also present in some GPR87, GPR171, and GPR34 orthologs which are not activated by ADP. This does not rule out that these residues are involved in nucleotide binding of e.g. P2Y12 but it implicates additional positions which determine ligand specificity. In-depth structure-function analysis e.g. by mutagenesis studies are required to identify key positions and their structural properties.
Physiological relevance of P2Y12-like receptors
Functional characterization of P2Y12-like receptors in heterologous expression systems
Signal transduction of the ADP receptor P2Y12 via pertussis toxin-sensitive Gi proteins and adenylyl cyclase inhibition is well established [22]. Similar Gi protein-coupling specificity was found for P2Y13 [22, 24], P2Y14 [43], and lyso-PS receptor GPR34 [31, 33]. Because adenylyl cyclase inhibition assays are usually less sensitive and robust several other experimental setups were established to measure function of P2Y12-like GPCR. It has been shown that Gα15 and Gα16 can be activated by a wide variety of GPCR [44]. The ability of Gα16 to bypass the selectivity of receptor/G protein interaction was also useful to measure activation of P2Y13 [45]. It has been demonstrated that replacement of the four or five C-terminal amino acids of Gαq with the corresponding Gαi residues (referred to as GΔ6qi4 [46]) confers the ability to stimulate the PLC-β pathway onto Gi-coupled receptors [47]. Successful heterologous expression and activation by applying chimeric Gαqi4 has been demonstrated for P2Y12–14 and lyso-PS receptor GPR34 [25, 33, 48].
We have previously shown that P2Y12 as well as the lyso-PS receptor GPR34 display increased basal activity in functional assays when compared with other GPCR [33]. This high basal activity can be either discussed as the natural ground state of the receptor activity equilibrium or as the effect of receptor agonists that are present in the cell culture medium or that are released from cells. Analyzing mutations in the highly conserved DRY motif of P2Y12 we recently showed that basal activity is abolished in the DHY mutant but agonist-induced activation remains intact (Fig. 4). We conclude that the basal activity of the ADP receptor is rather a genuine property of this receptor and not due to continuous stimulation by agonists. High basal activity appears to be a general feature of all members of the P2Y12-like receptor group. In our initial ortholog screen we identified two GPR87/P2Y14 receptors, carp GPR87/P2Y14 types 1a and 1b (AY241103, AY241102), which differ in only ten amino acid positions. Interestingly, carp GPR87/P2Y14 type 1b displays higher basal activity and studies are ongoing to identify residues that promote constitutive activity.
Fig. 4High basal activity is a genuine property of P2Y12. Basal activity of the wild-type P2Y12 (DRY motif) and a mutant P2Y12 (DHR motif) was determined in transiently transfected COS-7 cells. Gi coupling of P2Y12 was rerouted to IP production by co-transfection of a chimeric G protein GΔ6qi4 [46]
Because many standard mammalian expression systems endogenously express P2Y receptors clear-cut functional studies are difficult to perform. One exception is the 1321N1 human astrocytoma cell line which does not express P2Y receptors. However, transient expression of GPCR in 1321N1 cells is limited by low transfection efficiency and stable transfection of these cells is usually required. Heterologous expression of GPCR in yeast was initially established for large-scale purification of receptor proteins. These advances in the expression of heterologous GPCR in the yeast Saccharomyces cerevisiae have led to the development of sensitive and selective assays of ligand-induced GPCR activation (reviewed in [49]). To facilitate a more systematic genetic analysis of GPCR function, e.g., by saturating random mutagenesis, we took advantage of a yeast expression system in which parts of the mammalian GPCR signaling system (GPCR and chimeric G protein) are linked to a modified yeast pheromone pathway (Fig. 5) [50]. The coupling of receptor activity to the genetically engineered yeast pathway allows for rapid and economical screening of substance libraries and randomly modified receptor libraries. Functional studies in yeast may have an advantage especially for P2Y receptors because one can (at least partially) circumvent specific problems in working with nucleotide receptors (see above). Only two NTPDases (GDA1 and YND1/APY1) have been found within the entire yeast genome which are mainly expressed in the Golgi apparatus [51]. Although conversion of nucleotides by nucleotidases and ectoapyrases occur also in yeast and one cannot exclude conversion of extracellular nucleotides by yeast enzymes, there is no endogenous plasma membrane P2Y receptor in yeast which can mediate and, therefore, interfere with transmembrane signaling of heterologously expressed P2Y receptors in yeast. We and others have successfully used this system for expression and functional study of P2Y12 [52], P2Y14 [53], and lyso-PS receptor GPR34 (unpublished data). As shown in Fig. 6a, P2Y12-expressing yeast cells grow only in the presence of the agonist 2-methylthioadenosine 5′-diphosphate (2MeS-ADP).
Fig. 5Functional expression and in vitro evolution of GPCR in yeast. Genetically modified yeast cells are transformed with a mammalian GPCR. Following agonist activation and constitutive receptor activity, the receptor couples to a chimeric G protein [backbone yeast G protein Gpa1 in which the C-terminal five amino acids were replaced by the respective mammalian G protein sequence (e.g., from the Gi protein)]. Activation of the chimeric G protein enables yeast cells to grow on histidine-free medium by utilizing parts of the yeast mating pathway [49]. Gpa yeast ortholog of the mammalian G protein alpha subunit, Ste4 yeast ortholog of the mammalian G protein beta subunit, Ste18 yeast ortholog of the mammalian G protein gamma subunit, Ste12 transcription factor that is activated by a MAP kinase signaling cascade, Far1 cell cycle regulator that directly inhibits the yeast cyclin-dependent kinase Cdc28-ClnFig. 6Functional expression and random mutagenesis of P2Y12 in yeast. a The human P2Y12 receptor was transformed into modified yeast (see Fig. 4). The agonist 2-methylthioadenosine 5′-diphosphate (2MeS-ADP) induces a robust yeast cell growth. b The entire coding region of the human P2Y12 was subjected to random mutagenesis and transformed yeast cells were selected for growth in agonist-free U−/H−medium. Colonies that grow under this condition contain a constitutively active P2Y12. Exemplarily, mutations of a triple mutant (L115Q/F177S/R224G) were individualized and tested separately for constitutive activity. The data indicate that L115Q mainly contributes to the constitutive activity of the triple mutant
Constitutively active GPCR are useful tools in studying the action of inverse agonists and activation mechanisms in GPCR. A constitutively active P2Y12 was generated by replacing the endogenous C terminus with the corresponding part of the human P2Y1 receptor [54]. Pharmacological evaluation of several P2Y12 antagonists revealed AR-C78511 (an adenosine derivative) as a potent P2Y12 inverse agonist. Traditional mutagenesis approaches are limited to screen for activating mutations because the number of mutant proteins that can be investigated is usually relatively small, primarily because of technical reasons and the time and costs needed to generate and analyze large numbers of mutant receptors. To circumvent these limitations, yeast has emerged as a highly useful host for the in vivo reconstitution of mammalian GPCR. Therefore, we applied a random mutagenesis and screening approach in yeast to identify key residues in maintaining ground stage of the human P2Y12. PCR-based random mutagenesis was optimized to induce approximately four mutations in 1 kbp. Several dozen clones were selected and are now under in-depth investigation. For example, we identified a triple mutant P2Y12 (Leu115Gln/Phe177Ser/Arg224Gly), and individual characterization revealed Leu115Gln (TMD3) to be mainly responsible for constitutive activity in the yeast expression system (Fig. 6b). These promising results in applying the yeast expression system await further efforts to identify functionally relevant determinants in P2Y12-like receptors.
Establishing in vivo function of P2Y12-like receptors using mouse models
Selective receptor ligand and receptor-deficient animal models are suitable tools to evaluate the physiological relevance of distinct GPCR. Enormous efforts have been undertaken in the development of selective and clinically useful ADP and other nucleotide receptor ligands. But except for P2Y12, dissection of the physiological relevance of all other P2Y12-like GPCR is at the very early stage. Since the pharmacological properties of ligands have been reviewed in detail elsewhere [2] we only shortly summarize available data on receptor-deficient animal models.
Based on the clinical success of irreversibly bound P2Y12 antagonists, such as clopidogrel, the pivotal role of ADP in arterial thrombogenesis is well established. Although the combined action of P2Y1 and P2Y12 is necessary for the full platelet aggregation response to ADP, mice deficient for P2Y12 already display reduced platelet adhesion/activation, thrombus growth, and stability [18, 55]. Except for the altered platelet functionality P2Y12-deficient mice appear normal under standard laboratory conditions. In consent with this finding, we occasionally identified a frameshift (T insertion at base pair position 667) in the P2Y12 coding sequence of several individuals of the Asian house mouse (inbred laboratory strain of Mus musculus castaneus from of the Laboratoire Génome Populations Interactions Adaptation at the Universite Montpellier, France) during ortholog screening which showed no obvious phenotype in standard laboratory captivity. The platelet function was, however, not tested. The identified P2Y12 inactivation in some Asian house mice may reflect either natural polymorphisms present in the wild population or a new polymorphism that has been introduced during captivity. To distinguish between these possibilities, we analyzed the position 667 in P2Y12 from 88 wild M. m. castaneus trapped in Taiwan [41]. All contained the intact P2Y12 allele indicating that the inactive P2Y12 allele is very rare or absent in this wild M. m. castaneus population and favors the hypothesis of its acquisition in captivity (unpublished observations).
The fact that receptor deficiency is per se compatible with viability and fertility appears to be true also for other P2Y12-like receptor-deficient species and mouse models. For example, mice individually lacking GPR82 and GPR34 are viable, fertile, and produced viable offspring (own unpublished observation). Further, there are several vertebrate classes and species naturally deficient in distinct P2Y12-like receptors. For example, P2Y13 is absent in fish genomes sequenced so far, GPR82 is not present in pufferfish genomes, and GPR171 is absent in pufferfish and Xenopus genomes.
It appears that many functions of P2Y12-like receptors are more distinct and their disclosure requires specific challenging conditions. Indeed, P2Y12-deficient mice revealed an unexpected phenotype when specifically challenged. CNS injury is accompanied by release of nucleotides, serving as signals for microglial activation or chemotaxis. Microglia cells express several purinoceptors, including P2Y12. Microglia in P2Y12-deficient mice showed significantly diminished directional branch extension toward sites of cortical damage in the living mouse. These results imply that P2Y12 is a primary site at which nucleotides act to induce microglial chemotaxis at early stages of the response to local CNS injury [56].
Variants of P2Y12-like receptor genes within human populations
Activating and inactivating mutations in GPCR have been made responsible for more than 30 different human diseases [57]. As expected from its pivotal role in platelet activation inactivating mutations in P2Y12 can cause a congenital bleeding disorder. Only a few missense (Met1Arg, Pro258Thr, Arg256Gln, Arg265Trp) and frameshifting (frameshift at amino acid position 240) mutations have been reported [19, 58–60]. Unexceptionally, the missense mutations found are at highly conserved positions and cause an impairment of receptor function. There are no other human diseases identified yet which are associated with dysfunction of P2Y12-like receptors.
The antithrombotic effect of clopidogrel is considerably variable and the P2Y12 gene was screened for possible sequence variants. Five nucleotide variations were found in the human P2Y12 gene, two of them silent substitutions in the coding region [61]. Several studies were initiated to investigate the impact of P2Y12 polymorphisms on atherosclerosis [62, 63] and clopidogrel efficiency in preventing neurological events [64] showing no or some association to one haplotype. In single nucleotide polymorphism (SNP) projects, such as HapMap [65], SeattleSNP etc., silent variations have been identified at amino acid position Val4, Asn6, Gly12, and Phe182 of P2Y12. For example the C/T variation at amino acid position Phe182 is only found in the African population whereas the G/T variation at position Gly12 is present only in European and Asian populations. One missense mutation (Glu330Gly; receptor C terminus) has been detected in SNP projects. This variant is absent in European and Asian populations but displays an allelic frequency of about 15% in the African population. The functional relevance of this missense mutation has not been studied yet but a Gly at position 330 is naturally found in many other P2Y12 orthologs including primate P2Y12 almost ruling out a specific input in receptor function.
One silent variant (C/T at Ile59) and one substituting variant (T/C at Met158Thr) were identified in the coding region of P2Y13. The Thr158 variant is less frequent in African populations (~4%) when compared to European (~21%) and Asian (~18%) populations. Met158 is not conserved and a Thr at this position is found in the dolphin (Tursiops truncates).
Only two silent substitutions (A/G at Ala35 and T/C at Phe240) have been identified in P2Y14 so far. The GPR171 gene contains two more frequent silent substitutions (T/C at Tyr19, A/C Thr58) and one missense variant (A/G at Ile283Val, frequency about 6%) which is located within the DPXXY motif of TMD7. Ile283 is quite conserved among vertebrate GPR171 orthologs. The functional relevance of Val283 has not been studied yet but it occurs naturally in the zebrafish ortholog.
In the human GPR87 gene three silent polymorphisms (G/A at Pro46, G/A at Leu179, C/T at Tyr355) and one nonsynonymous polymorphism (C/T at Thr205Met) are present. The position 205 is not conserved and Met205 is found in several rodents. Only rare silent substitutions in the coding regions have been found in GPR82 (C/T at Asp313) and in GPR34 (A/G at Val296) [34].
SNP stochastically occur in individual genomes and can amount to a reasonable frequency in populations by drift but also by selection. There are several approaches and methods which are suitable to distinguish between drift and selection. Population genetic models predict that selection can leave “footprints” in closely linked genomic regions. Several methods were developed to detect signatures of selective sweeps in genomic sequences [66]. All methods require data on allele variation (mainly SNP) within populations. Large genome-wide analyses have scanned the human genome for signatures of positive selection on the basis of nonsynonymous and synonymous substitution ratios or single nucleotide polymorphism (SNP) data. Several loci which contain GPCR genes have been identified using such methods, but the P2Y12-like receptor-containing loci showed no strong signatures of recent positive selection in these studies [67–70].
Conclusion
P2Y12-like receptors which are grouped mainly by phylogenetic relations have been an inherent part of the vertebrate GPCR repertoire since more than 450 Myr. Although sharing features in respect to structural determinants and signal transduction the activating ligands are heterogeneous such as nucleotides, nucleotide derivatives, leukotrienes, and phospholipids. We are at the very beginning of understanding the physiological importance of the individual members. The nature of the ligands, first functional data, and expression of several members in migrating cells point at functions in immunologic response and tissue damage response. Doubtless, upcoming receptor-deficient mouse models and selective receptor ligands will help to unveil the functions of P2Y12-like receptor members. Further, one should consider combined receptor-deficient mouse models within this group but also with other P2Y receptors to uncover phenotypes which are hidden by receptor redundancy, e.g., in the case of ADP receptors.

evolution
nucleotide receptor
g protein-coupled receptor
orphan gpcr


Letrozole in the extended adjuvant setting: MA.17

Relapse after completing adjuvant tamoxifen therapy is a persistent threat for women with hormone-responsive breast cancer. Third-generation aromatase inhibitors, such as letrozole, provide a new option for extended adjuvant hormonal therapy after 5 years of tamoxifen. MA.17 was conducted to determine whether letrozole improves outcome after discontinuation of tamoxifen. Postmenopausal women with hormone receptor-positive breast cancer (N = 5,187) were randomized to letrozole 2.5 mg or placebo once daily for 5 years. At a median follow-up of 30 months, letrozole significantly improved disease-free survival (DFS; P < 0.001), the primary end point, compared with placebo (hazard ratio [HR] for recurrence or contralateral breast cancer 0.58; 95% confidence interval [CI] 0.45, 0.76] P < 0.001). Furthermore, letrozole significantly improved distant DFS (HR = 0.60; 95% CI 0.43, 0.84; P = 0.002) and, in women with node-positive tumors, overall survival (HR = 0.61; 95% CI 0.38, 0.98; P = 0.04). Clinical benefits, including an overall survival advantage, were also seen in women who crossed over from placebo to letrozole after unblinding, indicating that tumors remain sensitive to hormone therapy despite a prolonged period since discontinuation of tamoxifen. The efficacy and safety of letrozole therapy beyond 5 years is being assessed in a re-randomization study, following the emergence of new data suggesting that clinical benefit correlates with the duration of letrozole. MA.17 showed that letrozole is extremely well-tolerated relative to placebo. Letrozole should be considered for all women completing tamoxifen; new results from the post-unblinding analysis suggest that letrozole treatment should also be considered for all disease-free women for periods up to 5 years following completion of adjuvant tamoxifen.
Introduction and rationale
There is a persistent risk of breast cancer recurrence following primary treatment [1–3]. Initially, patients with hormone receptor-positive (HR+) breast tumors have a lower risk of recurrence than those with HR− tumors, but with longer follow-up, the opposite may be the case [3, 4]. For example, Saphner showed that the significantly higher hazard of recurrence in HR− versus HR+ patients in the time period 0–12 years (P < 0.00001) could be explained by the higher risk of recurrence in years 0–5 for HR− patients (P < 0.0001). However, between years 3 and 4, the hazard of recurrence for HR− and HR+ patients crossed, and beyond 5 years was actually higher for HR+ patients (P = 0.00002) [4]. These data clearly indicate the need for continuous hormonal treatment for women with HR+ tumors.
The benefits of adjuvant hormonal treatment with tamoxifen were first demonstrated in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 trial [5]. This large randomized, double-blind, placebo-controlled trial involving patients with node-negative, HR+ breast cancer demonstrated a significant prolongation of disease-free survival (DFS) among women treated with tamoxifen for 5 years, as compared with those receiving placebo. Updated results with longer follow-up demonstrated that the 5-year benefit in DFS with tamoxifen persisted through at least 10 years of follow-up, and a statistically significant survival benefit was also observed [6]. However, since the optimal duration of tamoxifen therapy was not known, patients who had completed 5 years of tamoxifen therapy and were disease-free were re-randomized to receive placebo or tamoxifen. Results published with a follow-up of 7 years after reassignment demonstrated a disadvantage in patients who continued tamoxifen compared with those who discontinued: DFS was 78 vs. 82%, respectively (P = 0.03), and overall survival (OS) was 91 vs. 94% (P = 0.07). Thus, extending tamoxifen treatment beyond 5 years was not deemed beneficial [7] nor recommended [8] when the MA.17 trial was initiated.
While clearly in the best interest of patients, discontinuation of tamoxifen after 5 years creates a therapeutic dilemma because of the persistent risk of breast cancer recurrence. Relapse or appearance of new tumors after completion of tamoxifen therapy is relatively common in patients with HR+ tumors [1, 2, 4, 7]. The Oxford meta-analysis found that more than half of breast cancer recurrences and two thirds of breast cancer deaths occur after 5 years of adjuvant tamoxifen [3]. In the NSABP B-14 trial, the average annual rate of breast cancer recurrences was 8.9 per 1,000 patients who discontinued tamoxifen at 5 years [7]. Patients in whom tamoxifen is discontinued therefore require an alternative treatment option to provide continuing protection from recurrence.
The loss of efficacy seen with long-term tamoxifen therapy may result from the emergence of a hormone-independent tumor phenotype [9, 10] or the induction of hypersensitivity to estradiol [11, 12]. Tamoxifen is thought to be more susceptible than aromatase inhibitors (AIs) to this adaptive change because of its intrinsic agonist properties [12]. Furthermore, data from the NSABP B-14 trial suggest that residual tumor cells may become tamoxifen-resistant, and that continued use of tamoxifen might in fact stimulate their proliferation [6, 13].
The development of highly potent and selective third-generation AIs provided a new hormone therapy option for patients with HR+ breast cancer [14–16]. It is suggested in independent studies that Letrozole (Femara®) is the most potent of the AIs as determined by in vitro assays [17] and clinical studies of total body aromatization [18]. A recent study has demonstrated that the more complete inhibition of aromatase achieved by 2.5 mg of letrozole than by 1 mg of anastrozole results in a greater degree of suppression of estradiol [19]. The clinical efficacy of letrozole was initially demonstrated in patients with HR+ metastatic breast cancer. In this setting, first-line therapy with letrozole was shown to significantly improve outcome compared with tamoxifen therapy [20, 21]. While OS was not significantly improved (34 vs. 30 months, respectively), an exploratory analysis of the patients that did not cross over showed a longer survival benefit for letrozole-treated patients (35 vs. 20 months) [22]. Second-line therapy with letrozole has also demonstrated significant clinical benefits in patients with HR+ metastatic breast cancer in whom tamoxifen has failed [23], and in one randomized trial demonstrated a significantly higher response rate than anastrozole in this population [24].
Data from in vivo models using MCF-7 cells transfected with the aromatase gene have shown that letrozole is more effective than tamoxifen and devoid of the agonist action observed with the selective estrogen receptor modulator [25, 26]. Although the mechanisms of estrogen blockade are different for letrozole and tamoxifen, combining the two agents did not increase antitumor activity in the MCF-7 xenograft model [25]. The authors proposed that sequential administration of tamoxifen and letrozole would be a more effective strategy [25]. This hypothesis was recently confirmed in vivo [27]. Using the MCF-7 model, it was demonstrated that tumors progressing on tamoxifen responded to second-line letrozole therapy, but tumors that progressed on letrozole did not respond to second-line treatment with tamoxifen or fulvestrant [27].
The evident need to continue hormone therapy after discontinuation of tamoxifen in patients with HR+ primary breast cancer and the potential efficacy of AIs in tumors resistant to tamoxifen provided the rationale for testing adjuvant letrozole after 5 years of tamoxifen. This paper reviews the key results from the landmark MA.17 trial [28, 29] and discusses the advantages of letrozole treatment after the recommended 5 years of tamoxifen therapy have been completed. To date, letrozole is the only AI approved in the United States and Europe for extended adjuvant therapy.
Trial design and patients
MA.17 was a phase III, randomized, double-blind, placebo-controlled trial of letrozole as extended adjuvant therapy in postmenopausal women with primary breast cancer who had completed approximately 5 years of adjuvant tamoxifen therapy [28, 29]. The aim of the trial was to determine whether letrozole improves outcome after the discontinuation of adjuvant tamoxifen therapy. The MA.17 trial was led by the National Cancer Institute of Canada Clinical Trials Group and included the North American Breast Intergroup and the Breast International Group. Institutional review boards of participating institutions approved the study protocol, and all patients gave written informed consent.
Patient population
The trial enrolled 5,187 postmenopausal women with early-stage breast cancer in whom 5 years of tamoxifen (range 4.5–6 years) therapy had been completed less than 3 months before enrollment. Eligible women had to have histologically confirmed, HR+ primary breast cancer. HR+ tumors were defined as estrogen receptor-positive (ER+) or progesterone receptor-positive (PgR+) as determined by a level of 10 fmol/mg of protein or a positive immunohistochemical analysis. Women were defined as being postmenopausal if they were aged at least 50 years at the start of adjuvant tamoxifen therapy, were <50 years of age at the start of tamoxifen therapy but postmenopausal at the initiation of tamoxifen therapy, were <50 years at the start of tamoxifen therapy but had undergone bilateral oophorectomy, were premenopausal and <50 years at the start of tamoxifen therapy but became amenorrheic during chemotherapy or treatment with tamoxifen, or were any age but had postmenopausal levels of luteinising hormone or follicle-stimulating hormone prior to the study. All women had a good performance status and life expectancy of at least 5 years.
Randomized trial design
Eligible women were randomly assigned to receive treatment with letrozole (2.5 mg) or placebo orally daily for 5 years (see Fig. 1). Women were stratified according to the tumor HR status (positive or unknown), lymph-node status (negative, positive, or unknown), and receipt or nonreceipt of previous adjuvant chemotherapy. Exploratory sub-analyses were based on these stratification factors and two additional covariates (criteria for the definition of postmenopausal status at the start of tamoxifen treatment and duration of tamoxifen treatment).
Fig. 1MA.17 randomized trial design
End points and rules for interim analyses
The primary end point of the trial was DFS, defined as the time from randomization to the earliest recurrence of the primary disease (in the breast, chest wall, or nodal or distant metastatic sites) or the development of a new primary breast cancer in the contralateral breast. Secondary cancer and death without a recurrence or a diagnosis of contralateral breast cancer were not included as events. The trial was powered to detect a 2.5% improvement in 4-year DFS with letrozole (from 88 to 90.5%). Two interim analyses were scheduled, and stopping rules were specified a priori for interim monitoring [30].
The secondary efficacy end points of the trial were OS (defined as the time from randomization to death from any cause), annual incidence rate of contralateral breast cancer, long-term safety and tolerability, and overall and menopause-specific quality of life (QOL). In addition, distant DFS (DDFS), defined as the time from random assignment until the first observation of distant metastasis, was included as a secondary efficacy end point in the final analysis [29].
QOL and long-term safety were assessed as secondary end points [31]. Adverse events were graded according to the Common Toxicity Criteria of the National Cancer Institute (version 2.0). QOL was assessed with the Medical Outcomes Study 36-Item Short Form General Health Survey (SF-36) and the Menopause-Specific Quality of Life questionnaire [32, 33]. The effects of letrozole on lipid profile and bone mineral density (BMD) were assessed annually in companion studies to MA.17 [34, 35].
Efficacy of letrozole as extended adjuvant therapy
A total of 5,187 patients were randomized to either letrozole (n = 2,593) or placebo (n = 2,594). Because of noncompliance, 10 patients in the letrozole and seven in the placebo arm were excluded from all analyses, leaving 5,170 patients (2,583 on letrozole and 2,587 on placebo) in the time to event analysis (50 patients deemed ineligible for several reasons and 33 with major protocol violations were included in the analysis). The final safety analysis excluded 21 patients who never received study medication, yielding a final safety population of 5,149 patients, 2,572 receiving letrozole and 2,577 receiving placebo [29]. Based on the 43% reduction in recurrence risk (P = 0.00008) with letrozole seen in the first interim analysis at 2.4 years’ median follow-up [28], the data and safety monitoring committee recommended that the MA.17 trial be discontinued early, and the participants were informed of the results. The trial was unblinded in October 2003, and patients on placebo were given the opportunity to switch to letrozole. Updated efficacy results after a median follow-up of 30 months confirmed the significant clinical benefits of letrozole as extended adjuvant therapy [29]. The updated trial results and recent additional efficacy analyses of MA.17 are summarized below.
Letrozole significantly improves outcome
At 30 months’ follow-up, letrozole significantly improved DFS, the primary end point, compared with placebo (see Fig. 2) [29]. The 4-DFS for patients receiving letrozole was 94.4%, compared with 89.8% for patients receiving placebo. The hazard ratio for recurrence or contralateral breast cancer was 0.58 (95% confidence interval [CI] 0.45, 0.76; P ≤ 0.76), representing a 42% reduction in risk for letrozole relative to placebo. The updated analysis also showed that letrozole produced a statistically significant improvement in DDFS (hazard ratio = 0.60; 95% CI 0.43, 0.84; P = 0.002), which may be regarded as a more meaningful end point than overall DFS; women with distant metastases inevitably die of breast cancer, and an improvement in DDFS may therefore translate into longer overall survival [36, 37]. Letrozole treatment non-significantly prolonged time to contralateral breast cancer incidence, resulting in a 37.5% relative risk reduction compared with placebo [29].
Fig. 2Kaplan–Meier curves for disease-free survival in the updated analysis of MA.17. N, number at risk; S, survival percent, with 95% confidence intervals (CIs) in parentheses. Reprinted from ref. [29] with permission
The prospectively planned subgroup analysis showed that letrozole significantly improved DFS in all patients, irrespective of nodal status. The reduction in risk of recurrence in node-positive tumors was 39% (hazard ratio = 0.61; 95% CI 0.45, 0.84), and 55% in those with node-negative tumors (hazard ratio = 0.45; 95% CI 0.27, 0.73). While OS was not significantly improved (hazard ratio = 0.82; 95% CI 0.57, 1.19; P = 0.3), letrozole significantly improved OS in patients with node-positive tumors (hazard ratio = 0.61; 95% CI 0.38, 0.98; P = 0.04) (see Fig. 3), and this was the first survival advantage demonstrated by an AI in early breast cancer.
Fig. 3Forest plots of the treatment effect (letrozole vs. placebo), in terms of overall survival, in subgroups defined by hormone receptor status, lymph node status, previous chemotherapy, menopausal criteria, and duration of tamoxifen treatment. For each subgroup, the hazard ratio for death from any cause is plotted as a solid square, and the area of the square is proportional to the variance of the estimated effect. The length of the horizontal line through the square indicates the 95% confidence interval (CI). The arrow at the end of the horizontal line indicates that the confidence interval is larger than the scale of the figure. Reprinted from ref. [29] with permission
Additional MA.17 analyses
Optimal duration of extended adjuvant letrozole
The final MA.17 database, including all events up to the date of unblinding, was analyzed to examine the relationship between duration of treatment and outcomes [38, 39]. Data from this analysis have provided further evidence to support an extended duration of letrozole, as this cohort analysis found that the longer patients are exposed to extended adjuvant letrozole (at least out to 48 months), the greater the benefit [39].
The risk of disease recurrence increased over time in the placebo group, whereas in patients receiving letrozole, risk appeared to peak at around 2 years of treatment and decrease thereafter. In the overall patient population, hazard ratios for events in DFS and DDFS progressively decreased over time, favoring letrozole, with the trend being significant (P < 0.0001 and P = 0.0013, respectively). The trend for OS was not significant but was always <1. In the 2,360 patients with node-positive status, hazard ratios for DFS, DDFS, and OS all decreased over time, with tests for trend all showing significance (P = 0.0004, 0.0005, and 0.038, respectively). Considering the 2,568 patients with node-negative status, the hazard ratios for DFS decreased over time, with the test for trend being significant (P = 0.027), whereas the hazard ratios for DDFS and OS showed no significant change over time (see Table 1).
Table 1Analysis of the hazard ratios for disease recurrence over time between the letrozole and placebo arms of MA.17Month after randomizationNo. at risk (letrozole/placebo)Hazard rate (letrozole)Hazard rate (placebo)Hazard ratio (letrozole vs. placebo)a122,425/2,4090.000930.001800.52 (0.40–0.64)241,555/1,5300.001050.002360.45 (0.33–0.56)36768/7230.000900.002610.35 (0.21–0.48)48244/2310.000590.003060.19 (0.04, 0.34)Reprinted from ref. [38] with permission from ElsevieraHazard ratios <1 indicate values in favor of letrozole
MA.17 ITT analysis
The intent-to-treat (ITT) analysis at 54 months’ follow-up looked at all outcomes, including all events before and after the unblinding, based on the original randomization of letrozole versus placebo; it did not take into account whether or not patients in the placebo group switched to letrozole at the unblinding of the data. The results further showed that patients randomized initially to letrozole had fewer DFS events than those initially randomized to placebo [40]. Publication of the final analysis of this data are awaited, but these provisional results highlight the strong beneficial effect of extended adjuvant therapy with letrozole when started immediately after tamoxifen.
Impact of HR status on clinical benefit
A retrospective analysis was conducted to determine whether HR status had an effect on the outcome of letrozole in the extended adjuvant setting [41]. ER and PgR positivity were defined as ≥10 fmol/mg protein or positive by immunocytochemical analysis. Preliminary results from 4,635 patients, based on local testing of HR status, showed that the reduction in risk of recurrence with letrozole compared with placebo was greatest in women with the most hormone-dependent tumors. The final results of this analysis await publication. These results should be interpreted cautiously, as this was an unplanned, retrospective analysis, and receptor levels were measured locally. Furthermore, from the outset, the analysis of outcomes in all other subgroups besides the ER+/PgR+ (n = 3,809) patients was weakened by the low numbers of patients in these groups (e.g., ER+/PgR−, n = 636) [42].
MA.17 post-unblinding analysis
The trial was unblinded in 2003 because DFS values were met (stopping boundary nominal significance, P = 0.0008) and patients were given the opportunity to cross over. Post-unblinding analysis of MA.17 has provided additional efficacy data on patients who had crossed over from placebo to letrozole (n = 1,655), comparing them with patients who elected no treatment at the time of the unblinding (n = 613) [43]. These patients had not received any hormonal therapy after discontinuing tamoxifen. Data were adjusted for baseline patient and disease variables including tumor size, nodal status, and prior adjuvant chemotherapy. A preliminary analysis suggests that letrozole has significant clinical benefits in patients in whom treatment with the AI is started after a prolonged period since the discontinuation of tamoxifen. The publication of these results is eagerly awaited as they may effect patient care worldwide.
MA.17 re-randomization
A re-randomization of all patients completing letrozole to receive a further 5 years of letrozole or placebo is under way to confirm this finding [40, 44]. An amendment to this protocol allows women completing 5 years of any AI to be re-randomized to a further 5 years of letrozole or not regardless of prior tamoxifen or its duration. The MA.17 re-randomization study should provide additional insights into the efficacy and safety of extending letrozole therapy beyond 5 years [45].
Safety of letrozole in the extended adjuvant setting
The women included in this trial had been disease-free for approximately 5 years during treatment with tamoxifen and, therefore, the safety and tolerability of continued hormone treatment with letrozole was an important consideration when the MA.17 trial was designed. Furthermore, early unblinding of the trial has not prevented the collection of long-term safety data, and additional sub-studies are providing useful information on the safety profile of letrozole in the extended adjuvant setting. The safety of AIs is discussed in detail in the paper by Dr. Perez in this supplement.
The MA.17 trial showed that letrozole is extremely well tolerated relative to placebo. The most common adverse events reported were secondary to estrogen suppression and included hot flashes, myalgia, arthralgia, alopecia, and newly diagnosed osteoporosis. The increase in newly diagnosed osteoporosis (8.1% for letrozole vs. 6% for placebo; P = 0.003) [29] was predictable from the potent suppression of estrogens by third-generation AIs [17] and the association between estrogen levels and bone turnover [46–48]. Of note, no significant difference in clinical fracture rate was seen between letrozole and placebo groups (5.3 vs. 4.6%; P = 0.25) [29].
MA.17B is a companion study designed to compare the effects of letrozole (n = 122) and placebo (n = 104) on BMD in the L2–L4 (posteroanterior) region of the spine and hip [35]. At 24 months, patients receiving letrozole had a significant decrease in total hip BMD (−3.6 vs −0.71%; P = 0.044) and lumbar spine BMD (−5.35 vs. −0.70%; P = 0.008). Further follow-up is necessary to evaluate the long-term clinical implications of this modest increase in bone resorption and reduction in BMD in the spine and hip with letrozole compared with placebo. Prophylactic use of the bisphosphonate zoledronic acid is being studied as a means to prevent BMD loss [49, 50]. Results from two clinical trials have indicated that early use of zoledronic acid effectively prevents BMD loss in women receiving adjuvant letrozole [49, 51].
Estrogen has a beneficial effect on lipid profiles, and it has been suggested that AIs may have a relatively unfavorable effect. However, MA.17L, a substudy of MA.17 (n = 347), demonstrated that letrozole does not significantly alter serum cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, or lipoprotein (a) compared with placebo [34]. This is in agreement with the results of the main MA.17 trial, which showed no difference in hypercholesterolemia rates between placebo and letrozole [28]. Importantly, in the updated analysis of the MA.17 dataset, there were no significant differences between the letrozole and placebo arms in the incidence of hypercholesterolemia (16% in each arm; P = 0.79) or cardiovascular events (5.8 vs. 5.6%; P = 0.76) [29].
The QOL substudy was conducted in 3,612 patients treated in MA.17 (1,813 letrozole and 1,799 placebo) [31]. The analysis demonstrated that letrozole did not have an adverse impact on overall QOL, as determined by SF-36, which is an important and reassuring finding, as the extension of letrozole treatment for up to 10 years in this setting is now being tested [44].
Conclusions
In recent years, considerable progress has been made in developing more effective hormonal treatments for women with breast cancer and improving the efficacy demonstrated with tamoxifen [3]. The pioneering MA.17 trial has demonstrated the need for extended adjuvant therapy after 5 years of tamoxifen to reduce the risk of recurrence in postmenopausal women with HR+ breast cancer. On the basis of this trial, letrozole was approved as extended adjuvant therapy, and it is currently the only AI approved for this indication [22, 52].
MA.17 demonstrated that extended adjuvant therapy with letrozole provides women further protection against relapse after the completion of tamoxifen. These findings support the concept that distant micrometastases that have survived 5 years of tamoxifen therapy remain highly estrogen-sensitive and responsive to extended adjuvant letrozole treatment. This is an important clinical benefit in view of the persistent risk of relapse beyond 10 years in patients with HR+ tumors [4]. In fact, the cohort analysis showed that in the placebo group, there was actually an increasing risk of disease recurrence over time after discontinuing prior tamoxifen [40]. The preliminary MA.17 ITT data suggest that the strongest beneficial effect is still obtained when starting letrozole within 3 months of completing tamoxifen [38], while the post-unblinding results suggest that women with hormone-dependent breast cancer who are prescribed letrozole following a prolonged delay after completing tamoxifen may experience a significant improvement in outcome [43]. Therefore, if a patient misses the chance to start letrozole within 3 months post tamoxifen, these results suggest that there is still a benefit to initiating letrozole therapy for up to 5 years following the discontinuation of tamoxifen. Importantly, women in all risk categories benefited in terms of reduced risk for recurrence of their cancer. Thus, in both node-positive as well as node-negative women, there was a strong improvement in DFS, and “low risk” status of the primary tumor should not preclude consideration of extended adjuvant therapy with letrozole. Extended therapy with adjuvant letrozole should therefore be considered for all women currently completing tamoxifen. In addition, in women who have completed tamoxifen within the last 5 years, introduction of letrozole for 5 years can be discussed because although we do not have level 1 evidence for benefit in this setting as yet, our post-unblinding analysis of MA17 strongly supports the potential for benefit in these women.
Among the clinical questions left unanswered are the optimal duration of letrozole and the long-term safety of extended adjuvant therapy in women leading a normal healthy lifestyle. Preliminary results of a cohort study analysis of MA.17 provide support for the use of extended adjuvant letrozole for at least up to 4 years [40, 53]. The MA-17 re-randomization study will assign patients completing 5 years of letrozole to a further 5 years of letrozole or placebo and should provide data on the efficacy and safety of extending letrozole therapy beyond 5 years [45].
In conclusion, HR+ breast cancer presents an unremitting threat that may require life-long hormone therapy. The optimal hormone treatment strategy is evolving based on the results of landmark clinical trials in the initial adjuvant [54, 55], sequential adjuvant [56], and extended adjuvant settings [28, 29]. The optimal agent, sequence of treatments, or combination of treatments will be able to provide the greatest improvement in OS with minimal acute and long-term toxicity. MA.17 has demonstrated that letrozole is highly effective and extremely well tolerated when given in the extended adjuvant setting. The results of the pivotal MA.17 trial have changed current clinical practice to extend letrozole protective therapy in thousands of breast cancer patients currently receiving 5 years of adjuvant tamoxifen. The benefits of long-term adjuvant letrozole treatment clearly outweigh any adverse events in postmenopausal women who have survived breast cancer after initial adjuvant therapy with tamoxifen. Long-term side effects and risks continue to be monitored and taken into account when any individual patient is being considered for extended adjuvant therapy.

letrozole
ma.17
tamoxifen
breast cancer
aromatase inhibitors