Missense Mutations in TCF8 Cause Late-Onset Fuchs Corneal Dystrophy and Interact with FCD4 on Chromosome 9p

Article (PDF Available)inThe American Journal of Human Genetics 86(1):45-53 · January 2010with25 Reads
DOI: 10.1016/j.ajhg.2009.12.001 · Source: PubMed
Fuchs corneal dystrophy (FCD) is a degenerative genetic disorder of the corneal endothelium that represents one of the most common causes of corneal transplantation in the United States. Despite its high prevalence (4% over the age of 40), the underlying genetic basis of FCD is largely unknown. Here we report missense mutations in TCF8, a transcription factor whose haploinsufficiency causes posterior polymorphous corneal dystrophy (PPCD), in a cohort of late-onset FCD patients. In contrast to PPCD-causing mutations, all of which are null, FCD-associated mutations encode rare missense changes suggested to cause loss of function by an in vivo complementation assay. Importantly, segregation of a recurring p.Q840P mutation in a large, multigenerational FCD pedigree showed this allele to be sufficient but not necessary for pathogenesis. Execution of a genome-wide scan conditioned for the presence of the 840P allele identified an additional late-onset FCD locus on chromosome 9p, whereas haplotype analysis indicated that the presence of the TCF8 allele and the disease haplotype on 9p leads to a severe FCD manifestation with poor prognosis. Our data suggest that PPCD and FCD are allelic variants of the same disease continuum and that genetic interaction between genes that cause corneal dystrophies can modulate the expressivity of the phenotype.
Missense Mutations in TCF8 Cause
Late-Onset Fuchs Corneal Dystrophy
and Interact with FCD4 on Chromosome 9p
S. Amer Riazuddin,
Norann A. Zaghloul,
Amr Al-Saif,
Lisa Davey,
Bill H. Diplas,
Danielle N. Meadows,
Allen O. Eghrari,
Mollie A. Minear,
Yi-Ju Li,
Gordon K. Klintworth,
Natalie Afshari,
Simon G. Gregory,
John D. Gottsch,
and Nicholas Katsanis
Fuchs corneal dystrophy (FCD) is a degenerative genetic disorder of the corneal endothelium that represents one of the most common
causes of corneal transplantation in the United States. Despite its high prevalence (4% over the age of 40), the underlying genetic basis of
FCD is largely unknown. Here we report missense mutations in TCF8, a transcription factor whose haploinsufficiency causes posterior
polymorphous corneal dystrophy (PPCD), in a cohort of late-onset FCD patients. In contrast to PPCD-causing mutations, all of which are
null, FCD-associated mutations encode rare missense changes suggested to cause loss of function by an in vivo complementation assay.
Importantly, segregation of a recurring p.Q840P mutation in a large, multigenerational FCD pedigree showed this allele to be sufficient
but not necessary for pathogenesis. Execution of a genome-wide scan conditioned for the presence of the 840P allele identified an addi-
tional late-onset FCD locus on chromosome 9p, whereas haplotype analysis indicated that the presence of the TCF8 allele and the
disease haplotype on 9p leads to a severe FCD manifestation with poor prognosis. Our data suggest that PPCD and FCD are allelic vari-
ants of the same disease continuum and that genetic interaction between genes that cause corneal dystrophies can modulate the expres-
sivity of the phenotype.
FCD represents the most common form of genetic disor-
ders of the corneal endothelium.
The disorder affects
as much as 4% of the population over the age of 40 and
accounts for a significant fraction of the corneal transplan-
tation performed in the United States every year.
cally, FCD is marked by the development of guttae, excres-
cences of Descemet membrane that appear in the fourth or
fifth decade and increase in number over time.
As the
disease progresses, visual acuity decreases secondary to
corneal edema and endothelial cell loss, with end-stage
disease evidenced by the formation of painful epithelial
FCD is genetically heterogeneous, exhibiting an auto-
somal-dominant mode of inheritance with variable pene-
trance and expressivity. The rare form of early-onset FCD
is causally associated with mutations in COL8A2 (MIM
120252), whereas the more common late-onset FCD has
been localized to three loci: FCD1, FCD2, and FCD3 on
chromosomes 13, 18, and 5, respectively.
clinically distinct, corneal endothelial dystrophies share
clinical features suggesting that genes implicated in one
corneal dystrophy may also harbor mutations liable for
other dystrophies. This premise was strengthened when
pathogenic mutations in SLC4A11 (MIM 610206), a borate
transporter in which loss-of-function mutations cause
autosomal-recessive congenital hereditary endothelial
dystrophy (CHED2 [MIM 217700]), were identified in
sporadic late-onset FCD patients.
Therefore, we hypoth-
esized that TCF8 (MIM 189909), a transcription factor
shown to be causally associated with another corneal
dystrophy, PPCD (MIM 609141),
may contribute to
the genetic load of late-onset FCD.
Here, we report five missense mutations in TCF8 associ-
ated with late-onset FCD, which appear to be causal
because of a loss of protein function based on an in vivo
complementation assay. One of these mutations, encoding
a p.Q840P change, was present in both a sporadic FCD
patient and in a large family and was transmitted in
a manner consistent with an autosomal-dominant trait.
However, the 840P allele could not explain FCD in all
patients, suggesting the presence of a second pathogenic
allele in this pedigree. A genome-wide scan conditioned
to the presence/absence of the TCF8 mutant allele identi-
fied a new locus for late-onset FCD, FCD4 on chromosome
9p subsequent genetic and clinical analyses suggested that
although mutations in each of TCF8 and FCD4 are suffi-
cient for pathogenesis, genetic interaction between these
two loci can lead to a more severe form of the disease.
Our data represent the first familial evidence for mutations
that cause late-onset FCD, supporting the hypothesis that
several corneal dystrophies, although clinically distinct,
share the same molecular etiology. Additionally, our data
suggest that the quality and quantity of mutations in
FCD-associated loci can have a profound impact on the
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;
Center for Corneal Genetics,
Cornea and External Disease Service, Wilmer Eye Institute, Johns Hopkins Hospital, Baltimore, MD 21205, USA;
Center for Human Genetics,
of Ophthalmology, Duke University Medical Center, Durham, NC 27710, USA;
Center for Human Disease Modelin g, Department of Cell Biology, Duke
University, Durham, NC 27710, USA
*Correspondence: katsanis@cellbio.duke.edu
DOI 10.1016/j.ajhg.2009.12.001. ª2010 by The American Society of Human Genetics. All rights reser ved.
The American Journal of Human Genetics 86, 45–53, January 8, 2010 45
clinical presentation of the phenotype, which in turn will
probably impact patient management.
Material and Methods
Family members were recruited through a proband with FCD pre-
senting to our clinics at Johns Hopkins University or Duke Univer-
sity. Extended pedigrees were subsequently developed through
interviews and patients were examined in locations proximal to
their area of residence. Recruitment, examination, and procedures
for DNA sample collection were approved by the Institutional
Review Boards for Human Subjects Research at the Johns Hopkins
University School of Medicine and Duke University Medical
Center, respectively, according to the Declaration of Helsinki.
Each study participant provided written consent.
Determination of Phenotype and Disease Severity
Individuals underwent detailed ophthalmic examination includ-
ing slit-lamp biomicroscopy. Severity was graded on a modified
scale according to Krachmer and colleagues with >12 central guttae
in either eye (grade 1).
Progression of confluence was defined by
three grades: horizontal width less than 2 mm (grade 2), from 2 to
5 mm (grade 3), and >5 mm (grade 4). The development of stromal
and/or epithelial edema elevated this score to grade 5, and grade 6 is
disease severe enough to require keratoplasty.
An Affymetrix (Affymetrix, Santa Clara, CA) 5.0 SNP array was
used for genome-wide linkage analyses. 500 ng of genomic DNA
was used in a multiplexed SNP genotyping assay according to
manufacturer’s instructions. Arrays were then scanned in a Gene-
Chip Scanner 3000 7G (Affymetrix). The output signal files were
checked for quality control (QC) with the Affymetrix BRLMM
analysis tool 2.0 (BAT 2.0). The default threshold score was set at
0.05 and was used as a cutoff for SNPs to be excluded. Cell files
were processed further to generate genotypes with the BAT 2.0
Affymetrix genotyping console and locally written Perl scripts.
Linkage Analyses
SNP genotypes were incorporated into the family pedigree files to
generate PED files with Plink software.
Pedigrees were analyzed
assuming an autosomal-dominant mode of inheritance, 0.02
disease allele frequency, and 90% penetrance by MERLIN algo-
SNP allele frequencies from an ethnically matched
control population were obtained from HapMap. The critical
interval on 9p was delineated by genotyping STR markers; primers
and conditions available upon request. Two point linkage analyses
were performed with the FASTLINK version of MLINK from the
LINKAGE Program Package.
Maximum LOD scores were calcu-
lated with ILINK under an autosomal-dominant model with
a 0.02 disease allele frequency and 90% penetrance. The marker
order and distances between the markers were obtained from the
Marshfield database and the NCBI chromosome 9 sequence maps.
Equal allele frequencies were assumed for the initial genome scan,
while for fine mapping allele frequencies were derived from 96
unrelated and unaffected individuals.
Mutation Screening
Primer pairs for individual exons of TCF8 were designed with
Primer3. The sequences and amplification conditions are available
upon request. Each amplicon was purified and sequenced bidirec-
tionally with Big Dye Terminator Ready Reaction Mix according to
manufacturer’s instructions (Applied Biosystems).
Construction of the TCF8 Vector
We cloned the TCF8 ORF into a pCS2þ plasmid as follows. The
3375 bp TCF8 ORF was amplified from HEK293T cDNA in two
separate amplification reactions. In the first step, the 1875 bp frag-
ment was amplified, digested with BamHI and BglII restriction
endonucleases, and cloned into BamHI/BglII-digested pCS2þ
plasmid. The orientation of the clone was detected by restriction
digestion with BamHI, BglII, and XhoI. In the second step, a
1775 bp fragment was amplified, digested with BglII and XhoI
restriction endonucleases, and cloned into BglII/XhoI digested
pCS2þ plasmid harboring the N-terminal TCF8 fragment in the
correct orientation. The sequence of TCF8 was confirmed by Big
Dye Terminator Ready reaction mix according to manufacturer’s
instructions (Applied Biosystems).
Generation of Site-Directed Mutants
All five missense mutations identified in this study, as well as
a previously identified mutation N696S,
were cloned in the
TCF8-pCS2þ vector with QuikChange Site-Directed Mutagenesis
Kit according to manufacturer’s instructions (Stratagene, La Jolla,
CA). The presence of each mutation was confirmed with bidirec-
tional sequencing. Additionally, the entire TCF8 ORF for each clone
was sequenced to rule out the presence of any mutations that may
have been incorporated during the mutagenesis procedure.
In Vitro Transcription
The pCS2þ plasmids harboring the wild-type and mutant alleles
of TCF8 were digested with NotI to linearize the plasmids. These
linearized plasmids were used for in vitro transcription with
mMessage mMachine transcription kit utilizing the SP6 promoter
according to the manufacturer’s instruction (Ambion Austin TX).
In Vivo Analyses
We used an antisense translation blocking morpholino (MO) to
suppress translation of zebrafish tcf8. The MO was injected into
one- to two-cell stage embryos. The exact concentration of MO
necessary was determined by a dose-response curve. Embryos
were raised to somite stages (approximately 14 hr) and scored for
early developmental defects. Embryo phenotypes were rescued
by coinjecting mRNA encoding full-length wild-type human
TCF8 mRNA and the efficiency of rescue of the zebrafish tcf8MO
was measured. Approximately 100 embryos were injected with
either MO alone, MO and wild-type TCF8 mRNA, or MO and
mRNA bearing one of each of the missense mutations and were
scored blind to the injection cocktail.
Mutations in TCF8 Are Sufficient to Cause FCD
The association of SLC4A11 with late-onset FCD
the possibility that genes that cause other corneal endothe-
lial dystrophies might also contribute to FCD. To investi-
gate this hypothesis, we focused on TCF8, a transcription
factor that has been causally linked to PPCD, another
endothelial dystrophy with some phenotypic similarities
to FCD.
We therefore sequenced the entire coding
46 The American Journal of Human Genetics 86, 45–53, January 8, 2010
region of TCF8 in 192 unrelated adult-onset FCD patients
of northern European descent. In addition to a number
of known SNPs, whose frequency in our cohort was
indistinguishable from reported HapMap data, we also
identified four unique alleles encoding the missense
changes p.N78T, p.Q810P, p.Q840P, and p.A905G. Each
of these variants affect evolutionary conserved residues
(Figure 1) and was not found in 560 unrelated, ethnically
matched control chromosomes or in HapMap samples of
any ethnicity. Additionally, computational predictions
with both SIFT
and PolyPhen
suggested that they
were pathogenic, raising the strong possibility that
missense alleles in TCF8 can contribute to late-onset
FCD. To confirm these data, we sequenced a second, inde-
pendent cohort of 192 sporadic samples of Caucasian
descent, ascertained under the same clinical criteria. We
found three missense changes: a novel p.P649A that is
also evolutionarily conserved and computationally pre-
dicted to be pathogenic (Figure 1) and p.N78T and
p.Q840P, each of which was also found in the first cohort
with no evidence for a shared ancestral haplotype. Six of
the seven FCD patients were sporadic and as such, segrega-
tion analysis was not possible. The seventh patient bearing
the recurring p.Q840P mutation, however, belongs to
a large, multigenerational FCD family, acronymed TH. Tar-
geted recruitment within TH (Figure 2) enabled us to
further identify 12 individuals (seven females, five males)
who fulfill the FCD phenotypic criteria (Table 1). A 13
individual (Figure 2; individual IV:10) exhibited early signs
of FCD; however, the patient did not have sufficient
guttae for a positive diagnosis. In view of her younger
age (38 years) and the number of guttae present bilaterally
(<12), she was designated as a subclinical case.
Identification of a Fourth FCD Locus
Segregation analysis showed that the p.Q840P allele is
sufficient but not necessary for disease pathogenesis.
None of the seven unaffected individuals carry the 840P
allele, whereas the mutation is present in 7/12 affecteds
(Figure 2), suggesting that a second, independent genetic
event might account for the phenotype in the other five
patients. To investigate this possibility, we performed
a genome-wide screen by genotyping all family members
with an Affymetrix 5.0 array. Approximately 481K SNPs
passed quality control and were considered for linkage.
However, because of the unattainable computational
needs required to perform linkage with the entire marker
set, we selected every 5
accepted SNP to simplify the
data while maintaining uniform coverage across the
genome and performed two-point linkage on ~96K SNPs.
Initial analyses under various disease models failed to
detect LOD scores even above 2.0 when considering the
entire family. We therefore took into consideration the
TCF8 p.Q840P allele and recomputed linkage conditional
to this mutation. This model predicated that (1) all affected
individuals negative for the p.Q840P mutation must
Figure 1. Sequence Analyses and Evolutionary Assessment of Late-Onset FCD Mutants
(A) Sequence chromatograms of the five TCF8 mutations (c.232A>G, c.1945C>G, c.2429A>C, c.2519A>C, c.2714C>G) identified in
late-onset FCD patients.
(B) Illustration of the evolutionary conservation of Asn78, Pro649, Gln810, Gln840, and Ala905 in other TCF8 orthologs.
The American Journal of Human Genetics 86, 45–53, January 8, 2010 47
harbor a pathogenic allele elsewhere in the genome; and (2)
affected individuals heterozygous for the p.Q840P muta-
tion may not necessarily harbor the pathogenic allele,
with the exception of individuals with affected offspring
who lack the p.Q840P mutation. Under this model, we
found suggestive linkage to a single region in the genome
on chromosome 9p (highest two-point LOD score of 2.43
with rs1410041). Importantly, a ‘reverse’ genome scan
was also consistent with a two-locus model. When we
conditioned for the 9p risk allele (affected individuals or
individuals with affected offspring negative for the 9p allele
must harbor a second pathogenic allele elsewhere in the
genome, whereas the rest of the affected individuals may
not necessarily conceal this allele), we obtained the highest
genome-wide two-point LOD score of 1.93 at TCF8
(rs1148233), close to the theoretical maximum.
Because SNPs are biallelic, they have reduced informa-
tiveness. Therefore, we genotyped all members of family
TH with a series of 10 evenly spaced STR microsatellite
markers (Figure 2). We observed LOD scores of 3.09 and
3.20 at q ¼ 0 with markers D9S168 and D9S256, respec-
tively (Table 2), close to the theoretical maximum attain-
able with this family, thereby defining a fourth FCD
locus, FCD4. Recombinants place the mutation in a 14 Mb
interval between D9S1681 proximally and D9S1684
distally. Reconstruction of the disease haplotype was
consistent with a two-locus model, in that all affected indi-
viduals negative for the p.Q840P TCF8 mutation harbor
the disease-transmitting 9p22.1-p24.1 haplotype (Fig-
ure 2). Notably, individual IV:10, with a borderline diag-
nosis, carries the disease haplotype as predicted by the
presence of preclinical FCD findings. After considering
her as affected, the LOD score is elevated to 3.48 (Table 2).
Genetic Interaction between TCF8 and FCD4
Within family TH, five affected individuals are heterozy-
gous for both the p.Q840P mutation and the disease haplo-
type on 9p22.1-p24.1 (Figure 2). We therefore wondered
whether the presence of two pathogenic alleles influences
the clinical phenotype and severity of the disease. To
compare across the same age, we examined the clinical
records of individuals III:5, III:7, III:9, and III:11 (all
ages > 70). Individuals III:5, III:7, and III:9 harbor both
p.Q840P mutation and the FCD4 disease allele, whereas
individual III:11 harbors only the p.Q840P mutation.
The clinical records confirmed that all three individuals
with both mutations have a severe FCD phenotype (modi-
fied Krachmer grading of 6 bilaterally) and have undergone
corneal transplantations, bilaterally. In sharp contrast to
his siblings, individual III:11 has a milder FCD phenotype
(Krachmer grading of þ2.5 bilaterally). Progression of
disease for individuals in family TH with a single patho-
genic allele (TCF8 p.Q840P or disease haplotype on 9p)
shows as a linear relationship between age and severity
Figure 2. Pedigree of the Family TH Linked to the FCD4 Locus
Symbols: filled symbol, affected; diagonal line through a symbol, deceased; question mark, affectation status known. Haplotypes of 12
adjacent chromosome 9p microsatellite markers are shown, with alleles forming the disease-bearing haplotype shaded black. The TCF8
mutation; c.2519A>C identified in family TH is shown at the bottom.
48 The American Journal of Human Genetics 86, 45–53, January 8, 2010
with an r
value of 0.84 (Figure 3). Notably, the severity of
individuals III:5, III:7, and III:9, harboring pathogenic
alleles in both TCF8 and FCD4, is not consistent with the
progression pattern of family TH, and based on these
observations, we predict that individuals IV:4 and IV:6
(both in their 40s at time of the examination) will develop
a severe FCD phenotype compared with other affected
The fact that three affected individuals in family TH
underwent a corneal transplant was surprising because
the majority of the families in our cohort rarely have
multiple affecteds that had corneal transplants. Therefore,
Table 1. Clinical Characteristics of Affected Individuals of Family TH
Individual ID Sex
Age at Time of the
Examination (Years)
Age at Time of the Penetrating
Keratoplasty (Years)
FCD Krachmer
Grading TCF8 Mutation
FCD4 Disease
III:5 F 78 71 PK bilaterally p.Q840P yes
IV:1 M 55 N.A. OD; þ1.0/ OS; þ1.5 p.Q840P no
IV:2 F 57 N.A. OD; þ2.5/ OS; þ2.5 wild-type yes
III:7 F 76 70 PK bilaterally p.Q840P yes
IV:4 F 49 N.A. OD; þ1.0/ OS; þ1.0 p.Q840P yes
IV:5 M 53 N.A. OD; þ1.0/ OS; þ1.5 wild-type yes
IV:6 M 46 N.A. OD; þ1.0/ OS; þ1.0 p.Q840P yes
IV:7 F 42 N.A. OD; þ1.0/ OS; þ1.0 wild-type yes
III:9 M 72 68 PK bilaterally p.Q840P yes
IV:8 F 47 N.A. OD; þ1.0/ OS; þ1.0 wild-type yes
IV:9 F 45 N.A. OD; þ1.0/ OS; þ1.0 wild-type yes
IV:10 F 37 N.A. trace bilaterally wild-type yes
III:11 F 70 N.A. OD; þ2.5/ OS; þ2.5 p.Q840P no
Individuals III:5, III:7, and III:9 (Figure 1) had bilateral corneal transplants. Penetrating keratoplasty was performed on each eye separately and only once the
severity of disease had reached 6 on the modified scale proposed by Krachmer and colleagues.
Abbreviations: PK, penetrating keratoplasty; F, female; M,
male; N.A., not applicable.
Table 2. Two-Point LOD Scores Generated with 9p STR Markers
Marker cM Mb 0.00 0.01 0.05 0.10 0.20 0.30 Z
D9S1686 12.50 4.63 0.672 0.658 0.598 0.567 0.520 0.357 0.672 0.00
0.670 0.656 0.596 0.565 0.519 0.356 0.670 0.00
D9S1681 18.60 5.27 0.484 0.321 0.091 0.216 0.346 0.500 0.500 0.30
1.417 1.225 0.646 0.448 0.223 0.155 0.155 0.30
D9S281 15.33 6.71 0.092 0.047 0.357 0.436 0.501 0.475 0.501 0.20
0.094 0.046 0.358 0.436 0.502 0.476 0.502 0.20
D9S168 20.40 10.57 3.094 3.038 2.807 2.689 2.507 1.862 3.094 0.00
3.375 3.314 3.067 2.939 2.744 2.049 3.375 0.00
D9S256 22.80 11.01 3.201 3.145 2.916 2.798 2.617 1.972 3.201 0.00
3.482 3.422 3.175 3.048 2.854 2.160 3.482 0.00
D9S1869 26.50 14.26 1.988 1.969 1.874 1.816 1.719 1.321 1.988 0.00
2.268 2.246 2.133 2.066 1.955 1.508 2.268 0.00
D9S1684 34.42 19.60 5.918 2.870 1.394 0.839 0.745 0.232 0.232 0.30
0.204 0.185 0.125 0.102 0.075 0.022 0.022 0.30
D9S1846 37.58 21.62 3.221 2.397 1.403 1.148 0.873 0.375 0.375 0.30
4.299 2.220 1.169 0.919 0.658 0.221 0.221 0.30
LOD scores were calculated at different q values for each marker with individual IV:10 as unaffected (top) and affected (bottom row).
The American Journal of Human Genetics 86, 45–53, January 8, 2010 49
we wondered whether the presence of pathogenic alleles at
two loci might have contributed to the higher incidence of
corneal transplants in family TH. To examine this possi-
bility, we interrogated our entire cohort setting the
following two stringent criteria: (1) we restricted our anal-
yses to families with three or more affecteds; and (2) we
only considered affecteds with bilateral corneal trans-
plants. We were able to examine 33 families and found a
remarkably low frequency of bilateral transplants (9/246)
which, despite the relatively modest sample size, was
significantly different compared to 3/12 in family TH
¼ 10.618; p ¼ 0.0006). Even under reduced stringency
criteria, where all transplant events were considered in our
cohort, the enrichment in family TH remained significant
¼ 8.17; p ¼ 0.0042). As such, the most parsimonious
explanation for these observations is that in family TH
the p.Q840P allele interacts with the FCD4 causal muta-
tion to modulate the severity of the phenotype.
In Vivo Functional Assessment of TCF8 Mutations
Taken together, our genetic data implicate missense muta-
tions in TCF8 in the development of late-onset FCD. All
mutations affect conserved residues and have been found
exclusively in patients (two of them recurrent). Impor-
tantly, phasing pedigree TH for the presence of the
p.Q840P allele maps a new FCD locus supportive of a
two-locus model. Although TCF8 null mutations have
been shown to cause PPCD, an earlier onset endothelial
corneal dystrophy, we have determined that missense
alleles in FCD are of potential mechanistic consequence.
We considered two possibilities: (1) loss-of-function alleles
lead to PPCD and dominant-negative or gain-of-function
mutations cause FCD, or (2) both disorders can be induced
by relative TCF8 loss. To investigate these possibilities and
to obtain further evidence for the functional relevance of
the mutated residues, we took advantage of an in vivo
complementation assay in zebrafish. Upon reciprocal
BLAST, we identified the sole ortholog of TCF8 in the
zebrafish genome (81% similarity at protein level), also
known as kheper. Previous studies have shown that expres-
sion of dominant-negative tcf8 RNA results in severe
defects during zebrafish gastrulation and neurulation.
Therefore, under the reasonable premise that morpholino
(MO)-induced tcf8 suppression should reproduce such
phenotypes, we initiated a series of in vivo complementa-
tion studies. Specifically, to interrogate the mechanisms
by which mutations in TCF8 cause FCD in humans, we
sought to use these developmental defects as a functional
readout and ask whether (1) suppression of endogenous
tcf8/kheper produces similar phenotypes early in develop-
ment; (2) whether wild-type (WT) human TCF8 mRNA
can rescue such phenotypes; (3) whether expression of
human TCF8 mRNA bearing each of the five point
mutations can rescue the phenotype; and (4) whether
expression of the same mutants might induce early devel-
opmental defects (indicative of a dominant-negative or
gain-of-function effect).
To suppress expression of tcf8 in embryos, we designed
a translational blocking morpholino (tbMO). Injection of
2.5 ng of tbMO at the one- to two-cell stage resulted in
defects in early development consistent with published
observations in approximately 80% of embryos,
could be categorized into two severity classes. Class I
embryos survived gastrulation but had shortened body
axis and pronounced detachment of cells along the dorsal
axis at mid-somitic age, whereas class II embryos exhibited
complete disruption of embryo morphology during gastru-
lation (Figure 4A). These phenotypes could be rescued effi-
ciently by coinjection of human WT TCF8 mRNA. Coin-
jection of 100 pg of human mRNA with 2.5 ng of tbMO
resulted in a complete rescue of the morphant phenotypes
(97.5% of embryos, n ¼ 100–150 embryos scored blind)
(Figure 4B). To determine whether mutations in TCF8
result in disruption of protein function, we coinjected
each mutant mRNA and assessed its ability to rescue mor-
phant phenotypes. This assay further supported our
human genetics evidence in that each of the five muta-
tions reduced or abolished TCF8 function. Two mutations,
p.N78T and p.Q810P, partially rescued the MO phenotype,
indicating that these are probably hypomorphic, whereas
rescue with human TCF8 mRNA encoding either 649A,
840P, or 905G were indistinguishable from tbMO-injected
embryos, suggesting that these alleles are severe (Figure 4B).
The assay is not promiscuous; injection of tbMO with
human mRNA encoding an N696S mutation reported
previously in a Chinese FCD patient and reported to be
fully rescued the morphant phenotype (Fig-
ure 4B). Notably, though injection of wild-type mRNA
Figure 3. Progression of Late-Onset FCD with Age
in Family TH
The diamonds represent the individuals with single
pathogenic allele (TCF8 p.Q840P or disease haplo-
type on 9p), whereas the triangles symbolize individ-
uals harboring both pathogenic alleles. The age of
each individual was recorded at the time of the
examination or at the time of bilateral keratoplasty.
50 The American Journal of Human Genetics 86, 45–53, January 8, 2010
resulted in defects in early development, injection of each
of the mutant mRNAs alone did not produce the same
severity of defects, suggesting that these mutations are
unlikely to exert a dominant-negative or gain-of-function
effect (Figure 4B). As such, although caution must be exer-
cised in translating the function of alleles in a develop-
mental assay to late-onset corneal degeneration pheno-
types, it is likely that a loss-of-function mechanism
underlies the FCD-associated TCF8 mutations.
Here, we report five novel missense mutations in TCF8
identified in two cohorts of patients diagnosed with late-
onset FCD. These mutations affect evolutionary conserved
alleles and were absent from ethnically matched control
samples. Additionally, we identified a novel locus for late-
onset FCD, FCD4, in a large family, with the critical interval
mapped to chromosome 9p. Haplotype analyses refined
the interval to a 14.3 Mb (15.8 cM) on 9p24.1-22.1 with
a maximum two-point parametric LOD score of 3.20.
Although the maximum LOD score of 3.20 is only slightly
higher than the traditional limiting value of 3.0, it repre-
sents the maximum theoretical obtainable value for this
family. Our data reinforce the notion that there is extensive
heterogeneity for late-onset FCD, contrary to early-onset
that is causally associated with COL8A2, with TCF8 contrib-
uting as much as 2% to the genetic load (7/384 unrelated
patients), with no evidence for shared haplotypes among
unrelated patients with the same mutation. Based on
genetic and mutational data from all known FCD loci,
FCD2 remains the most common locus, with a predicted
contribution of ~25%, followed by SLC4A11, in which we
and others have found mutations in 3%–4% of sporadic
late-onset FCD patients.
Each other mapped FCD locus
represents, at present, a private mutation.
Mutations in TCF8 have been associated previously with
a dystrophy of the corneal endothelium. Inter-
estingly, all mutations reported in PPCD either are
nonsense mutations or result in premature termination of
TCF8; in contrast, we found only missense mutations in
Figure 4. Suppression of tcf8 Produces Specific Defects in Zebrafish Embryos
(A) Embryos injected with MO against tcf8 have early developmental defects including short body axes and detachment of cells along
the dorsal embryonic axis (arrowhead). These defects can be rescued by coinjection with full-length human mRNA.
(B) Proportions of unaffected embryos are rescued almost completely in coinjected embryos as compared to those injected with MO
The American Journal of Human Genetics 86, 45–53, January 8, 2010 51
late-onset FCD patients. We have encountered this
phenomenon before: MKS1 (MIM 609883), one of the genes
that causes Meckel-Gruber syndrome (MKS [604896]), a
neonatal lethal condition, is always found to be null in
Meckel patients. Under the hypothesis that hypomorphic
mutations in that locus might cause a less severe pheno-
type, we were able to show that residual activity at this locus
is causally related to Bardet-Biedl syndrome.
Given the
fact the late-onset FCD represent a milder pathogenic
phenotype compared with PPCD, the discovery of only
missense mutations suggests that functionally deficient
TCF8 alleles are responsible for late-onset FCD phenotype,
whereas nonsense mutations lead to PPCD, most probably
because of haploinsufficiency.
The molecular mechanism of TCF8 pathogenicity
leading to two different corneal dystrophies remains
unclear. TCF8 is a transcription factor acting in some
instances as an enhancer and in other instances as a
The two zinc finger domains present in the
TCF8 define the DNA-binding specificity. Ikeda and
colleagues investigated the DNA-binding properties of
TCF8 protein and identified G/TT/GCACCTGT and C/
TACCTG/TT as the optimal binding sites for the N- and
C-terminal zinc finger motifs, respectively.
Krafchak and colleagues identified these consensus se-
quences present in the promoter regions of COL4A1 (MIM
120130), COL4A2 (MIM 120090), COL4A3 (MIM 120070),
COL4A5 (MIM 303630), COL4A6 (MIM 303631), and
Although TCF8 transcripts have been detected
in corneal samples,
it remains to be seen whether defi-
ciency in the DNA-binding properties of TCF8 leads to
late-onset FCD.
Our data provide the first familial evidence for muta-
tions in adult-onset FCD and indicate that loss of function
of TCF8 can induce a range of corneal dystrophy pheno-
types. Recently, it has been proposed that loss-of-function-
mutations in SLC4A11, a gene mutated in CHED2, might
also contribute to adult-onset FCD.
Taken together, these
data suggest the presence of a continuum across several
clinically distinct corneal dystrophies, where the nature
of mutations might determine the severity and onset of
the phenotype, a phenomenon we have encountered in
other disorders.
As such, genes identified in CHED2,
PPCD, and FCD represent bona fide candidates for all three
clinical presentations. Moreover, our data also suggest that
genetic interactions between FCD loci probably interact to
modify the expressivity of the disorder; understanding the
molecular basis of such interaction will be important both
in terms of disease mechanism and also in the context of
patient management and prognosis.
We thank all family members for their enthusiastic participation
in this study. This study was supported in part by National Eye
Institute Grants R01EY016835 (J.D.G.) and R01EY016514
(G.K.K.), the Kwok Research Fund (J.D.G.), and the National Insti-
tute of Child Health and Development Grant R01HD04260 (N.K.).
The Gottsch and Katsanis laboratories contributed equally to this
Received: September 18, 2009
Revised: November 21, 2009
Accepted: December 1, 2009
Published online: December 24, 2009
Web Resources
The URLs for data presented herein are as follows:
International HapMap Project, http://www.hapmap.org
Marshfield Clinic Research Foundation, http://www.
National Center for Biotechnology Information, http://www.ncbi.
Primer3, http://frodo.wi.mit.edu/primer3
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The American Journal of Human Genetics 86, 45–53, January 8, 2010 53
    • "e l s e v i e r . c o m / l o c a t e / v i s r e s also been implicated in the pathogenesis of Fuchs endothelial corneal dystrophy (Mehta et al., 2008; Riazuddin et al., 2010). ZEB1 also plays a significant role in the development of the neural tube and its derivatives, as an absence of ZEB1 gene expression is associated with severe defects in neurodevelopment, such as exencephaly and a failure of neural tube closure in a mouse model (Liu et al., 2008). "
    [Show abstract] [Hide abstract] ABSTRACT: Posterior polymorphous corneal dystrophy (PPCD) is a dominantly inherited disorder of the corneal endothelium that has been associated with mutations in the zinc-finger E-box binding homeobox 1 gene (ZEB1) gene in approximately one-third of affected families. While the corneal dystrophies have traditionally been considered isolated disorders of the corneal endothelium, we have recently identified two cases of maldevelopment of the corpus callosum in unrelated individuals with PPCD. The proband of the first family was diagnosed shortly after birth with agenesis of the corpus callosum and several other developmental abnormalities. Karyotype, FISH and whole genome copy number variant analyses were normal. She was subsequently diagnosed with PPCD, prompting screening of the ZEB1 gene, which identified a novel deletion (c.449delG; p.(Gly150Alafs∗36)) present in the heterozygous state that was not identified in either unaffected parent. The proband of the second family was diagnosed several months after birth with thinning of the corpus callosum and PPCD. Whole genome copy number variant analysis revealed a 1.79 Mb duplication of 17q12 in the proband and her father and brother, neither of whom had PPCD. ZEB1 sequencing identified a novel deletion (c.1913-1914delCA; p.(Ser638Cysfs∗5)) present in the heterozygous state, which was also identified in the proband's affected mother. Thus, we report the first two cases of the association of PPCD with a developmental abnormality of the brain, in this case maldevelopment of the corpus callosum.
    Full-text · Article · Apr 2014
    • "SLC4A11 (NM_032034.3), and ZEB1 (NM_030751.5) genes were either taken from the literature [7,17,19] or were designed using the ExonPrimer and Primer3 [35] tools available online, and were checked for off-target sequence homology using the BLAT algorithm [36] all PCR amplifications used 30 ng input genomic DNA per primer pair. The suffixes " .2 "
    [Show abstract] [Hide abstract] ABSTRACT: Fuchs endothelial corneal dystrophy (FECD) is a genetically heterogeneous disorder that has been primarily studied in patients of European or Asian ancestry. Given the sparse literature on African Americans with FECD, we sought to characterize the genetic variation in three known FECD candidate genes in African American patients with FECD. Over an 8-year period, we enrolled 47 African American probands with FECD. All participants were clinically examined with slit-lamp biomicroscopy, and when corneal tissue specimens were available, histopathologic confirmation of the clinical diagnosis was obtained. The coding regions of known FECD susceptibility genes collagen, type VIII, alpha 2 (COL8A2); solute carrier family 4, sodium borate transporter, member 11 (SLC4A11); and zinc finger E-box binding homeobox 1 (ZEB1 [also known as TCF8]) were Sanger sequenced in the 47 probands using DNA isolated from blood samples. Twenty-two coding variants were detected across the COL8A2, SLC4A11, and ZEB1 genes; six were nonsynonymous variants. Three novel coding variants were detected: a synonymous variant each in COL8A2 and SLC4A11 and one nonsynonymous variant in ZEB1 (p.P559S), which is predicted to be benign and tolerated, thus making its physiologic consequence uncertain. Variation in the COL8A2, SLC4A11, and ZEB1 genes is present in only a small fraction of our African American cases and as such does not appear to significantly contribute to the genetic risk of FECD in African Americans. This observation is on par with findings from previous sequencing studies involving European or Asian ancestry patients with FECD.
    Full-text · Article · Dec 2013
    • "The pathogenesis of the disease is complex and not known completely and possibly involves interaction between genetic and environmental factors [19]. The early-onset FECD may be associated with mutations in the COL8A2 gene, encoding the í µí»¼2 subtype of collagen VIII [20], whereas late-onset FECD may be associated with mutations in the SLC4A11 gene, which encodes a zinc finger transcription factor and the TCF8 gene encoding sodium borate transporter [21] [22] [23]. "
    [Show abstract] [Hide abstract] ABSTRACT: Oxidative stress may play a role in the pathogenesis of keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Iron may promote the stress by the Fenton reaction, so its homeostasis should be strictly controlled. Transferrin is essential for iron homeostasis because it transports iron from plasma into cells. The malfunction of transferrin, which may be caused by variation in its gene (TF) variation, may contribute to oxidative stress and change KC and FECD risk. To verify this hypothesis we investigated the association between three polymorphisms of the TF gene, g.3296G>A (rs8177178), g.3481A>G (rs8177179), and c.-2G>A (rs1130459), and KC and FECD occurrence. Genotyping was performed in blood lymphocytes in 216 patients with KC, 130 patients with FECD and 228 controls by PCR-RFLP. We studied also the influence of other risk factors. The A/A genotype and the A allele of the g.3296G>A polymorphism were associated with KC occurrence, while the G allele was negatively correlated with it. We observed a decrease in KC occurrence associated with the A/G genotype of the g.3481A>G polymorphism. We did not find any association between the c.-2G>A polymorphism and KC. No association was found between all three polymorphisms and FECD occurrence.
    Full-text · Article · Nov 2013
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