A Missense Mutation G2320R in the Thyroglobulin Gene Causes Non-goitrous Congenital Primary Hypothyroidism in the WIC-rdw Rat
Paul S. Kim1,
Ming Ding1,
Shekar Menon,
Cha-Gyun Jung,
Ji-Ming Cheng,
Tomomi Miyamoto,
Bailing Li,
Sen-ichi Furudate and
Takashi Agui
Division of Endocrinology (P.S.K., S.M., B.L.) Department of
Medicine University of Cincinnati and Veterans Affairs Medical
Center Cincinnati, Ohio 45267
Graduate Program in Cell
and Molecular Biology (P.S.K., S.M.) Department of Cell Biology
University of Cincinnati College of Medicine Cincinnati, Ohio
45267
Center for Experimental Animal Science (M.D.,
C.-G.J., J.-M.C., T.M., T.A.) Nagoya City University Medical
School Nagoya, Aichi 467-8601, Japan
Department of
Laboratory Animal Science (S.-i.F.) Kitasato University School of
Medicine Sagamihara, Kanagawa 228-8555, Japan
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ABSTRACT
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A convincing line of evidence is being developed
that the congenital nongoitrous hypothyroidism and dwarfism observed in
the WIC-rdw rat may indeed be caused by a primary defect in
thyroid hormonogenesis. In support of this hypothesis, several recent
reports have shown the presence of elevated molecular chaperone levels
in the WIC-rdw thyrocytes, the endoplasmic reticulum of
which was markedly dilated, suggesting a defect in intracellular
protein transport. Here the studies were undertaken to identify the
precise molecular defect in the WIC-rdw rat. First, the
genetic linkage analysis revealed that the rdw locus was on
rat chromosome 7 and was identical to the thyroglobulin
(Tg) gene locus. Moreover, the Tg protein level was reduced
in the WIC-rdw thyroid despite a similar level of the
Tg gene transcripts that were indistinguishable in their
size from the normal. Next, the complete sequencing of the
rdw and the normal rat Tg cDNAs revealed a single
nucleotide change, G6958C, resulting in a G2320R missense mutation in a
highly conserved region of the Tg molecule. Finally, transient
expression of the intact Tg cDNA containing the rdw
mutation in the COS-7 cells showed no detectable Tg in the secreted
media, indicating a severe defect in the export of the mutant Tg.
Together, our observations suggest that a missense mutation, G2320R, in
the Tg gene is responsible for the rdw mutation in the
WIC-rdw rat.
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INTRODUCTION
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A vast majority of inherited congenital hypothyroid goiter (CHG),
characterized by mental retardation and abnormal growth, is caused by a
deficiency in one of the components of hormonogenesis in the thyroid,
including thyroid peroxidase, sodium iodide symporter, or thyroglobulin
(Tg). In humans, the overall incidence of congenital hypothyroidism is
1:3,0004,000 newborns, caused either by thyroid dysgenesis
(
80%) or dyshormonogenesis (
20%). Although uncommon,
qualitative or quantitative defects of Tg are an established cause of
CHG (1). Although the precise molecular mechanism has not been well
established in most cases, at least in several reports, a common defect
appears to be the presence of misfolded mutant Tg that accumulates
inside the cell, unable to reach its final destination (2, 3). Normal
Tg must first be folded and assembled into its proper tertiary and
quaternary structure in the rough endoplasmic reticulum (ER) before it
can be exported along the distal secretory pathway, ultimately to an
extracellular space known as the colloid lumen, where it is iodinated
and stored. Thus, perturbations in the folding of nascent Tg can often
lead to defective intracellular transport of Tg (4). During the past,
the structural properties of the Tg have been well characterized (5): a
660-kDa glycoprotein that is secreted as a homodimer to serve as the
unique peptide backbone on which thyroid hormones are synthesized. As
the major secretory product of the thyrocytes, Tg typically accounts
for as much as 50% of total protein in the thyroid gland.
The WIC-rdw rat, established from a closed colony of
Wistar-Imamichi (WIC) rats as a spontaneous mutant exhibiting
congenital dwarfism (rdw), is inherited as an
autosomal recessive (6). Although the initial reports of reduced
circulating levels of both GH and PRL suggested hypopituitarism in the
WIC-rdw rat (7, 8, 9, 10), elevated TSH levels and the reduced
level of T3 and T4 pointed
toward a primary defect in thyroid hormone production (11). The latter
has been supported by several recent studies which showed the
restoration of not only their normal growth but also their normal serum
levels of all pituitary hormones after the administration of
T4 (8), feeding of extracts of the bovine thyroid
(S-i. Furudate, unpublished data), or normal thyroid transplantation
(S-i. Furudate, unpublished data). A recent morphological study of
WIC-rdw thyrocytes further revealed dilated ER and reduced
secretory granules as well as very low levels of Tg in the colloid
lumen (12). These and other observations have strongly suggested that
the dwarfism is attributable to a primary defect of the thyroid and not
of the pituitary. Moreover, it was recently reported (13) that the
molecular chaperones were markedly elevated in the WIC-rdw
rat thyroid, similar to the cog/cog mouse that was defective
in Tg export (4), thus exhibiting many features of an endoplasmic
reticulum storage disease [ERSD (3)]. Interestingly, in a dramatic
contrast to most human patients and animal models of CHG, histological
analysis revealed a surprisingly hypoplastic thyroid gland that was
smaller than the normal control despite elevated circulating levels of
TSH in the WIC-rdw rat (11).
In the present study, in identifying the gene responsible for the
observed phenotype, the rdw locus was mapped to the rat
chromosome (Chr) 7 and found to be identical to the Tg gene
locus, thus prompting our investigation to focus on the Tg
gene. Additional studies revealed that the Tg protein level was
reduced in the WIC-rdw thyroid, yet the transcripts of the
Tg gene were similar in size and quantity, suggesting a
possible defect in the Tg molecule. Consequently, a search
for the mutation of the Tg gene revealed a single nucleotide
(nt) substitution that cause a Gly to Arg change at a position that is
highly conserved in other species including the human, mouse, and
bovine. Here we provide solid evidence that a missense mutation in the
Tg gene is indeed responsible for the nongoitrous congenital
primary hypothyroidism in the WIC-rdw rat.
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RESULTS
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Chromosomal Mapping of the rdw and Tg
Gene Loci
As the first step in identifying the gene responsible for the
WIC-rdw phenotype, we started with the chromosomal mapping
of the rdw locus using 138 (BN x
WIC-rdw)F1 x WIC-rdw
backcrosses (BC). The rdw locus was mapped to the rat Chr 7,
7.6 centimorgans (cM) downstream of the microsatellite locus
D7Rat19 and 6.6 cM upstream of the D7Rat136 (Fig. 1
). The rat Tg gene locus has
already been assigned to Chr 7 using somatic cell hybrid analysis,
although its detailed location was unknown (14). Since it was reported
that the mutations in the Tg gene caused Tg deficiency in
several animal models, we continued to map the rat Tg gene
locus using the same BC genetic panel. Using previously elucidated
information on the exon-intron structure of the rat Tg gene
(15), Tg gene introns were sequenced, revealing a G to A
substitution at nt 764 in intron 2 of the WIC-rdw rat
Tg gene as compared with that of the BN rat. This nt
substitution caused loss of a recognition site for the restriction
enzyme Cac8I. When the PCR product of the Tg gene
intron 2 (1,050 bp) from the BN rat was digested with Cac8I,
five DNA fragments (394, 356, 245, 35, and 20 bp) were observed as
predicted by the sequence analysis of intron 2, which contained four
Cac8I recognition sites. In Fig. 2
, lane 2, only three bands (394, 356,
and 245 bp) were observed in the BN normal rat sample, since two short
fragments (20 and 35 bp) ran off the gel (not shown). However, in the
homozygous WIC-rdw rat samples, the band of 245 bp
(open arrow) was converted to a 280-bp band (245 + 35 bp;
closed arrow) due to the loss of one of the Cac8I
sites (Fig. 2
, lane 3). The genotype of the Tg
gene was further determined by the presence of the 245-bp and 280-bp
bands in all the BC samples (Fig. 2
, lanes 47). These mapping results
indicated that the Tg gene locus was identical to the
rdw locus (Fig. 1
). The full-length sequence of the rat
Tg gene intron 2 has been submitted to GenBank (accession
number, AF221623).

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Figure 1. Linkage Mapping of the rdw and
Tg Gene Loci in Rat Chr 7
The numbers on the left of the line
indicate genetic distance [centimorgans (cM)] between each locus
listed on the right.
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Figure 2. Determination of the Tg Gene
Genotypes in BC Rats
Genomic DNAs were amplified by PCR with intron 2-specific primers,
treated with the restriction enzyme Cac8I, and then
subjected to 6% PAGE. Closed and open
arrows indicate WIC-rdw (280 bp) and BN (245 bp)
alleles, respectively. Lane 1, Hae digests; lane 2, BN
rat; lane 3, WIC-rdw rat; lanes 4 and 5, representative
BC rats with homozygous WIC-rdw genotype; lanes 6 and 7,
representative BC rats with F1 genotype. The band sizes
(bp) are listed on the left.
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Examination of Tg Gene Expression in
the WIC-rdw Rat Thyroid
When compared with the normal control, the expression of the
Tg gene was modestly up-regulated in the WIC-rdw
rat thyroid. More importantly, as shown in Fig. 3
, the transcripts of the Tg
gene were normally detected in the WIC-rdw thyroid. The
similar size and abundance of the WIC-rdw rat Tg
mRNA (
8.5 kb) in comparison to the F344 wild-type (F344-wt) normal
control further excluded the possibility of a gross gene deletion, a
quantitative defect in transcription, or Tg mRNA
instability.

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Figure 3. Northern Blotting of the Tg Gene
Transcripts in the WIC-rdw and F344-wt Rats
mRNAs isolated from the WIC-rdw and F344-wt rat thyroid
homogenates were run on 1% agarose gel and analyzed by Northern blot
analysis using a Tg cDNA fragment containing the 5'-end
to detect the full-length Tg gene transcript ( 8.5
kb). Both the 28S and 18S RNAs (not shown) ran faster than the
Tg mRNA. ß-Actin mRNA levels were also measured on a
separate gel that was loaded with equal aliquots of the mRNA extracts.
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Next, the steady-state level of the Tg protein in the
WIC-rdw rat thyroid was examined by SDS-PAGE and Coomassie
staining (Fig. 4A
). The 330-kDa band
representing the intact Tg was significantly reduced in the
WIC-rdw rat. This band was verified to be the Tg protein by
loading the purified human and mouse Tg sample on the same gel (not
shown) and by Western blotting using antirat Tg antibody (Fig. 4B
). Of
note, several lower mol wt bands were significantly elevated in the
WIC-rdw rat thyroid, which likely represent GRP94 (Fig. 4A
,
p95) BiP/GRP78 (Fig. 4A
p75), and other ER resident proteins (not
shown) as reported previously (13). The elevated levels of these ER
molecular chaperones, which are essential in assisting in the folding
of many nascent polypeptides, in both the heterozygous and homozygous
rat thyroids (Fig. 4B
, lanes 2 and 3, respectively), further
suggested that the WIC-rdw rat appeared to exhibit an
ERSD-like feature previously described in the cog/cog mouse
(4) and human patients with CHG (16). Together, these observations were
consistent with the possibility that the rdw mutation could
be a point mutation in the Tg gene as in the
cog/cog mouse or several human families (2).
Complete Sequencing of the Tg cDNAs of Normal and
WIC-rdw Rats
First, incomplete Tg cDNAs were isolated from the two
cDNA libraries generated from the F344-wt normal and the
WIC-rdw rat thyroids by a plaque-hybridization clone
screening method using 967-bp 5'-end and 930-bp 3'-end rat
Tg cDNA fragments as probes. The remaining sequence was
obtained from the products of RT-PCR amplification using flanking
primer sets that were synthesized based on mouse and human
Tg cDNA sequence information (Table 1
). From these, the complete sequence of
the WIC-rdw rat Tg cDNA was determined (Fig. 5
) and compared with that of the F344-wt
rat (GenBank accession number, AF221622). Although the full-length
sequence information on the human, mouse, and bovine Tg
cDNAs are available (17, 18, 19), that of the normal rat Tg
cDNA has not yet been reported. Therefore, it was necessary to sequence
the normal (F344-wt) rat Tg cDNA in its entirety as well.
The rat Tg cDNA contained a 8,307-bp open reading frame
(ORF), which encoded a protein containing 2,768 amino acids, showing
92.5%, 77.7%, and 75.2% homology with the mouse, human, and bovine
Tg cDNA, respectively. The predicted primary structure of
the rat Tg showed 90.4%, 73.3%, and 71.0% homology with those of the
mouse, human, and bovine Tg, respectively (not shown). As shown in
Figs. 5
and 6
, there was a single nt
substitution, G6958C, that caused a single amino acid substitution,
G2320R, in the C-terminal region of the protein. The glycine at
position 2320 and the surrounding residues are highly conserved among
other species (Fig. 7
), suggesting that
the rdw mutation likely represents a missense mutation
associated with a structural and/or functional alteration of the Tg
polypeptide and not just a single nt polymorphism (SNP). Highly
homologous to the intact acetylcholinesterase (AChE), the C-terminal
fifth of the Tg protein, which contains the rdw mutation,
was previously hypothesized to contain domain(s) that may be
structurally important in the Tg dimerization (20). In support of this
hypothesis, G2320 (Fig. 7
, arrowhead) appeared to be
absolutely conserved not only in other Tgs and AChE but also in other
functionally unrelated proteins that share primary structural
similarities.

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Figure 5. The Complete Primary Structure of the Rat
Tg Deduced from Tg cDNA Sequences
The complete primary structures of rat Tg deduced from
Tg cDNA sequences were obtained for F344-wt normal and
WIC-rdw rat, which differed only at position 2,320. The
complete rat Tg cDNA encodes a total of 2,768 amino
acids. At the N terminus, a 20-residue signal sequence peptide
(underlined) is followed by a strictly conserved
sequence NIFEY- that contains the hormonogenic Tyr-5. Arg (R) at
position 2,320 found in the rdw Tg is shown
enlarged in bold.
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Figure 6. The Identification of the WIC-rdw
Mutation
The nt sequences surrounding the WIC-rdw mutation (nt
nos 69676951) show transversion 6,958G>C (arrow;
shown here in the antiparallel direction) in the WIC-rdw
Tg cDNA but not in the WIC-+/+ Tg cDNA.
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Figure 7. Comparison of the Residues Surrounding G2320R
Alignment of the WIC-rdw rat, F344 rat, mouse, bovine,
and human Tg aa sequences shows that this region is highly conserved.
The arrowhead indicates the G2320R mutation found in the
WIC-rdw rat and the positions of G2320 (in the normal
rat Tg sequence) that is strictly conserved in all species as well as
in AChE and other functionally unrelated proteins. Surrounding
conserved amino acids are shown in bold (allowing for
similar residues, i.e. aspartate for glutamate,
threonine for serine, and hydrophobic aa or methionine for alanine) and
underlined in the Wic-rdw rat.
Abbreviations: Wic-rdw, Tg from the
WIC-rdw homozygous rat; NL, neuroligin; CaE, rat
carboxylesterase ES-10; Bu ChE, butyryl cholinesterase; T AChE, Torpedo
acetylcholinesterase.
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Absence of the rdw Mutation in Other
Rat Inbred Stains
Made possible by the fact that the G6958C nt substitution in the
WIC-rdw rat Tg gene caused a loss of the
recognition site for the restriction enzyme, NIaIV, the
occurrence of this substitution in other rat inbred strains was
examined by digesting RT-PCR products (260 bp) containing this region
with NIaIV (Fig. 8
). In
contrast to the RT-PCR product from the WIC-rdw rat
Tg, which was the only one resistant to NIaIV,
those from other inbred strains including the WIC-+/+ rat were cleaved
into the two expected fragments of 117 and 143 bp (Fig. 8
, lanes 23
and 58). Consistent with its genotype, the WIC-rdw/+ rat
showed both undigested and digested bands (Fig. 8
, lane 9).

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Figure 8. Detection of a Missense Mutation at nt 6,958 in the
Tg Gene
The 260-bp Tg cDNA fragments (nt 6,815 7,075) containing the
location of the rdw mutation were generated using corresponding
flanking primers using thyroids removed from six different normal
stains of rat (BN, F344-wt, BBDR, WKY, WKAH, and WIC-(+/+), as well as
from the WIC-rdw homozygote and the WIC-rdw/+ heterozygote rats. The
presence of G6958C mutation renders the 260-bp cDNA resistant to
Nia; 260-bp RT-PCR products were digested with
Nia, subjected to 6% PAGE, and visualized by ethidium
bromide staining.
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Transient Expression of Full-Length Mouse Tg
cDNA Containing the rdw Mutation
Using site-directed mutagenesis, an intact normal mouse
Tg cDNA (20) was changed to contain the rdw
mutation. The COS-7 cells were then transiently transfected with each
of the Tg cDNAs that contained either the rdw or
the cog mutation and compared with the cells that were
transfected with the normal Tg cDNA and the untransfected
cells. After overnight chase in serum-free media, the cells were lysed
under denaturing conditions and analyzed by reducing SDS-PAGE. When the
cellular Tg contents were examined by immunoblotting using anti-Tg
antibodies, similar amounts of Tg bands were observed in all the
transfected cell lysates but not in the untransfected control (Fig. 9A
). In contrast, only the normal Tg was
found in the secreted media, suggesting that the rdw
mutation prevented the normal export of Tg (Fig. 9B
) by rendering the
protein incompetent for intracellular transport, very similar to the
cog Tg (Fig. 9
). As in other models of defective Tg
trafficking, the rdw Tg was retained inside the ER in both
primary WIC-rdw thyrocytes and COS-7 cells that were
transfected with the Tg cDNA containing the rdw
mutation (our unpublished data).

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Figure 9. Transient Expression of Tg cDNAs in
COS-7 Cells
COS-7 cells grown in serum containing media to 70% confluency were
transiently transfected with full-length normal, cog,
and rdw Tg cDNAs, which were generated by site-directed
mutagenesis. Forty eight hours after transfection, the cells were
chased in serum-free media for 20 h. Both the cell lysates (A) and
the secreted media (B) were analyzed by reducing SDS-PAGE followed by
Western blot using anti-Tg antibody.
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DISCUSSION
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The mutants exhibiting dwarfism, which was found in a
Wistar-Imamichi rat closed colony, inherited as an autosomal recessive
was named rdw (rat dwarf) and established as an inbred
strain, WIC-rdw. In this study, we showed that the
WIC-rdw phenotype was due to a missense mutation in the
Tg gene. Several lines of evidence suggested that this
mutation was truly responsible for the rdw phenotype. 1)
Recombination between the Tg gene and rdw locus
was not observed in 138 BC, since the rdw locus was
identical to the rat Tg gene locus. 2) In a direct
comparison, the only significant difference in the nt sequence between
the F344 normal vs. WIC-rdw rat Tg
cDNA ORF was the G6958C substitution. The predicted Gly
Arg
substitution occurred at aa position 2,320, which was strictly
conserved not only in other rat and mouse inbred strains but also in
other mammalian species including human and bovine. 3) The parental
normal rat strain, WIC-+/+, showed no substitution at codon 2,320, and
the WIC-+/rdw rats showed heterozygous genotype at codon
2,320. 4) Transient expression of the full-length mouse Tg
cDNA containing the rdw mutation, showing the intracellular
presence of the intact Tg which was unable to be secreted, provided the
confirmation that rdw mutation was indeed responsible for
the observed phenotype.
Northern blotting and SDS-PAGE analysis of the WIC-rdw rat
thyroid homogenates revealed that the transcripts of the mutated
Tg gene were not different from those of the wild type with
respect to their size and amount. In fact, the Tg mRNA level was
modestly elevated in the WIC-rdw thyrocytes, probably
reflecting the increased thyroid stimulating effect of the elevated
circulating TSH level, yet the amount of the 330-kDa Tg protein was
reduced in the WIC-rdw thyroid. Several likely explanations
merit consideration. For one, it may be that the missense substitution
from a smaller neutral aa, glycine, to a larger basically charged aa,
arginine, within a highly conserved domain, may have caused a
substantial conformational change in the C-terminal region of Tg
protein. As for most other disease-causing mutant proteins, such change
often leads to structural instability that renders the protein highly
susceptible to aggregation and/or proteolysis. This was in part
supported by the experimental observation that the rdw Tg
was quite prone to proteolysis when the thyroid follicles were lysed
under nondenaturing conditions, even in the presence of a full battery
of lysosomal protease inhibitors (our unpublished data). On the
other hand, when the thyroid follicles were lysed in denaturing and
reducing buffer at boiling temperature, one of the most potent
conditions for inhibiting lysosomal protease activities, a greater
amount of the rdw Tg was recovered after SDS-PAGE. To verify
this hypothesis, further experiments utilizing limited proteolysis will
be helpful.
Another plausible explanation for the reduced level of Tg protein in
the WIC-rdw thyroid is the inhibition of the translation
initiation by the presence of the malfolded proteins in the ER, as a
part of the unfolded protein response (UPR). In all eukaryotes, the
accumulating malfolded or misfolded proteins in the ER are increasingly
bound by several molecular chaperones including BiP/GRP78.
Consequently, as the available level of free or unbound BiP/GRP78
falls, a transmembrane kinase known as IRE-1p/ERN-1p is activated,
triggering the ER-UPR pathway that results in the transcriptional
induction of multiple ER chaperone genes (21). These molecular
chaperones then act to reduce the potential harm posed by the misfolded
mutant proteins that are prone to aggregation (3). At the same time,
the ER-UPR pathway activates another ER resident transmembrane protein
kinase or PERK/PEK, which phosphorylates the eukaryotic translation
initiation factor or eIF2
that eventually leads to the attenuation
of translation (22). Together, the elevated levels of ER chaperones and
the reduction in the continued synthesis of the misfolded mutant
proteins serve to minimize the toxic accumulation of potentially
harmful aggregates of misfolded proteins, thereby enhancing the
probability of cell survival.
Several mutations in the human (2, 23, 24, 25, 26, 27), mouse (20), bovine (28),
and goat Tg genes (29) have been elucidated at the molecular
level. It is interesting to compare the rdw mutation with
the recently identified cog mutation in the congenital
goiter mouse (20). The cog Tg gene also contained a single
nt change that resulted in a missense mutation, L2263P, located near
the N-terminal side of the rdw mutation. Both mutations are
associated with the marked accumulation of the chaperone proteins,
observed in other ERSDs involving mutant secretory proteins (3).
Additional similarities include normal sizes and amounts of the
Tg gene transcripts in both mutant thyrocytes, full-length
Tg proteins that exhibited increased susceptibility to proteolysis, as
well as the decreased synthesis and impairment of intracellular
transport of both mutants. Moreover, although not as striking as the
conservation of residues flanking the cog mutation, the
region surrounding G2320, especially on its C-terminal side, appears to
be also well conserved (Fig. 7
) not only among Tgs from different
species but also among other homologous proteins. Since the latter,
which include neuroligin, a novel neuronal cell surface protein
important in cell-cell interactions (30) and AChE, are functionally
unrelated to Tg, it may be that the homology stems from their
conformational similarities. In this case, substituting a positively
charged arginine for a smaller, hydrophobic G2320, which appears to be
absolutely conserved (Fig. 7
, arrowhead), may lead to
structural instability. Confirming such a possibility will require
additional studies.
Finally, despite many similarities between the rat and mouse models of
Tg deficiency, there is one important difference that stands out
between the two models: the size of the their thyroid glands. In the
cog/cog mouse (and in several human patients with CHG), the
constant TSH stimulation that occurs in primary hypothyroidism
leads to the development of massive goiter that eventually compensates
for the quantitative defect in hormonogenesis. It appears that a very
small fraction of the cog Tg, which has been shown to be a
temperature-sensitive mutant (4), is able to reach the distal secretory
pathway to form thyroid hormones. The absence of this compensatory
response in the WIC-rdw rat may in part explain the severity
of the dwarfism and infertility in the adult rat, compared with the
cog/cog mouse. All previously reported mutations in the
Tg gene in other species caused goitrogenesis, yet the
rdw mutation was associated with a hypoplastic thyroid gland
(11, 12). Although the reason for this phenomenon remains unknown, it
is intriguing to consider the possibility that the rdw
mutation may be toxic to the host thyrocytes. Hence, comparing the
intracellular fates of the cog and the rdw Tgs as
well as their interactions with the essential components of the ER-UPR
pathway may provide new insights into how mutant proteins exert their
toxic effects on the host cells. On the contrary, cytotoxic effect of
the rdw mutation would be difficult to reconcile with the
autosomal recessive nature of the rdw phenotype. Additional
experiments are clearly needed to determine the mechanism by which
WIC-rdw heterozygous rat thyrocytes expressing the mutant Tg
protein avoid the same fate observed in the homozygous rat. Along this
line,
1-antitrypsin (AAT) deficiency, which is generally regarded as
an autosomal recessive, has been observed as an autosomal dominant in
some patients as well as in the transgenic animal model (3, 31).
Although most of these patients suffer from juvenile pulmonary
emphysema, only the patients with a specific mutation in the
AAT gene known as the Z-variant are additionally at risk for
hepatocellular damage leading to liver cirrhosis. Thus, the
susceptibility of each mutant to intracellular degradation may hold the
key in determining the potential cellular toxicity of the various
mutant proteins.
In conclusion, we have shown that the G2320R missense mutation is
indeed responsible for the congenital hypothyroidism and dwarfism in
the WIC-rdw rat, which will be useful for future
investigation of the relationship of Tg protein structure with its
stability, transport, and glycosylation mechanisms.
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MATERIALS AND METHODS
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Rat Crosses and the Mapping of the rdw Locus
Backcrosses (BC) were produced by crossing 138 (BN x
WIC-rdw)F1 x WIC-rdw rats.
Male WIC-rdw rats transplanted with normal thyroids were
used for crossing, since WIC-rdw rats are infertile. The
rdw phenotypes were identified based on body weights at 8
weeks of age; at this time, the animals were killed and their thyroids
and livers were quickly removed for subsequent experiments. DNAs were
extracted from the liver and pooled into two groups, +/rdw
and rdw/rdw genotypes. PCR was performed first with 60
microsatellite marker primers (Research Genetics, Inc.,
Huntsville, AL), which had been selected as distributed in all
autosomal chromosomes and showed polymorphism between BN and
WIC-rdw rats. Since some microsatellite markers localized in
Chr 7 showed linkage with the rdw genotype, other
microsatellite markers localized in Chr 7 were further examined.
Linkage analysis was performed using Map Manager v2.6.5.
Mapping of the Rat Tg Gene Locus
The Tg gene intron 2 was amplified by PCR with
primers 5'-AGCAGGATGAATATGTTCCA-3' and 5'-ATCCACACACCAGCAAGATT-3'.
Amplified DNA fragments that were digested with 0.25 U of
Cac8I (New England Biolabs, Inc., Beverly, MA)
were analyzed in 6% polyacrylamide gels containing 90
mM Tris-borate and 2 mM
EDTA. DNA bands were visualized by staining with 0.5 µg/ml ethidium
bromide solution. Genotypes of the Tg gene were determined
by the presence or absence of 280-bp and 245-bp bands (see Fig. 2
).
Linkage analysis was performed using Map Manager v2.6.5.
Detection of the Tg Gene Transcripts
The Tg gene transcripts were detected by Northern
blotting. Poly(A)+ RNAs were purified from total
RNAs of F344-wt and WIC-rdw rat thyroids using oligo(dT)
cellulose type 7 (Amersham Pharmacia Biotech,
Buckinghamshire, UK). Aliquots of 2 µg of
poly(A)+ RNA were denatured in 50% formamide,
2.2 M formaldehyde at 65 C for 10 min,
electrophoresed in 1% agarose gels containing 2.2
M formaldehyde, and transferred onto
Hybond-N+ membranes (Amersham Pharmacia Biotech). The poly(A)+ RNAs were
immobilized on the membranes by heating at 80 C for 2 h.
Prehybridization and hybridization of the membranes were performed in
buffer containing 50% formamide, 5 x standard saline citrate
solution (SSC), 5 x Denhardts solution, and 0.5% SDS at 45 C
for 16 h. The 5'-end of the Tg cDNA fragment was
amplified by RT-PCR and used as a probe. The probe was labeled with
-32P dCTP using a Rediprime Kit (Amersham Pharmacia Biotech). Membranes were washed in buffer containing
0.1 x SSC and 0.1% SDS and exposed to X-AR film
(Kodak, Rochester, NY) at -70 C for 24 h.
Detection of the Tg Protein
Freshly removed thyroids were homogenized under denaturing
condition in solution containing 4% SDS, 2% mercaptoethanol, and 10
mM Tris, pH 6.8, and boiled for 5 min. Different aliquots
of thyroid homogenates (
50 mg/ml protein) were analyzed by reducing
4 or 4.5% SDS-PAGE. Protein bands were visualized by staining with
Coomassie brilliant blue R-250. Parallel samples were also subjected to
SDS-PAGE and transferred to nitrocellulose before examination for
intact Tg by immunoblotting with antirat Tg and antimouse Tg antibodies
(4, 20) and secondary antibody conjugated with horseradish peroxidase.
ECL chemiluminescence method (Amersham Pharmacia Biotech,
Arlington Heights, IL) was used for the band detection.
Determination of the Tg cDNA Sequence
The sequences of 5'- and 3'-ends of the Tg cDNA were
determined by sequencing the cDNA clones obtained from the cDNA
libraries of the F344 and WIC-rdw rat thyroids. The two cDNA
libraries were generated using a ZAP-cDNA Gigapack Gold Cloning Kit
(Stratagene, La Jolla, CA) according to the
manufacturers instruction. The clones were screened by the
plaque-hybridization method with probes of 5'- and 3'-end fragments of
the rat Tg cDNA, of 967 and 930 bp in size, respectively.
The rest of the Tg cDNA sequence was determined by
sequencing TA clones containing the rat Tg cDNA fragments,
which had been amplified by RT-PCR with the primers shown in Table 1
.
Primers were synthesized according to the sequences of the murine and
human Tg cDNAs. TA cloning was performed using a TA Cloning
Kit (Invitrogen, Carlsbad, CA) in accordance with the
manufacturers instructions. Sequencing was performed with a
DSQ-1000 automatic sequencer (Shimazu, Kyoto, Japan) using a
primer cycle sequencing kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturers
instructions.
Detection of the rdw Mutation by
Restriction Enzyme Digestion
RT-PCR was performed with total RNAs extracted from thyroids as
templates and sense (5'-CAACACCTCCTCAAATCAGT-3', nt 6,8156,835) and
antisense (5'-GTCCAGTAGCCCCCAGTTGC-3', nt 7,0557,075) primers. PCR
products were digested by incubation with 1 U of NiaIV
(New England Biolabs, Inc., Beverly, MA) at 37 C for
16 h and electrophoresed in 6% polyacrylamide gels containing 90
mM Tris-borate and 2 mM
EDTA. DNA bands were visualized by staining with 0.5 µg/ml ethidium
bromide solution.
Site-Directed Mutagenesis
QuickChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA) was used to make the G to C
point mutation in the full-length normal mouse Tg cDNA (20)
in pBK-CMV vector (Stratagene) according to the
manufacturers instruction. The two primers T15'-GCT GAC CAT TGA TCG
CTC CAT CCT GGC-3' and T25'-GCC AGG ATG GAG CGA TCA ATG GTC AGC-3',
both containing the desired mutation and each complementary to the
opposite strands of the vector, were used in a PCR reaction. Four
Tg cDNAs were isolated and sequenced to determine the
presence of the point mutation. The presence of the rdw
mutation, G6958C, in the new Tg cDNA was confirmed by a
second sequencing.
Transient Transfections of COS-7 Cells
COS-7 cell were grown in DMEM containing 10% FBS to
approximately 7080% confluency before transfection with 1 µg of
each plasmid identified as pBK-CMV-normal Tg,
pBK-CMV-cog Tg, or pBK-CMV-rdw Tg using
Lipofectamine reagent (Life Technologies, Inc.) in
serum-free DMEM, according to the instructions. After 5 h
incubation, the transfection mixture was washed and replaced with fresh
DMEM containing 10% FBS for an additional 48 h. The cells were
then chased overnight in serum-free DMEM and lysed in denaturing buffer
containing 4% SDS. The secreted medium collected during chase was
precipitated with 10% TCA on ice for 1 h before centrifugation at
14,000 x g for 10 min at 4 C. After washing with 100%
ethanol, the obtained pellet was resuspended in denaturing cell lysis
buffer. Both the cell lysates and the secreted media were subjected to
reducing 4% SDS-PAGE before the Western blot analysis using anti-Tg
antibody.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Y. Fujii and H. Iwase, Department of the Second
Surgery, Nagoya City University Medical School, for the human thyroid
tissue and Ms. M. Yasuda, Nagoya City University Medical School, for
assistance with genetic linkage analysis of the rdw.
Parts of this work were supported by NIH Grant DK-52076 (P.S.K.) and
Veterans Affairs Merit Award (P.S.K.).
 |
FOOTNOTES
|
---|
Address requests for reprints to: T. Agui, Center for Experimental Animal Science, Nagoya City University Medical School, Mizuho-ku, Nagoya, Acihi 467-8601, Japan. E-mail: t.agui{at}med.nagoya-cu.ac.jp
1 These authors contributed equally. 
Received for publication July 21, 2000.
Revision received September 12, 2000.
Accepted for publication September 13, 2000.
 |
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