REPORT
UBE2A, Which Encodes a Ubiquitin-Conjugating Enzyme, Is
Mutated in a Novel X-Linked Mental Retardation Syndrome
Rafaella M. P. Nascimento, Paulo A. Otto, Arjan P. M. de Brouwer, and Angela M. Vianna-Morgante
We report a mutation of UBE2A/HR6A, which encodes a ubiquitin-conjugating enzyme (E2), a member of the ubiquitin
proteasome pathway, as the cause of a novel X-linked mental retardation (XLMR) syndrome that affects three males in
a two-generation family. A single-nucleotide substitution, c.382CrT in UBE2A, led to a premature UAG stop codon
(Q128X). As a consequence, the predicted polypeptide lacks the 25 C-terminal amino acid residues. The importance of
this terminal sequence for UBE2 function is inferred by its conservation in vertebrates and in Drosophila. UBE2A mutations
do not appear to significantly contribute to XLMR, since no UBE2A mutations were identified in 15 families with
nonsyndromic and 4 families with syndromic idiopathic XLMR previously mapped to intervals encompassing this gene.
This is the first description of a mutation in a ubiquitin-conjugating enzyme gene as the cause of a human disease.
Monogenic X-linked mental retardation (XLMR) has been
estimated to affect ∼10% of mentally retarded males.1,2
Mutations in 59 genes on the X chromosome have been
implicated in familial mental retardation (Greenwood Genetic Center), and they represent about one-third of the
X-linked genes demonstrated to be mutated in human
monogenic diseases.3 With the identification of genes involved in XLMR, a picture emerges indicating that some
genes are mutated in both syndromic and nonsyndromic
mental retardation.4 However, mutations in such genes account for only a small proportion of XLMR-affected families and males with sporadic mental retardation.1 The
FMR1 gene, mutated in the fragile X syndrome, is the most
noticeable exception, with a prevalence of 2%–2.5% in
cohorts of mentally retarded males5 and affecting roughly
one-quarter of XLMR-affected families.6 Therefore, many
genes involved in XLMR still await identification.
Here, we report a nonsense mutation in UBE2A, which
encodes a ubiquitin-conjugating enzyme (E2) in the proteasome pathway of protein degradation, as the cause of
a novel XLMR syndrome. Ubiquitination of proteins and
their degradation constitute a major mechanism in the
regulation of protein levels in mammalian cells. In addition, ubiquitination is recognized to have pleiotropic functions in the regulation of various cellular processes, such
as control of transcription factor activity,7 receptor internalization,8 and histone modifications, which modulate
chromatin structure.9
The described family includes three mentally retarded
males in two generations, related through their clinically
normal mothers (fig. 1 and table 1). Informed consent was
obtained from every participating individual or from his
or her guardian(s), and the study was approved by The
Ethics Committee on Research on Human Subjects of the
Institute of Biosciences, University of São Paulo, São Paulo.
Physical examination was performed on the three affected
males. The family provided information about the patients (i.e., pregnancy and condition at birth, developmental milestones, intellectual and adaptive functioning)
and made medical records available. The patients’ mothers
were clinically unaffected and did not show any overt
intellectual or adaptive impairment; I-2 is a housewife, II2 is the head of a school for mentally impaired children,
and II-4 is a nutritionist. At age 46 years and 9 mo, II-3
developed acute myeloid leukemia. Chromosome studies
of cultured blood lymphocytes—prometaphase G-banding of individuals II-3, III-2, and III-3 and in situ hybridization of subtelomeric probes (Chromoprobe MultiprobeT System [Cytocell]) of individual II-3—did not reveal any
alterations. The result of molecular testing of patient III2 for fragile X syndrome was negative.
On the basis of the family pedigree, we assumed an Xlinked pattern of inheritance for this previously undescribed mental retardation syndrome (fig. 1). This assumption was further strengthened by our finding that the
presumptive obligate carriers had completely skewed X
inactivation in leukocytes, as demonstrated by the methylation status of the CAG repeat of the androgen receptor
gene10 (data not shown). Indeed, skewed X-chromosome
inactivation appears to be characteristic of carriers of many
gene mutations involved in XLMR.11 These observations
prompted us to search for the X-linked gene involved in
the syndrome. Given the small size of the family, an exclusion-mapping strategy was performed. Using DNA extracted from peripheral blood leukocytes, we genotyped
46 microsatellite loci throughout the X chromosome (fig.
2), to locate regions of common descent in the three patients. In the initial mapping, 18 microsatellite loci ∼10
cM apart (ABI PRISM Linkage Mapping Set MD-10 [Applied Biosystems]) were amplified by PCR with fluorescent-
From the Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo (R.M.P.N.; P.A.O. A.M.V.-M.);
and Department of Human Genetics, Radboud University Medical Centre, Nijmegen, The Netherlands (A.P.M.d.B.)
Received May 15, 2006; accepted for publication June 12, 2006; electronically published July 3, 2006.
Address for correspondence and reprints: Dr. Angela M. Vianna-Morgante, Departamento de Genética e Biologia Evolutiva, Instituto de Biociências,
Universidade de São Paulo, Caixa Postal 11461, 05422-970 São Paulo, SP, Brazil. E-mail: avmorgan@ib.usp.br
Am. J. Hum. Genet. 2006;79:549–555. 䉷 2006 by The American Society of Human Genetics. All rights reserved. 0002-9297/2006/7903-0018$15.00
www.ajhg.org
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Volume 79 September 2006
549
Figure 1. A, The three affected males, II-3 (aged 46 years and 7 mo), III-2 (aged 19 years and 11 mo), and III-3 (aged 5 years and
4 mo). B, Two-generation genealogy of the study family, showing affected males related through their clinically normal mothers. C,
Chromatograms of the sense-strand sequence from I-2 (obligate carrier) and II-3 (affected male). A c.382CrT mutation in UBE2A exon
6 was identified as the cause of the syndrome.
labeled primers, and the amplified fragments were analyzed on a MegaBACE 1000 automated sequencer with the
MegaBACE Genetic Profiler software (Amersham Bioscience–GE Healthcare). The other 28 markers were selected
from the National Center for Biotechnology Information
(NCBI) database, with regard to their location on the X
chromosome and level of heterozygosity; PCR was performed according to standard conditions, and, after electrophoresis on 6% denaturing polyacrylamide gels, the
amplified products were visualized by silver staining. Genotyping of 36 markers spaced at ∼5 cM disclosed three
loci, at Xq23-25, harboring alleles shared by all three affected males, but their inheritance from a common ancestor could not be determined. A further 10 loci 1 Mb
apart within the defined Xq23-q25 region were analyzed.
An ∼15-Mb segment could be delimited by the excluded
550
markers DXS8088 (Xq23) and DXS1047 (Xq26.1); all alleles within this defined region were shared by the affected
males, and alleles at DXS8053, DXS8081, and DXS8057
were proven to be identical by descent (fig. 2). This candidate segment contained 86 known genes (NCBI); three
of them had been previously associated with mental re-
Figure 2. Pedigree of the affected males and genotypes of the
46 analyzed markers, from Xpter to Xqter. The legend is available
in its entirety in the online edition of The American Journal of
Human Genetics.
The American Journal of Human Genetics
Volume 79 September 2006
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Table 1.
Clinical Findings for the Mentally Retarded Patients
Patient
Characteristic
Age at examination
Birth weight (percentile)
Height (percentile)
Weight (percentile)
Head circumference (percentile)
Hair whorls
Wide face
Midface hypoplasia
Synophris
Up-slanted palpebral fissures
Ocular hypertelorism
Low nasal bridge
Large mouth with down-turned corners and thin lips
Short, broad neck
Low posterior hairline
Widely spaced nipples
Small penis
Small, flat feet, with dorsum swelling
Onychodystrophya
Marked generalized hirsutism
Myxedematous appearance
Dry skin
Seizures
Severe speech impairment
White matter hypodensityc
a
b
c
II-3
III-2
III-3
46 years and 7 mo
50th
!3rd
197th
198th
⫹
⫺
⫺
⫹
⫹
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
Not examined
19 years and 11 mo
90th–97th
10th–25th
90th
198th
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹b
⫹
5 years and 4 mo
1 97th
10th
197th
50th
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹b
⫹
After puberty.
Absent speech.
Determined by magnetic resonance imaging.
tardation: LAMP2 (lysosomal associated membrane protein 2), mutated in patients with multisystem glycogen
storage disease—Danon disease (MIM 300257), an X-linked
dominant disorder affecting predominantly cardiac and
skeletal muscles—and also found to be mutated in primary
cardiomyopathy12,13; GRIA3 (glutamate receptor, ionotrophic, AMP 3), interrupted in a female carrier of a balanced
chromosomal translocation t(X;12)(q24;q15) who presented with bipolar disorder and mental retardation14; and
AGTR2 (angiotensin II receptor, type 2), disrupted in a
balanced translocation t(X;7)(q24;q22) in a female with
moderate mental retardation15 and also mutated in males
with variable mental retardation.16 On the basis of the
clinical features associated with these mutations and those
present in our patients, we considered AGTR2 the best
candidate gene. Mutation screening was performed by direct sequencing on a MegaBACE 1000. The AGTR2-only
coding exon was amplified with primers described elsewhere,16 and no mutations were found. We then performed a candidate-gene search (Genatlas) and found 30
genes within the candidate interval that were expressed
in brain. However, only one of those genes, UBE2A/HR6A,
was expressed in both brain and lymphocytes. Because of
the skewed X-inactivation in the patients’ mothers, which
likely represents the survival/proliferation advantage of
lymphocytes with the active normal allele in a woman
with mutation, we sequenced this gene directly. The six
coding exons of UBE2A were amplified, with flanking
www.ajhg.org
intronic primer pairs (table 2) that we designed using
Primer3 software,17 and were sequenced. A c.382CrT substitution leading to a premature UAG stop codon (Q128X)
was detected in all three affected males and in their mothers (fig. 1). The sister of one of the affected males (III-1,
with a rather random X-inactivation pattern, as documented by the methylation status of the AR gene [data
not shown]) did not carry this mutation.
We then screened for UBE2A mutations in 19 affected
The American Journal of Human Genetics
Table 2. Primer Pairs Designed
to Amplify the Coding
Sequence of the UBE2A Gene,
with Use
of Primer3 Software
Exon
UBE2A-1F
UBE2A-1R
UBE2A-2F
UBE2A-2R
UBE2A-3F
UBE2A-3R
UBE2A-4F
UBE2A-4R
UBE2A-5F
UBE2A-5R
UBE2A- 6F
UBE2A-6R
Primer Sequence
(5r3)
cgtggggctttaatgacata
aaccttcgggaagacagaca
catgcgggacttcaagaggt
ccaaacattttcccctaccc
ccgggacatccatttgtagt
cagaggcaggttcctaagca
cctctctaccctgtatctttgcat
ggcaccacaaaatacacagga
tgggaagcaacataggaatctt
aggtgtgagcgactgtaccc
tgttttgcattaaggaactgaca
gggaggtgacaaacacatca
Volume 79 September 2006
551
males from XLMR-affected families that were collected by
the Euro-MRX Consortium, previously mapped to intervals encompassing this gene. Four families had syndromic
and 15 families had nonsyndromic idiopathic XLMR. The
phenotypes, linkage intervals, and maximum LOD scores
of the Euro-MRX families are summarized in table 3. No
mutations were detected in these patients.
UBE2A/HR6A is a ubiquitination pathway gene that encodes E2. The E2 conjugases, in conjunction with ligating
enzymes (E3), mediate the attachment of ubiquitin molecules to proteins, thus targeting them for degradation by
the proteasome complex. UBE2A is one of the two human
orthologues of the Sacharomyces cerevisiae RAD6/UBC2
gene. In humans and other mammals, the gene is duplicated with one X-linked (UBE2A) copy and one autosomal
(UBE2B) copy.23 The coding regions of human UBE2A and
UBE2B paralogues share 80% identity and produce proteins with 96% amino acid identity and a seven-residue
difference in their 152 amino acids. The UBE2A mutation
in our patients introduces a premature stop codon and
abolishes the 25 C-terminal amino acids of the protein.
The great importance of this sequence for UBE2 function
can be inferred from its high conservation in both vertebrates and Drosophila (fig. 3).
The high conservation of UBE2A and UBE2B amino acid
sequences raises the question of function specificity or
redundancy of these proteins, which are ubiquitously expressed, although the ratios between these proteins vary
significantly in different cells and tissues.24 The fact that
double Ube2a/Ube2b (hr6a/hr6b) knockout mice are not
viable indicates that these genes are crucial for development, and viability was demonstrated to depend on the
presence of at least one functional allele, by the construcTable 3.
Families from the Euro-MRX Consortium Screened for Mutations in UBE2A
Family
Phenotypea (MRX Number)
D004
L022
L025
L037
L048
N005
N043
N108
P004
T011
T013
T014
T025
T048
T052
L056
MRX
MRX (MRX35)
MRX
MRX (MRX70)
MRX
MRX
MRX
MRX
MRX
MRX (MRX61)
MRX (MRX62)
MRX
MRX
MRX
MRX
MRXS (spastic paraplegia, macrocephaly, hypotonia, and
developmental delay)
MRXS (hypotonia, ataxia, and areflexia)
MRXS (microcephaly, epilepsy, and developmental delay)
MRXS (short stature, microcephaly, and facial dysmorphy)
N032
P014
T019
tion of different knockout mice.25 However, the Ube2aand Ube2b-knockout mice differ at least in reproductive
performance. Male-limited sterility is exhibited by Ube2bknockout26 but not Ube2a-knockout25 mice. In contrast,
whereas Ube2b-knockout females are fertile,26 Ube2a-knockout females fail to produce offspring in spite of normal
ovulation; the absence of the UBE2A/HR6A protein in oocytes prevents embryonic development beyond the twocell stage.25 Since UBE2B protein levels, compared with
those of UBE2A,24 are high in spermatids of wild animals
and the opposite is observed in oocytes,25 the infertility
phenotypes might result from a dose-dependent effect in
the germ cells. However, other observations point to different functional properties of UBE2A and UBE2B. The
polyubiquitination of the cyclophilin CYC4/hCyP-60 requires UBE2B (but not UBE2A),27 and UBE2A (not UBE2B)
was found to interact with Rfp14 (ret finger protein-like
4) in a yeast two-hybrid screen.28
The affected males present a neurodevelopmental disorder. A number of studies have addressed the function
of ubiquitination during neuronal development. Nerve
growth factor (NGF)–induced neurite outgrowth from rat
pheochromocytoma cells (PC12) is concurrent with increased levels of ubiquitin-protein conjugates and coincides with up-regulated activities of ubiquitin-conjugating
enzymes but not with enhanced ubiquitin-dependent proteolysis; neurite outgrowth is accelerated by blocking ubiquitin-dependent proteolysis, and such outgrowth is inhibited by a dipeptide inhibitor of E3-dependent ubiquitination. These data imply that ubiquitination and ubiquitin-dependent proteolysis are positive and negative
regulators of neurite outgrowth, respectively.29 Down-regulation of UBE2B mRNA in PC12 cells leads to a reduction
Maximum LOD
Score
Flanking Markers
Reference
1.20
2.41
1.50
2.10
1.30
1.03
1.14
1.51
.68
3.51
2.23
1.20
1.00
.60
2.20
2.18
DXS993 and DXS8043
DXS178 and HPRT
DXS424 and Xqter
DXS8063 and DXS1047
DXS991 and DXS1047
DXS424 and DXS292
DXS8076 and DXS1108
DXS1169 and DXS8067
DXS1217 and DXS1062
DXS135 and DXS737
DXS458 and DXS737
MAOB and DXS425
DXS1214 and DXS1212
DXS993 and DXS737
DXS990 and DXS8057
DXS8054 and DXS1001
…
Gu et al.18
Claes et al.19
Claes et al.20
…
…
…
…
…
…
Raynaud et al.21
Raynaud et al.21
…
…
…
…
6.97
.60
2.96
DXS1231 and DXS1001
DXS986 and DXS1047
DXS178 and DXS292
…
…
Raynaud et al.22
NOTE.—All families have males with mental retardation in at least two generations. Obligate carrier females are not affected.
a
MRX and MRXS p nonsyndromic and syndromic XLMR, respectively.
552
The American Journal of Human Genetics
Volume 79 September 2006
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Figure 3. Alignment of UBE2 protein sequences, showing the high amino acid sequence identity between the human protein and those of mouse (100%), zebrafish (96%–100%),
and Drosophila (85%–87%). The consensus ubiquitin-binding cysteine- and serine-phosphorylation sites are underlined. In the box, the highly conserved segment of the protein
corresponds to the sequence abolished by the c.382CrT mutation (Q128X), which creates a premature stop codon. The identical amino acid sequence of UBE2A in the vertebrates
is shown in bold. (Information is based on the NCBI database and GenBank.)
of NGF-induced neurite length, and pharmacological inhibition of ubiquitin-dependent protein degradation was
shown to significantly reduce axonal length and branching of adult sensory neurons in vitro.30 In Drosophila, synaptic development and function have been shown to be
regulated by ubiquitin-dependent mechanisms.31 Taken
together, these data point to an important role of the ubiquitin proteasome pathway in neuronal differentiation.
A few other human disorders have been recognized to
result from mutations in genes involved in ubiquitination
and proteasome function.32 Mutations in ligase genes from
the ubiquitin enzymatic pathway were identified as causative for Angelman syndrome (MIM 105830) (UBE3A), recessive juvenile Parkinson disease (MIM 600116) (PARK2),
autoimmune polyendocrinopathy syndrome type 1 (MIM
240300) (AIRE), and von Hippel-Lindau disease (MIM
193300) (VHL). To our knowledge, the UBE2A mutation
described here is the first in a ubiquitin-conjugating
enzyme gene to be associated with a human disease. As
in the case of UBE3A mutations causing Angelman syndrome,33,34 mutation of UBE2A leads to neurodevelopmental anomalies.
UBE2A mutations may be exclusive to the novel mental
retardation syndrome described here or may also cause
different clinical pictures, including nonsyndromic mental retardation, as reported for other genes involved in
XLMR.4 However, the failure to detect UBE2A mutations
in 19 idiopathic XLMR-affected families mapped to intervals encompassing UBE2A suggests that mutations in this
gene are not a common cause of XLMR, in keeping with
most XLMR genes identified to date.
Acknowledgments
We are indebted to the Euro-MRX Consortium for providing families for mutation screening. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo.
Web Resources
The accession numbers and URLs for data presented herein are
as follows:
Euro-MRX Consortium, http://www.euromrx.com/
Genatlas, http://www.genatlas.org/
GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for UBE2A
[accession number NP_003327], UBE2B [accession number
NP_003328], Ube2a [accession numbers NP_062642 and
NP_033484], ube2a [accession number NP_958430], ube2b [accession number NP_956013], and UbcD6 [accession number
NP_524130])
Greenwood Genetic Center, http://www.ggc.org/xlmr.htm (for
the X-linked Mental Retardation Database [August 2005])
NCBI, http://www.ncbi.nlm.nih.gov/
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi
.nlm.nih.gov/Omim (for Danon disease, Angelman syndrome,
recessive juvenile Parkinson disease, autoimmune polyendocrinopathy syndrome type 1, and von Hippel-Lindau disease)
Primer3, http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www
.cgi
554
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