A clinical and molecular-genetic analysis of Chinese patients with lattice corneal dystrophy and novel Thr538Pro mutation in the TGFBI (BIGH3) gene.

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c ? Indian Academy of Sciences
RESEARCH NOTE
A clinical and molecular-genetic analysis of Chinese patients
with lattice corneal dystrophy and novel Thr538Pro mutation
in the TGFBI (BIGH3) gene
PING YU1, YANGSHUN GU2, YUEHONG YANG1, XIAOYI YAN1, LILI CHEN2, ZHEN GE1, MING QI1,
JIANMIN SI3∗, and LEI GUO1,3∗
1Department of Medical Genetics, School of Medicine,2Department of Ophthalmology, The First Affiliated Hospital,
School of Medicine, and3Research Center for Modeling Human Diseases,
Zhejiang University, Hangzhou 310006, China
Lattice corneal dystrophy (LCD), characterized by the accu-
mulation of amyloid within the cornea, is one of the most
common inherited corneal diseases. Mutations in the human
transforming growth factor beta-induced gene (TGFBI), for-
merly designated BIGH3, have been shown to be associated
with different forms of LCD, including LCD type I (LCDI)
and type IIIA (LCDIIIA) (Munier et al. 2002). Here we
report the clinical and molecular-genetic analysis of LCD
in Chinese patients. Clinical evaluation showed presence
of corneal defects with variations in both age of onset and
severity of amyloid deposits in these patients.
analysis identified a novel A-to-C transversion at position
1612 (A1612C) in the TGFBI gene in a pedigree with LCDI.
The A1612C mutation is expected to result in a Thr538Pro
(T538P) substitution in TGFBI protein. Our study showed
that P501T mutation with its disequilibrium-linked polymor-
phism IVS10-3T→ C, previously identified only in Japanese
LCDIIIA patients (Tsujikawa et al. 2002), was also present
in Chinese LCDIIIA patients.
Sequence
Patients and methods
Subjects and ophthalmological examinations
As part of a genetic screening programme for vision impair-
ment, two members from one family (figure 1a) and three
sporadic cases with LCD were diagnosed at the Eye Clinic,
First Affiliated Hospital of Zhejiang University School of
Medicine. All cases were reexamined and ascertained by the
same ophthalmologists (Drs Yangshun Gu and Lili Chen),
and blood samples were collected. The corneas of some
∗For correspondence. Email: leiguo@yahoo.com (Lei Guo)
and sijm@163.com (Jianmin Si).
subjects were photographed with a slit-lamp camera. In-
formed consent was obtained from all subjects prior to their
participation in the study, in accordance with the regulations
of the Institutional Review Board and Ethics Committee of
the First Affiliated Hospital of Zhejiang University.
Molecular-genetic analysis
DNA samples were extracted from peripheral blood of the
five patients usingthe salting-outmethod describedbyMiller
(Miller et al. 1988). A group of 53 unrelated, healthy indi-
viduals were analysed as control. Based on the data of a
patient population analysis showing that there were four mu-
tational hotspot exons (exons 4, 11, 12 and 14) in the TGFBI
gene (Munier et al. 2002), we screened these exons using the
method of polymerase chain reaction – single strand confor-
mation polymorphism (PCR-SSCP). The primers used were
identical to those described in previous reports (Munier et
al. 1997; Korvatska et al. 1998). Thermal cycling was per-
formed on a GeneAmp PCR System 9700 with appropriate
conditions. PCR products were denatured and electrophore-
sis was performed in 8% nondenaturing polyacrylamide gels
with 6% glycerol or 5% sucrose in 1×TBE buffer at room
temperature. The gels were silver-stained. The PCR prod-
ucts with a mobility shift in gels were purified (Qiaquick
gel extraction kit, Qiagen) and sequenced on both strands by
using Mega BACE 1000 sequencer (Amersham Pharmacia
Biotech).
Results
Clinical manifestations
Clinical information of family A (figure 1b) and three spo-
radic cases with LCD is summarized in table 1.
Keywords. lattice corneal dystrophy; TGFBI (BIGH3); mutation; polymorphism.
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Ping Yu et al.
Molecular-genetic analysis
In family A, we found a shifted band on PCR-SSCP analy-
sis in the TGFBI gene exon 12 of the proband (III-1). Se-
quencing assay identified a novel mutation, ACA→CCA, at
codon 538 (figure 1c). The proband’s mother (II-2) showed
the same mutation (figure 1c). This mutation, confirmed by
sequencing both the sense and antisense strands, was never
presentin106chromosomes of 53unrelatedcontrol subjects.
Cases B and C were shown by PCR-SSCP and sequenc-
ing analysis to carry a common C417T/Arg124Cys mutation
in exon 4 of TGFBI gene. A Pro501Thr substitution, a previ-
ously reported common mutation in Japanese LCD patients,
was identified in exon 11 of TGFBI gene in case D (data not
shown).
We also identified four different single nucleotide poly-
morphisms (SNPs) in TGFBI gene.
(F540F) and 1416C/T (L472L), located in the coding regions
of the gene (figure 1d), are present in 16% and 13% of our
control subjects and of lower frequency than the reported
25% and 18% in an earlier study (Korvatska et al. 1998). The
other two SNPs, IVS10-3T→C and IVS12+23A→G, resid-
ing in introns 10 and 12, have frequencies of 3% and 6%
respectively. None of them cosegregates with the disease.
The SNPs 1620T/C
Discussion
In this study, we conducted a clinical and molecular-genetic
analysis in Chinese patients with LCD. Multiple forms of
LCD have been described (Munier et al. 2002), including
LCDI and LCDIIIA. Our subjects fall into two of them
based on their clinical features: LCDI (cases A, B and C)—
autosomal dominant, with early onset, characterized by thin
grayish, linear, branching deposits of amyloid material in
subepithelial and stromal layers of the cornea; and LCDIIIA
(case D)—autosomal dominant, with late onset, character-
ized by thick lattice lines extending across the cornea fre-
quently associated with superficial corneal erosions. While
cases A, B and C are categorized as LCDI, they varied in age
of onset and clinical manifestations. The patients of family A
and case C developed the symptoms much later than case B
and patients of previous reports. This suggests that not only
do different mutations in the TGFBI gene differently alter
its function, but also genetic modifiers and/or environmental
factors might affect clinical consequence of this disease.
We report here a novel mutation A1612C in the TGFBI
gene in a Chinese family with LCDI. The mutation, which is
a first-base alteration of codon 538 and results in threonine to
proline substitution, is different from the previously reported
mutation C1613G, which causes threonine to arginine sub-
stitution and LCDI/IIIA clinically (Munier et al. 2002). Two
different pathogenic mutations occur at the same codon, indi-
catingthatthreonine 538might be important tothe biological
function of the protein.
Two sporadic cases with LCDI have been associated with
the R124C mutation in our study. R124C alteration abol-
ishes a putative phosphorylation site, which might change
the tertiary structure of kerato-epithelin and lead to amyloid
conversion (Munier et al. 1997). R124C mutation has been
Figure 1. Identification of a new mutation in a family with LCDI. (a) Pedigree of family A with LCDI. Solid symbols indicate the affected
members, and open symbols indicate the unaffected members. The arrow indicates the proband. Asterisks indicate the members who were
examined. A diagonal line through the symbol indicates deceased members. (b) Slit-lamp photograph of the left eye of proband A-III-1
showing lattice-like lines in the central cornea. (c) Left: nucleotide sequence of TGFBI gene exon 12 of the proband A-III-1 (antisense
strand). Right: nucleotide sequence of TGFBI gene exon 12 of proband’s mother A-II-2 (antisense strand). Top lane: normal sequence;
bottom lane: mutated sequence. Arrows indicate an A-to-C transversion at the first nucleotide position in codon 538, which results in
a Thr538Pro substitution. (d) Domains of the TGFBI protein and the positions of the mutations and the SNPs. fasc1 to fasc4: internal
domains homologous to the Drosophila melanogaster fasciclin gene.
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Molecular study of TGFBI gene in Chinese LCD patients
Table 1. Summary of clinical manifestations in the subjects.
Case no.
A-III-1,
proband
Age
25
Sex
F
Age of onset
19
Vision
LE:20/100
RE: 20/70
Symptoms
Low vision and
photophobia in
both eyes for 5
years
Slit-lamp
examination
Numerous fine,
refractile lattice
lines and dots in
the anterior stroma
of the central
cornea in both eyes
Grafts are
transparent in both
eyes
Clinical
diagnosis
LCDI
A-II-2,
mother
55F 35 LE:N/A
RE:N/A
Low vision and
ocular pain in both
eyes for 25 years;
underwent
penetrating
keratoplasty in
right eye at age 47
years and in left
eye at age 49 years
Low vision, ocular
pain and
photophobia in
both eyes for 20
years
LCDI
B 26F5 LE:20/60
RE:20/50
Irregularity of the
epithelial surface
with fine branching
lattice lines in the
subepithelial and
anterior stroma in
both eyes
Irregularity of the
epithelial surface
with fine branching
lattice line in the
subepithelial and
anterior stroma in
both eyes
Thick, ropy
branching lattice
lines throughout
the cornea in both
eyes
LCDI
C22F19 LE:20/50
RE:20/60
Low vision and
photophobia in
both eyes for 3
years
LCDI
D 71M 68 LE:20/80
RE:20/100
Gradual reduction
in visual acuity in
both eyes for 3
years
LCDIIIA
found to be the cause of LCDI in a wide spectrum of eth-
nic groups (Munier et al. 1997). Among Japanese LCDI pa-
tients, 23% had the R124C mutation (Yoshida et al. 2002).
Further study is required to determine whether this site is a
mutation hotspot in Chinese LCD patients.
The Pro501Thr mutation, associated with LCDIIIA, was
reported previously only in western Japan, with an inci-
dence of about 6% (Yamamoto et al. 1998; Kawasaki et al.
1999) and reduced penetrance (Stewart et al. 1999; Ha et
al. 2002). Tsujikawa speculated that the Pro501Thr may be
a founder mutation rather than a hotspot since there was a
disequilibrium-linked polymorphism, IVS10-3T→C, in all
the Japanese patients with this mutation (Tsujikawa et al.
2002). Interestingly, our Chinese patient who carries the
Pro501Thr mutation also carries IVS10-3T→C. Thus, this
mutation might be a common ancestral mutation shared by
the two ethnic groups.
The TGFBI gene codes for the protein BIGH3, which
is highly conserved between species. This protein contains
an N-terminal secretory signal peptide, four 140-amino-acid
repeats (fasc1–4, figure 1d) with internal homology, and an
arg–gly–asp (RGD) motif at the C terminus. More than 25
mutations in the TGFBI gene have been identified in hun-
dreds of affected individuals of different ethnic groups (Endo
et al. 1999; Mashima et al. 2000; Kim et al. 2001; Chau et
al. 2003). Overexpression of mutated TGFBI gene in hu-
man corneal epithelial cells induces apoptosis (Morand et
al. 2003), indicating apoptosis might be a key element in
the pathophysiology of TGFBI-related corneal dystrophies.
However, the molecular mechanism underlying the disease
remains elusive and should be further studied.
Acknowledgements
This study was supported by Grant 02096 from Analysis and Mea-
surement Foundation of Zhejiang Province and Grant 30200097
from National Nature Science Foundation of China. Authors thank
the patients for their participation in this study.
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Ping Yu et al.
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