DOI: 10.1126/science.284.5414.665
Science 284, 665 (1999);
George Rice et al.
Xq28
Male Homosexuality: Absence of Linkage to Microsatellite Markers at
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into an SCF complex facilitates activation
of Cdc34’s intrinsic conjugating activity.
Rbx1 may also function as a ubiquitin carrier,
a possibility that remains to be tested.
Rbx1 is a highly conserved protein (15) that
plays an essential role in SCF function in part
by recruiting Cdc34. Consistent with this, rbx1
mutants have been independently found that are
synthetically lethal with cdc34 mutants (22).
The closest Rbx1 homolog in yeast is the essential
anaphase-promoting complex (APC)
subunit Apc11 (23), which may act in a parallel
fashion, binding to the Cdc53 homolog Apc2
and recruiting an E2 into the APC, making the
APC a distantly related member of the SCF
family of E3s. Sequences related to the R-box
motif in Rbx1 can be found in several other S.
cerevisiae proteins associated with ubiquitination,
including Hrd1, Rad18, and Ubr1, which
have been implicated in 3-hydroxy-3-methylglutary–
coenzyme A reductase ubiquitination
(24), DNA repair (25), and the N-end rule
pathway (26), respectively, and in four uncharacterized
open reading frames (Fig. 4F). Rad18
and Ubr1 form independent complexes with the
E2 Rad6 (27–29), which controls the N-end
rule pathway, DNA repair, and telomeric silencing
(25–27). Most R-box proteins are much
larger than Rbx1 and may themselves contain
substrate recognition domains, as has been
demonstrated for Ubr1 (29). In plants, the Prt1
R-box protein is genetically implicated in the
N-end rule pathway (30). In mammals, Mdm2,
a p53 E3, also contains an R-box–related motif
(14) that is required for its function (31). Currently,
ubiquitin-carrying HECT-domain proteins
are the only well characterized class of
E3s not linked to R-box proteins. Thus, E3s
may fall into two main classes: the SCF class
that require R-box proteins and the HECT class
that do not. The finding that mammalian Rbx1
is a component of the von Hippel–Lindau
(VHL)–Elongin B/C–Cul2 complex (15) suggests
that these complexes may also be involved
in the transfer of ubiquitin or ubiquitinlike
proteins.
References and Notes
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(1997).
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7. A. R. Willems et al., ibid., p. 453.
8. R. J. Deshaies, V. Chau, M. Kirschner, EMBO J. 14, 303
(1995).
9. Y. Barral, S. Jentsch, C. Mann, Genes Dev. 9, 399
(1995).
10. B. L. Schneider et al., Nature 395, 86 (1998).
11. N. Mathias et al., Mol. Cell. Biol. 16, 6634 (1996).
12. D. K. Skowyra, K. Craig, M. Tyers, S. J. Elledge, J. W.
Harper, Cell 91, 209 (1997).
13. Yeast extracts from isogenic YM4575 (GRR1) or
YM4576 (grr1D) strains (32) or CB018 ( pep4::HIS3)
were fractionated on DEAE-Sephacryl (33). Proteins
eluting with 250 and 500 mM KCl were collected (F250
and F500, respectively). Immunoblot analysis revealed
Cdc34 only in the F500 fraction. Twenty-microliter
ubiquitination reactions (30 to 60 min, 25¡C) contained
500 nM Cdc34, 300 nM E1, 2 mM adenosine triphosphate
(ATP), an ATP-regenerating system, 20 mM ubiquitin,
0 to 120 mg of F250, and 50 ng of active or
kinase-defective (K-) HA-Cln1/GST-Cdc28 (12). Cln1
autophosphorylation was performed at 25¡C in the
presence of 1 mM ATP for 30 min. Reaction mixtures
were separated by SDSÐpolyacrylamide gel electrophoresis
(SDS-PAGE), and products were visualized by
immunoblotting with ECL detection (Amersham). SCF
complexes were puriÞed from insect cells 40 hours after
infection with antibodies to tagged proteins (12). A
baculovirus encoding untagged Grr1 (in pVL1392) was
produced with Baculogold (Pharmingen). Rabbit anti-
Grr1 was generated with the peptide MDQDNNNHNDSNRL
(see legend to Fig. 4 for amino acid abbreviations).
RBX1 and rbx1-1 (16) extracts were prepared from cells
shifted to 38¡C for 16 hours. Strains (MATa RBX1::HIS3
ura3 leu2 trp1 lys2 his3D200 can1-100 cyh2) were
maintained by a plasmid containing either a wild-type
(pDK101) or mutant (pDK102) version of RBX1 in
pRS314.
14. D. K. Skowyra, D. M. Koepp, S. J. Elledge, J. W. Harper,
unpublished data.
15. T. Kamura et al., Science 284, 657 (1999).
16. RBX1 was disrupted by inserting the LEU2 gene into
the Nsi I site in the RBX1 open reading frame in
pDK100 (containing a genomic RBX1 fragment in
pRS316). A Sac IÐSph I fragment from pDK100 was
used to transform Y80 (MATa can1-100 ade2-1 his3-
11, 15 leu 2-3, 112 trp1-1 ura3-1) carrying pDK100
to generate the disruption strain. Strains that could
not grow without pDK100 were chosen, and integration
of the LEU2 gene was conÞrmed by Southern
(DNA) blot analysis. The rbx1-1 allele was generated
by polymerase chain reaction mutagenesis (34) with
primers ßanking the open reading frame. The plasmid
was rescued from a temperature-sensitive strain, retested,
and sequenced.
17. C. Connelly and P. Hieter, Cell 86, 275 (1996).
18. Baculoviruses encoding Rbx1 and MYC3-Rbx1 [in
pVL1392 or pHI-100 by universal plasmid subcloning
(35), respectively] were generated with Baculogold
(Pharmingen).
19. For SCF assembly, we coinfected Hi5 cells (2 3 106)
with recombinant baculoviruses. Forty hours later, cells
were lysed in 50 mM tris-HCl (pH 7.5) containing 100
mM NaCl, 0.5% NP-40, 2 mM dithiothreitol, 0.1 mM
Zn(OAC)2, and protease inhibitors (Boehringer-
Mannheim). Cleared lysates were immunoprecipitated
for 1 to 2 hours with 10 ml of beads containing immobilized
antibodies. Proteins were separated by SDSPAGE
and immunoblotted. HA-Cln1 and Cdc34 ubiquitination
was performed at 25¡C with 10 ml of agarose
beads containing SCF complexes with or without Rbx1
supplemented with 500 nM Cdc34, 300 nM E1, 4 mM
ATP, 20 mM ubiquitin (or 40 mM GST-UbRA), and 50 ng
of HA-Cln1/GST-Cdc28. Reaction mixtures were separated
by SDS-PAGE and immunoblotted.
20. A. Banerjee, L. Gregori, Y. Xu, V. Chau, J. Biol. Chem.
268, 5668 (1993).
21. F250 (1 mg) was fractionated over a Superdex 200
column (Pharmacia) in 50 mM tris-HCl (pH 7.5), 50
mM KCl. Pools of four fractions (0.25 ml each) were
concentrated with Centricon-10 and samples (50 mg
of protein) were used for assays (13).
22. M. Goebl, personal communication.
23. W. Zachariae et al., Science 279, 1216 (1998).
24. R. Y. Hampton, R. G. Gardner, J. Rine, Mol. Biol. Cell
7, 2029 (1996).
25. H. Huang, A. Kahana, D. E. Gottschling, L. Prakash,
S. W. Liebman, ibid. 17, 6693 (1997).
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(1990).
27. V. Bailly, S. Lauder, S. Prakash, L. Prakash, J. Biol.
Chem. 272, 23360 (1997).
28. V. Bailly, S. Prakash, L. Prakash, Mol. Cell. Biol. 17,
4536 (1997).
29. R. J. Dohmen, K. Madura K. B. Bartel, A. Varshavsky,
Proc. Natl. Acad. Sci. U.S.A. 88, 7351 (1991).
30. T. Potuschak et al., ibid. 95, 7904 (1998).
31. R. Honda, H. Tanaka, H. Yasuda, FEBS Lett. 420, 25
(1997).
32. J. S. Flick and M. Johnston, Mol. Cell. Biol. 11, 5101
(1991).
33. R. J. Deshaies and M. Kirschner, Proc. Natl. Acad. Sci.
U.S.A. 92, 1182 (1995).
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(1996).
35. Q. Liu, M. Z. Li, D. Leibham, D. Cortez, S. J. Elledge,
Curr. Biol. 8, 1300 (1998).
36. We thank M. Goebl for anti-Cdc34 and discussions,
M. Tyers for Cdc53, M. Johnson for strains, C. Correll
and R. Deshaies for HA-Grr1, and M. Li and P. Sen for
technical assistance. Supported by NIH grants
AG11085 (to J.W.H. and S.J.E.), GM54137 (to J.W.H.),
GM41628 (to R.C.C.), the Welch Foundation ( J.W.H),
the H. A. and Mary K. Chapman Charitable Trust
( J.W.C), and the Helen Hay Whitney Foundation
(D.M.K). S.J.E. and J.W.C. are Investigators with the
Howard Hughes Medical Institute.
25 February 1999; accepted 24 March 1999
Male Homosexuality: Absence
of Linkage to Microsatellite
Markers at Xq28
George Rice,1* Carol Anderson,1 Neil Risch,2 George Ebers1
Several lines of evidence have implicated genetic factors in homosexuality. The
most compelling observation has been the report of genetic linkage of male
homosexuality to microsatellite markers on the X chromosome. This observation
warranted further study and conÞrmation. Sharing of alleles at position
Xq28 was studied in 52 gay male sibling pairs from Canadian families. Four
markers at Xq28 were analyzed (DXS1113, BGN, Factor 8, and DXS1108). Allele
and haplotype sharing for these markers was not increased over expectation.
These results do not support an X-linked gene underlying male homosexuality.
Previous studies have suggested that there is
a genetic component in male sexual orientation.
These include controlled family studies
that have shown an increased frequency of
homosexual brothers of homosexual index
subjects as compared to heterosexual index
subjects (1, 2) and twin studies, which have
shown increased concordance for homosexu-
R E P O R T S
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al orientation in monozygotic as compared to
dizygotic twins (3). On the other hand, the
similar rates of male homosexuality in biological
and adoptive siblings of male homosexual
index subjects (3), coupled with methodological
uncertainties in family and twin
studies of homosexuality, suggest caution in
accepting a genetic-epidemiological basis for
homosexuality (4, 5).
The strongest support for a genetic component
in male sexual orientation came from
the studies of Hamer et al. (6, 7), who posited
the involvement of an X-linked gene at position
Xq28, based on family recurrence patterns
and molecular analysis of the X chromosome
in sibships in which there were multiple
brothers with homosexual orientation.
Specifically, Hamer and colleagues obtained
family history information from 76 gay male
index subjects and 40 gay brother pairs about
the sexual orientation of the first-, second-,
and third-degree relatives, with follow-up interviews
of a smaller proportion of relatives.
They reported increased rates of homosexual
orientation in the maternal uncles and male
cousins through maternal aunts, which was
suggestive of X-linked inheritance. Molecular
analysis of the X chromosome revealed an
excess of allele sharing in the region of Xq28
in 40 homosexual brother pairs (6) and, to a
lesser extent, in a follow-up study of 33
additional pairs (7).
However, the evidence for X linkage has
been questioned on theoretical and empirical
grounds (8, 9). Most would agree that male
homosexual orientation is not a simple Mendelian
trait. There would be strong selective
pressures against such a gene. Hamer’s identification
of a contribution from a gene near
Xq28 to homosexuality in some families that
were selected for X-linked transmission of
that trait might be fraught with type 1 (false
positive) error. This is important to consider,
given the irreproducibility of many linkage
reports for complex behavioral traits.
Given the political and social ramifications
of gene linkage in homosexuality, we
launched independent genetic studies of male
sexual orientation in Canada. Specifically, we
advertised in Canadian gay news magazines
(Xtra and Fugue) for families in which there
were at least two gay brothers. One hundred
and eighty-two individuals responded to the
advertisement. The respondents volunteered
information about the sexual orientation of
individuals in their families, including siblings,
parents, uncles, aunts, and first cousins,
although all members of the extended family
were not directly interviewed. The 182 families
included 614 brothers, 269 (44%) of
whom were homosexual. There were 148
families with two gay sons, 34 families with
three, and two families with four. The high
rate of sibling concordance reflects the nature
of the advertisement. The sample included
270 sisters, 49 (18%) of whom were said to
be gay. This rate is high compared to the
frequency of homosexual orientation in women
as ascertained in most population-based
studies, which suggest a sister concordance
rate of 14% (10).
Our molecular analysis was based on 52
gay sibling pairs from 48 families who were
willing to provide blood samples. Sexual orientation
was confirmed for all subjects at the
time of blood sampling by the direct questioning
of a gay interviewer. The index subject
read gay magazines and volunteered that
he was gay, and this observation was corroborated
by interviewing the gay brother. We
believe that the rate of false positives, as in
Hamer’s study, was low (6). The sample
included 46 families with two gay brothers.
There were two families with three gay brothers,
and these were considered as six pairs.
Four markers were analyzed (DXS1113,
BGN, Factor 8, and DXS1108), along a 12.5-
centimorgan (cM) region of Xq28. The methods
were as described (11). The allelic sharing
is shown in Table 1.
Genotyping was performed on DNA samples
from the brothers themselves without
genotyping of parents (alleles were identified
by comparison with population-based controls,
known as “identical by state,” rather
than by confirmation of maternal transmission,
known as “identical by descent”). Maternal
DNA was difficult to obtain. As controls,
we included an additional 33 sibling
pairs who were concordant for multiple sclerosis.
These were genotyped simultaneously
with the gay sibships (Table 2). Allele scoring
was performed independently by two
evaluators who were blind to the status of the
sibship.
A priori, a pair of brothers will share an
X-linked maternal allele, identical by descent
with probability 5 1/2. Therefore, for a
marker with heterozygosity H, under the null
hypothesis of no excess sharing, a brother
pair will share an allele identical by state with
probability 1/2 1 1/2 (1 – H) 5 1 – H/2. For
an X-linked trait-influencing locus in the region,
the sharing will be increased. For the
distal three markers taken as a haplotype, the
probability that brothers share the full haplotype
is approximately (1 – H/2) (1 – u)2,
where u is the recombination fraction in the
entire interval (2.5% for all three markers),
and H is the heterozygosity of the full haplotype
(which is 1 minus the product of the
homozygosities at the three loci, assuming
linkage equilibrium).
Table 1 shows no excess sharing for any
of the four markers tested nor for the haplotype
of the distal three loci. These results are
not consistent with an X-linked gene underlying
sexual orientation in this region of the
X chromosome.
We further analyzed these data with mul-
1Department of Clinical Neurological Sciences, University
of Western Ontario, 339 Windermere Road,
London, Ontario, Canada, N6A 5A5. 2Department of
Genetics, Stanford Medical School, Palo Alto, CA
94305Ð5120, USA.
*To whom correspondence should be addressed. Email
grice@julian.uwo.ca
Table 1. Allele sharing by state in 52 homosexual brother pairs.
Marker
Heterozygosity
Sharing Nonsharing
Chi
square
Observed Expected Observed Expected
DXS1113 0.7 34 33.80 18 18.20 0.00
BGN 0.82 30 30.68 22 21.32 0.04
Factor 8 0.68 35 34.32 17 17.68 0.04
DXS1108 0.74 32 32.76 20 19.24 0.05
Last three loci combined 0.99 24 26.96 28 25.61 0.44
Fig. 1. Multipoint map for Xq28. Multipoint lod
scores were calculated along the 12.5-cM region
for two values of ls (2.0, solid line; 1.5,
dashed line), where ls is the ratio for homosexual
orientation in the brothers of a gay
index subject, as compared to the population
frequency, that is attributable to a gene in this
region. Very strong exclusion is obtained for lod
scores ,Ð2.0, and strong exclusion is obtained
for lod scores ,21.0.
Table 2. Allele sharing in 33 control brother pairs.
Marker
Heterozygosity
Observed
sharing
Expected
sharing
Chi
square
DXS1113 0.7 23 21.45 0.34
BGN 0.82 22 19.47 0.80
Factor 8 0.68 20 21.78 0.43
DXS1108 0.74 23 20.79 0.63
R E P O R T S
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tipoint sib pair analysis by means of the
computer program ASPEX (12). Multipoint
lod (logarithm of the odds ratio for linkage)
scores were calculated along the 12.5-cM
region for two values of ls (2.0 and 1.5),
where ls is the ratio for homosexual orientation
in the brothers of a gay index subject, as
compared to the population frequency, that is
attributable to a gene in this region. Very
strong exclusion is obtained for lod scores
,–2.0, and strong exclusion is obtained for
lod scores ,21.0 (13). As depicted in Fig. 1,
ls values of two or greater can be very
strongly excluded. Values of 1.5 or greater
can also be strongly excluded. The lod scores
were clearly negative for all values of ls .
Hamer and colleagues described linkage
of male homosexuality to polymorphic markers
at Xq28 in 40 brother pairs. The sharing
was 33/40, deemed to be significant with a ls
value of 2.86 (6). In a follow-up study of 33
gay male sibling pairs (32 informative), 22
shared all the Xq28 markers (7). Our sample
comprised 46 sib pairs and 2 sib trios. The
sharing of distal Xq28 markers in the 46 sib
pairs was 20/46. For one of the sib trios, all
three brothers shared the same X chromosome;
for the other trio, two shared the same
X chromosome and the other was different.
Therefore, forming independent sib pairs by
picking two pairs out of three for each sibship
gives a total X-chromosome sharing of either
2 out of 4 or 3 out of 4. For comparison with
the results of Hamer et al., we used the more
favorable 3 out of 4, which gave a total of 23
out of 50 chromosomes shared for our sample.
This result was highly different statistically
from the first study of Hamer (6) (chi
square value 5 11.09, P ,0.001) but not
statistically different from the second study
(7) (chi square 5 3.21, P . 0.05). Combining
the two replication studies gives a total
sharing of 45 out of 82 (55%), which was not
significantly different from 50% (chi
square 5 0.78, P . 0.30). Also, the sharing
for the two replication studies combined is
significantly different from the original study
of Hamer et al. (chi square 5 7.74, P ,
0.01).
It is unclear from the original study to
what extent families were excluded to produce
the data set in which the positive linkage
analysis was reported. Families were excluded
if a father was gay or if there were any
first-degree lesbian relatives. By these precise
criteria, two sib pairs would have been
excluded from our study (one with a gay
father and one with two gay parents). For the
remaining pairs, the linkage evidence was the
same as for the entire group.
It is unclear why our results are so discrepant
from Hamer’s original study (6). Because
our study was larger than that of Hamer
et al., we certainly had adequate power to
detect a genetic effect as large as was reported
in that study. Nonetheless, our data do not
support the presence of a gene of large effect
influencing sexual orientation at position
Xq28.
Although we found no evidence of linkage
of sexual orientation to Xq28, these results
do not preclude the possibility of detectable
gene effects elsewhere in the genome.
References and Notes
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Epidemiol. 13, 117 (1996).
14. We are indebted to the gay men and women who
shared their family histories and their DNA to allow
the beginning of this work. B. Bass and M. Penman
helped launch the pedigree study. H. Armstrong, H.
Margalies, and K. Cousin provided expert technical
collaboration. The work was funded from our own
pockets.
9 November 1998; accepted 18 March 1999
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