Roberts syndrome is caused by mutations in ESCO2, a human homolog of yeast ECO1 that is essential for the establishment of sister chromatid cohesion.

Abstract

Roberts syndrome is an autosomal recessive disorder characterized by craniofacial anomalies, tetraphocomelia and loss of cohesion at heterochromatic regions of centromeres and the Y chromosome. We identified mutations in a new human gene, ESCO2, associated with Roberts syndrome in 15 kindreds. The ESCO2 protein product is a member of a conserved protein family that is required for the establishment of sister chromatid cohesion during S phase and has putative acetyltransferase activity.

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Roberts syndrome is caused by
mutations in ESCO2, a human
homolog of yeast ECO1 that is
essential for the establishment
of sister chromatid cohesion
Hugo Vega1,2, Quinten Waisfisz3, Miriam Gordillo2,4,
Norio Sakai2, Itaru Yanagihara5, Minoru Yamada5,
Djoke van Gosliga3, Hu ¨lya Kayserili6, Chengzhe Xu2,
Keiichi Ozono2, Ethylin Wang Jabs4, Koji Inui2& Hans Joenje3
Roberts syndrome is an autosomal recessive disorder
characterized by craniofacial anomalies, tetraphocomelia and
loss of cohesion at heterochromatic regions of centromeres and
the Y chromosome. We identified mutations in a new human
gene, ESCO2, associated with Roberts syndrome in 15
kindreds. The ESCO2 protein product is a member of a
conserved protein family that is required for the establishment
of sister chromatid cohesion during S phase and has putative
acetyltransferase activity.
Roberts syndrome (RBS; OMIM 268300) is characterized by pre- and
postnatal growth retardation, microcephaly, bilateral cleft lip and
palate, and mesomelic symmetric limb reduction1(Fig. 1a). RBS
chromosomes have a lack of cohesion involving the heterochromatic
C-banding regions around centromeres and the distal portion of the
long arm of the Y chromosome (known as premature centromere
separation, heterochromatin repulsion or puffing, or RS effect;
Fig. 1b)1,2. We identified seven families affected with RBS in two
isolated villages near Bogota ´, Colombia. After discovering a common
ancestor in the 18th century by genealogical studies of four families,
we began a genome-wide search for the disease-associated locus by
homozygosity mapping (Supplementary Methods online). Multi-
point linkage analysis using MAPMAKER-HOMOZ detected three
genomic regions (1q, 4q and 8p) with lod scores 45. GENEHUNTER
analysis across the three regions supported linkage only for 8p12–21.2
between markers D8S258 and D8S505. With additional families and
fine mapping, we confirmed a region of homozygosity in all 11
affected individuals studied (Fig. 1c). Multipoint analysis gave a
maximum lod score of 13.4 at D8S1839 (Fig. 1d).
Using a candidate gene approach, we screened the new transcript
LOC157570 containing D8S1839 and found eight different mutations
in 18 individuals with RBS from 15 families of different ethnic
backgrounds (Table 1 and Supplementary Fig. 1 and Supplementary
Methods online). We identified one missense mutation, resulting in
the amino acid substitution W539G at a highly conserved residue;
one nonsense mutation; and six frameshift mutations. In each family,
the mutation cosegregated consistently with disease status and was not
present in 100 control chromosomes. We characterized the gene,
ESCO2 (establishment of cohesion 1 homolog 2), using 5¢ and
3¢ RACE and RT-PCR. We obtained a cDNA of 3,348 nucleotides
with an open reading frame of 1,806 nucleotides. The coding sequence
is divided into 11 exons that span 30.3 kb, with the start codon in exon
2 and the stop codon in exon 11 (Supplementary Fig. 2 online).
Northern-blot analysis identified a predominant mRNA of B3.3 kb
in all human fetal tissues tested and in adult thymus, placenta
and small intestine (Supplementary Fig. 2 online). The predicted
polypeptide has 601 amino acids (pI ¼ 9.46, molecular weight ¼
68.3 kDa; Supplementary Fig. 3 online). ESCO2 is 492%, 77%, 57%
and 41% similar to the primate, rodent, chicken and fish orthologs,
respectively, indicating that it is evolutionarily conserved among
vertebrates (Supplementary Fig. 3 online). The ESCO2 C-terminal
portion was previously reported3,4as the first human sequence
identified with a substantial similarity to Saccharomyces cerevisiae
Eco1p (also called Ctf7p), an essential protein for establishment of
sister chromatid cohesion4,5. Members of the Eco1p family of
putative acetyltransferases consist of one highly conserved domain,
the Eco1p C-terminal domain, alone or fused to a variable
N-terminal extension4–8. The conserved C terminus includes the
Eco1p C2H2 Zn finger-like and acetyltransferase domains. The latter
domain is where the W539G mutation was found (Supplementary
Figs. 3 and 4 online). In Schizosaccharomyces pombe Eso1p, the C-
terminal domain is fused to an N-terminal extension with a DNA
polymerase Rad30p domain6. In humans, Efo1p has a linker histone
domain in the N terminus8. The N-terminal portion of ESCO2
does not have substantial similarity to those of Efo1p, Eso1p, Deco
(the ortholog in Drosophila melanogaster) or any other protein
in the database.
After DNA replication, sister chromatids are held together by
cohesin, a complex composed of Smc1, Smc3, Scc1, Scc3 and Pds5
(ref. 9). Deposition of cohesin onto chromatin depends on Scc2 and
Scc4 (ref. 9), but cohesin loading is not sufficient for sister-chromatid
cohesion. Eco1, Eso1 and Deco are required for the establishment of
Published online 10 April 2005; doi:10.1038/ng1548
1Instituto de Gene ´tica, Universidad Nacional de Colombia, Ciudad Universitaria, Bogota ´, Colombia.2Department of Developmental Medicine (Pediatrics) D-5,
Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.3Department of Clinical Genetics, VU University Medical Center,
Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.4McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, 733 North Broadway,
Baltimore, Maryland, 21205, USA.5Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, 840
Murodo-cho, Izumi, Osaka 594-1101, Japan.6Medical Genetics Department, Istanbul Medical Faculty, Istanbul University, Millet Cad., Capa 34390, Istanbul, Turkey.
Correspondence should be addressed to H.V. (hhvegaf@unal.edu.co) or E.W.J. (ejabs1@jhem.jhmi.edu).
468VOLUME 37 [ NUMBER 5 [ MAY 2005 NATURE GENETICS
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physical bridges between sisters during S phase, and mutations in the
genes encoding these proteins disrupt cohesion. In addition, these
mutants mis-segregate chromosomes, are sensitive to DNA damaging
agents and show mitotic arrest or delay4–7. The RBS cellular pheno-
type, which includes premature centromere separation, lagging chro-
mosomes, aneuploidy, micronuclei, decreased cell proliferation and
hypersensitivity to DNA-damaging agents1,2,10–12, closely resembles
that phenotype. The mitotic arrest or delay in Eco1, Eso1 and Deco
mutants requires a functional spindle checkpoint4–7. To investigate this
aspect in RBS, we examined the status of the mitotic checkpoint in
nocodazole-treated RBS cells. Normally, after spindle checkpoint
activation by agents such as nocodazole, Mad2 is recruited to kineto-
chores, securin is stabilized and sister-chromatid separation and
mitotic exit are prevented9. We found no difference in securin stabili-
zation between RBS cells and control cells (Supplementary Fig. 5 and
Supplementary Methods online). In addition, Mad2 was localized at
the kinetochore in chromosomes with or without premature centro-
mere separation and the DNA was not reduplicated (Supplementary
Fig. 5 online). These results show that the mitotic checkpoint activa-
tion is normal in RBS cells as in the yeast and fly mutants.
In yeast Eco1 mutants, cohesion is affected in both centromeres
and chromosome arms4, whereas Deco mutations in the fly
disrupt only cohesion at the centromeres7. ESCO2 mutations disrupt
cohesion specifically in heterochromatic regions but have little or
no effect at other chromosomal regions2(Fig. 1b). In humans,
the difference in cohesion release from arms versus centromeres
observed in mitosis may be the result of the differential regulation
of cohesin complexes13. Possibly, such differential regulation is deter-
mined when cohesion is established, as different members of the
family may be involved in the establishment of cohesion in different
chromatin domains.
RBS is the first human disorder identified to our knowledge in
which a chromatid-cohesion defect is associated with developmental
abnormalities. Defects in chromatid cohesion lead to mitotic spindle
checkpoint activation and impaired cell growth14. The RBS cohesion
defect could activate the mitotic spindle checkpoint, resulting in the
mitotic delay and the impaired cell proliferation observed in RBS
cells10,11. During embryogenesis, the loss of progenitor cells could
preclude a sufficient number of cells required for development of
structures affected in RBS.
D8S505
D8S1820
D8S1839
D8S1771
D8S258
D8S505
D8S1820
D8S1839
D8S1771
D8S258
D8S505
D8S1820
D8S1839
D8S1771
D8S258
D8S505
D8S1820
D8S1839
D8S1771
D8S258
D8S505
D8S1820
D8S1839
D8S1771
D8S258
D8S505
D8S1820
D8S1839
D8S1771
D8S258
Family 1
Family 2
FR5
1
1
1
6
1
HR12
3
2
HR11
1 3
2 3
3 1
6 7
4 4
FR4
1 2
FR2
1
4
5
FR3
3
3
3
6
4
3
3
2
2
2
22
4
7
3
3
4
– –
4
43
3 3
5
3 3
6
4 4
3
3
1
3
6
2
1
1
1
1
1
6
1
3
2
3
6
4
3
2
3
6
4
1
3
4
3
5
HR7HR8 HR9
1
2
3
6
4
HR10
1 1
3
3
3 6
4
HR1
1
4
5
4
2
HR2
3 3
1 3
3 3
6 7
2 6
3
1
1
3
1
2
3
4
3
3
3
6
4
S R5R6
– –
HR13
3 2
1 2
6
3 3
7 4
HR14
2
1 4
6 3
3
4 1
HR6
– –
1 2
3
7 7
HR4
1 3
2 1
3 3
6 7
1 4
HR3
3
R10
3 1
2 2
3 3
6 6
4 1
R12
2 2
D
– – – –
4 1 3 1 3 1
5 3 3 3 3 3
4 6 6 6 6 6
– – – – – –
R27 R28
1 1 1 1
2 2 2 2
3 3 3 3
6 6 6 6
4 4 4 4
R19
1 3
2
3 3
6 6
4 5
2
Family 3Family 4Family 5
2
5
3
– –
6
44
6 3
3 3
2 1
1
1 1
6
3
3
5
6
6
R31
2 3
1 1
6 6
3 3
4 7
Family 8 Family 7Family 6
R37
– –
2 2
3 3
6 6
R42
– –
2 1
6 6
3
R22
6 7
1 1
3
7 7
1
– –
– –
3
3
7
D8S260 D8S285 D8S505D8S1839 D8S1771 D8S258D8S1820
–6
–5
–4
–3
–2
–1
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
lod
ac
d
b
Figure 1 Clinical features, cytogenetics genotypes and multipoint linkage analysis of RBS. (a) Individual from family 7 with bilateral cleft lip and palate and
tetraphocomelia. (b) C-banding of metaphase chromosomes. Arrows show selected chromosomes with premature centromere separation. Arrowhead point to
‘splitting’ of the Y chromosome heterochromatic region. Open arrows show selected chromosomes with normal C-banded regions. (c) Families 1, 3–5 and 8
were used for the genome-wide linkage analysis. Heterozygosity in individuals R5, R6, R42 and R12 defined the critical region of 3.9 cM flanking
D8S1839. (d) lod score from MAPMAKER/HOMOZ. Marker order is telomere-D8S258-D8S1771-D8S1839-D8S1820-D8S505-centromere.
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This study was approved by the Institutional Review Boards of The
Johns Hopkins University and VU University Medical Center
(Amsterdam) and the Ad Hoc Ethics Committee-DIB of the
Universidad Nacional de Colombia. Informed consent was obtained
from all subjects involved in the study. Specific consent was obtained
from the parents to publish a photograph of their child.
URLs. The National Center for Biotechnology Information is available at
http://www.ncbi.nlm.nih.gov/. ClustalW is available at http://www.ebi.ac.uk/
clustalw/. JalView is available at http://www.jalview.org/. pSORT II is available
at http://psort.ims.u-tokyo.ac.jp/. Online Mendelian Inheritance in Man is
available at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db¼OMIM/.
GenBank accession number. ESCO2 cDNA, AY882862.
Note: Supplementary information is available on the Nature Genetics website.
ACKNOWLEDGMENTS
We thank the families with Roberts syndrome for their interest and cooperation;
D. Tomkins for R22 sample; Biobank at the Istituto Giannina Gaslini for R34
sample; and M. Camargo for technical support. H.V. was supported by
scholarships from the Japanese Ministry of Education, Culture, Sports,
Science and Technology and the Instituto Colombiano para el Desarrollo de la
Ciencia y la Tecnologia. M.G. is supported by the Smile Train Fellowship award
to the Center for Craniofacial Development and Disorders at Johns Hopkins
University. M.G. and E.W.J. were supported by the Louis H. Gross Foundation,
J.S. Sutland and L. and S. Pakula. Q.W. was supported by the Netherlands
Organization for Health Research and Development.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 11 January; accepted 24 February 2005
Published online at http://www.nature.com/naturegenetics/
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Table 1 ESCO2 mutations in individuals with RBS
Family OriginExon MutationType
Amino acid
changea
1–3, 6Colombia3 750_751insG
Homozygous
505C-T
Homozygous
1615T-G
NAd
252_253delAT
Homozygous
1457_1458delAG
Homozygous
1104_1105insA
Homozygous
877_888delAG
Homozygous
411_412insA
NAd
FrameshiftE251fsX30
4, 5, 7Colombia3 NonsenseR169X
8b
Canada10MissenseW539G
9c
Italy3Frameshift V84fsX7
10Turkey9FrameshiftK486fsX20
11Turkey6FrameshiftK369fsX34
12–14 Turkey4Frameshift R293fsX7
15Turkey3 FrameshiftK138fsX10
aThe affected amino acid residue is listed first, followed by ‘fs’ (to indicate a frameshift), ‘X’ (to
indicate a stop) and then a number indicating how many more residues occur before the stop.
bFrom ref. 15.cSamples from the Biobank at Istituto Giannina Gaslini, Genova, Italy.dParental
DNAs not available (NA).
Colombian families 1–3 and 6 have a common ancestor. Turkish families 11 and 13–15
are consanguineous.
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