VOLUME 43 | NUMBER 1 | JANUARY 2011 Nature GeNetics
Treacher Collins syndrome (MIM154500) is an autosomal-dominant
disorder of craniofacial development that occurs with an estimated
incidence of 1 in every 50,000 live births. TCS is characterized by a
combination of bilateral downward slanting of the palpebral fissures,
colobomas of the lower eyelids with a paucity of eyelashes medial to
the defect, hypoplasia of the facial bones, cleft palate, malformation of
the external ears, atresia of the external auditory canals, and bilateral
conductive hearing loss. A high degree of inter- and intra-familial
variation in the clinical phenotype is typically observed. The majority
of individuals with TCS are heterozygous for a mutation in TCOF1
(ref. 1). Although mutation detection rates as high as 93% have been
reported, a relevant subset of affected individuals in which the causa-
tive mutation has not been identified remains2–4.
To investigate the genetic basis of TCS in a 3-year-old boy who
was negative for a TCOF1 mutation, we performed a genome-wide
copy number analysis using an Affymetrix GeneChip 262K NspI SNP
array. We identified a 156-kb de novo deletion within chromosomal
region 13q12.2, extending from SNP A-4282134 (rs534150) to SNP
A-2305152 (rs1231044), that resulted in the deletion of the entire
POLR1D gene and exon 1 of LNX2 (Fig. 1 and Supplementary Fig. 1).
Subsequently, we sequenced POLR1D and LNX2 (Supplementary
Fig. 2) in this individual, his parents and in ten additional typi-
cal individuals with TCS who were negative for TCOF1 mutations
(Supplementary Methods). No mutation in the second POLR1D
allele of the propositus was identified; however, in one of the other
individuals, EsM42160, a heterozygous nonsense mutation was
detected that resulted in a premature stop codon at amino acid 87 in
exon 3 of POLR1D (p.Arg87X) (Fig. 1 and Supplementary Fig. 1).
To identify additional mutations in POLR1D, we sequenced a further
242 individuals with typical TCS or with clinical findings known
to be part of the TCS phenotypic spectrum who were negative for
TCOF1 mutations. Ten heterozygous nonsense mutations and seven
heterozygous missense mutations were detected in 20 index cases
(Table 1 and Supplementary Table 1). All seven missense mutations
are located in exon 3 of POLR1D and affect evolutionary conserved
amino acids in the RNA polymerase dimerization domain of POLR1D
(Supplementary Fig. 3), most likely disturbing dimerization of the
α subunits5. The p.Arg87X alteration, resulting from a CpG transi-
tion, was detected in three families and the p.Glu47Lys alteration was
detected in two unrelated families. These were the only recurrent
alterations we identified.
Analysis of the POLR1D dimerization domain in 280 unaffected
individuals did not reveal any sequence variants. In five affected
families (designated EsM6227, EsM3422, EsM6195, Man15 and
Man24), the identified mutation co-segregated with the TCS pheno-
type (Supplementary Fig. 4a). Notably, in families EsM5850,
EsM42160, Man33 and Man40, we discovered cases of nonpenetrance
(Supplementary Fig. 4b), which has been reported previously in TCS
but is not common with TCOF1 mutations6 (unpublished observa-
tions). As the mutations identified in nonpenetrant carriers were not
detected in unaffected controls, it is unlikely that these are nonpatho-
genic variants. We cannot rule out that, in addition to a POLR1D
mutation, the presence of a mutation at an unlinked locus is required
for expression of the TCS phenotype. However, segregation patterns
in the families Man33 and Man40 argue against biallelic inheritance.
Finally, a lack of penetrance may be due to a modifier gene that res-
cues the consequences of POLR1D mutations.
POLR1D encodes a subunit present in RNA polymerase I (PolI)
and RNA polymerase III (PolIII). Both of these polymerases are
involved in ribosomal RNA (rRNA) transcription, but PolIII mainly
synthesizes 5S rRNA and transfer RNA (tRNA). Together with RNA
polymerase II, which drives mRNA transcription, the three RNA
Mutations in genes encoding
subunits of RNA polymerases I and
III cause Treacher Collins syndrome
Johannes G Dauwerse1, Jill Dixon2, Saskia Seland3,
Claudia A L Ruivenkamp1, Arie van Haeringen1,
Lies H Hoefsloot4, Dorien J M Peters1, Agnes Clement-de Boers5,
Cornelia Daumer-Haas6, Robert Maiwald7, Christiane Zweier8,
Bronwyn Kerr2, Ana M Cobo9, Joaquín F Toral10,
A Jeannette M Hoogeboom11, Dietmar R Lohmann3, Ute Hehr12,
Michael J Dixon2,13, Martijn H Breuning1 & Dagmar Wieczorek3
1Center for Human and Clinical Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands. 2Faculty of Medical and Human Sciences, Manchester
Academic Health Sciences Centre, University of Manchester, Manchester, UK. 3Institut für Humangenetik, Universitaetsklinikum Essen, Essen, Germany.
4Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. 5Department of Pediatrics, Medical Center Haaglanden,
The Hague, The Netherlands. 6Praenatal-Medizin München, München, Germany. 7Medizinisches Versorgungszentrum für Laboratoriumsmedizin, Mikrobiologie und
Humangenetik, Mönchengladbach, Germany. 8Institute of Human Genetics, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany. 9Centre de
Référence Neuromusculaire, Hôpital Marin Assistance Publique-Hôpitaux de Paris (AP-HP), Hendaye, France. 10Hospital Universitario Central de Asturias, Oviedo,
Spain. 11Department of Clinical Genetics, Erasmus Medical Center Rotterdam, The Netherlands. 12Zentrum für Humangenetik am Universitaetsklinikum Regensburg,
Regensburg, Germany. 13Faculty of Life Sciences, University of Manchester, Manchester, UK. Correspondence should be addressed to J.G.D. (firstname.lastname@example.org).
Received 14 July; accepted 29 October; published online 5 December 2010; doi:10.1038/ng.724
© 2011 Nature America, Inc. All rights reserved.
Nature GeNetics? VOLUME 43 | NUMBER 1 | JANUARY 2011
polymerases consist of 14, 12 and 15 subunits, respectively. The
core of each enzyme is composed of two α- and two β-subunits. In
yeast, the α-subunits consist of two proteins, AC19 (encoded by the
POLR1D homolog) and AC40 (encoded by the POLR1C homolog),
which undergo a strong interaction7. Notably,
an essential role for AC40 and AC19 in the
assembly of the RNA polymerase enzyme
has previously been reported5,8. In view
of the strong interaction of POLR1D with
POLR1C, we hypothesized that the human
gene encoding AC40, POLR1C, was another
candidate gene likely to be mutated in TCS;
consequently, we sequenced this gene in the
252 individuals with TCS (Supplementary
Table 2). In individual EsM17985, who
had typical features of TCS, we detected a
nonsense mutation in exon 9 resulting in
the p.Lys327X alteration; however, we also
identified a missense mutation resulting
in the p.Arg279Gln alteration in exon 8
of POLR1C in the same individual. A
sequence analysis of the parents of individual
EsM17985 revealed that the mutation result-
ing in p.Lys327X was inherited from the
mother, whereas the mutation resulting in
p.Arg279Gln was inherited from the father.
Similarly, in a second unrelated individual,
Man42, we identified a stop mutation, result-
ing in p.Gly31ValfsX23, in exon 2 and a mis-
sense mutation, resulting in p.Arg279Trp, in
exon 8 of POLR1C. Sequence analysis of the
parents of this individual revealed that the
mutation resulting in p.Gly31ValfsX23 was
inherited from the mother, whereas the muta-
tion resulting in p.Arg279Trp was inherited
from the father. In both families, neither the mother nor the
father was affected. Notably, in a third individual and her more
severely affected brother, EsM5815, we found a 4-bp deletion in
intron 8 in the splice donor site (most likely resulting in skipping
table 1 mutations within POLR1D and POLR1C in individuals with treacher collins syndrome
Mutations within POLR1D,
resulting protein alterationIndividual
c.26+1, splice site mutation
Mutations within POLR1C,
resulting protein alteration
of mutation Familial
21EsM17985 c.836G>A, p.Arg279Gln;
A database of POLR1D and POLR1C mutations is maintained at the Leiden Open Variation Database (see URLs)15.
Exon 2Exon 1
Exon 2 Exon 1
Exon 4Exon 5 Exon 6Exon 7 Exon 8Exon 9
figure 1 Schematic representation of POLR1D and POLR1C and facial phenotypes of individuals with mutations within POLR1D and POLR1C .
Individuals with POLR1DI mutations are shown in c–f, and individuals with mutations in POLR1C are shown in g and h. (a) POLR1D
(ENST00000302979 in Ensembl), with the mutations found in the individuals in c–f shown above. (b) POLR1C (ENST00000372389 in Ensembl),
with the mutations found in the individuals in g and h shown above. (c) Individul Lei1, with a deletion in chromosome band 13q12.2. (d) Individual
EsM3422 with c.326–327del (p.His109fsX3). (e) Two affected brothers, EsM42160, with c.259C>T (p.Arg87X). (f) Affected mother and daughter,
EsM6227, with c.152T>G (p.Leu51Arg). (g) Individual EsM17985 with c.836G>A (p.Arg279Gln) and c.979A>T (p.Lys327X). (h) Individual Man42
with c.87delT (p.Gly31ValfsX23) and c.835C>T (p.Arg279Trp). We obtained written consent to publish photographs of all individuals shown.
© 2011 Nature America, Inc. All rights reserved.
VOLUME 43 | NUMBER 1 | JANUARY 2011 Nature GeNetics
of exon 8), and again, we detected the missense mutation in exon
8 resulting in p.Arg279Gln in exon 8. As the father of the index
case is deceased, no DNA was available for analysis; the healthy
sister did not carry any mutations. The unaffected mother and her
healthy sister carry the mutation resulting in p.Arg279Gln mutation.
Arginine 279 is an evolutionary highly conserved amino acid that
is located in the RNA polymerase dimerization domain of POLR1C
(Supplementary Fig. 3b). Mutations in codon 279 of POLR1C were
not detected in 272 unaffected individuals. In one control sample, we
observed a nonsense mutation, resulting in p.Arg303X. As we found
no other mutations in POLR1C, it is likely that this individual is an
unaffected mutation carrier. These observations, especially the seg-
regation within the third family, indicate autosomal recessive inherit-
ance of TCS in the three families described, as suggested previously9
(Supplementary Fig. 4c).
Previous analyses of Treacle, the protein encoded by TCOF1,
have indicated that this protein colocalizes with upstream bind-
ing factor (UBF), an RNA polymerase I transcription factor, in
the nucleolus, and that Treacle is involved in rRNA transcription
by interacting with UBF10. Furthermore, Treacle has an essential
role in ribosome biogenesis in vivo11. Haploinsufficiency of Tcof1
in mice results in craniofacial malformations that phenocopy
those observed in TCS. These anomalies arise as a consequence
of a deficiency in the number of cranial neural crest cells, which
results from reduced production of mature ribosomes in the neuro-
epithelium and neural crest during embryonic development. Thus,
in the absence of Treacle, insufficient ribosome biogenesis restricts
cell-cycle progression of these highly proliferative cell populations,
causing reduced proliferation, cell cycle arrest and high levels of
cell death11. On the basis of a reduction in Ubf activity observed
in Tcof1+/−embryos compared with their wildtype littermates, it
has been hypothesized that Treacle regulates proliferation directly
through Ubf; however, it has recently been shown that Treacle, but
not UBF, is essential for the nucleolar recruitment and retention
of the PolI transcriptional machinery12.
Mutations in POLR1D lead to haploinsufficiency of this gene, and
mutations in POLR1C lead to depletion of functional POLR1C. This
was demonstrated in individuals and families with TCS in the current
study. These observations indicate that diminished quantity of func-
tional PolI and/or PolIII results in an insufficient number of mature
ribosomes in the neuroepithelium and neural crest cells at a critical
time point during embryogenesis11. It has been hypothesized that this
causes apoptosis of progenitors of the first and second branchial arches,
resulting in the characteristic craniofacial abnormalities observed in
TCS13. Mutations in other genes that cause a diminished function of
PolI and/or PolIII result in a deficiency of rRNA and/or tRNA, and this
can cause persistent malfunction of bone marrow cells, for example,
in Shwachman-Diamond syndrome and Diamond-Blackfan anemia14.
Some individuals with the latter types of anemias also express a cranio-
facial phenotype that is consistent with TCS. Further analysis of the
ribosomal biogenesis pathway will hopefully identify further condi-
tions belonging to the ribosomopathies with or without a craniofacial
phenotype and will probably open new starting points for therapies.
URLs. Leiden Open Variation Database, http://www.lovd.nl.
Note: Supplementary information is available on the Nature Genetics website.
We express our sincere gratitude to the cases, their parents and other family
members for their participation in this study, as well as to the clinicians for sending
blood samples. We want to thank the Laboratory for Diagnostic Genome Analysis
(LDGA) in Leiden and the Forensisch Laboratorium voor DNA Onderzoek
(FLDO), as well as the TCS team in Essen and Regensburg for their excellent
technical assistance and Ivo Fokkema for his help with constructing the Leiden
Open Variation Databases (LOVD). This work was part of the CRANIRARE
Network funded through a grant from the German Ministry of Research and
Education to D.R.L. and D.W. (BMBF 01GM0802). The financial support of the
National Institutes of Health Research Manchester Biomedical Research Centre
and the Healing Foundation to J.D. and M.J.D. is gratefully acknowledged.
Mutation analysis: J.G.D., S.S., D.R.L.
Patient ascertainment including clinical data and/or TCOF1 mutation analysis:
J.D., A.v.H., L.H.H., D.J.M.P., A.C.-d.B., C.D.-H., R.M., C.Z., B.K., A.M.C., J.F.T.,
A.J.M.H., U.H., M.J.D., D.W.
Array analysis: C.A.L.R.
Manuscript writing: J.G.D., M.J.D., D.R.L., M.H.B., D.W.
Study design: J.G.D., A.v.H., D.W.
All authors contributed to the final version of the paper.
CoMPeTInG FInAnCIAL InTeReSTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturegenetics/.
Reprints and permissions information is available online at http://npg.nature.com/
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