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Original Studies |
Department of Laboratory Medicine and Clinical Genetics Unit (S.K.), Kyoto 606-8507 Japan; and the Department of Pediatrics, University of Manitoba (S.B., H.J.D.), Winnipeg, Manitoba, Canada R3A 1R9
Address all correspondence and requests for reprints to: Dr. Shinji Kosugi, Department of Laboratory Medicine, Kyoto University School of Medicine, 54 Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: kosugi{at}kuhp.kyoto-u.ac.jp
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Abstract |
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Arg; GGAAGA), in 10 patients examined in the
present study. All of the parents tested were heterozygous for the
mutation, suggesting that the patients were homozygous. The mutation
was located in the 10th transmembrane helix. Expression experiments by
transfection of the mutant NIS complimentary DNA into COS-7 cells
showed no perchlorate-sensitive iodide uptake, confirming that the
mutation is the direct cause of the iodide transport defect in these
patients. A patient who showed an intermediate saliva/serum technetium
ratio (14.0; normal, 
20) and was considered to have a partial or
less severe defect in the previous report (IX-24) did not have a NIS
gene mutation. It is now possible to use gene diagnostics of this
unique NIS mutation to identify patients with congenital hypothyroidism
due to an iodide transport defect in this family and to determine the
carrier state of potential parents for genetic counseling and arranging
rapid and early diagnosis of their infants. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We (17, 20) and others (15, 21) have identified a homozygous, missense,
and loss of function mutation of the NIS gene, T354P
(Thr354Pro), in nine Japanese patients. We also found
other missense and loss of function mutations of NIS [G543E
(Gly543Glu) homozygous mutation and a compound
heterozygous mutation of T354P/G93R (Gly93Arg)]
in three Japanese patients with ITD (19). In addition, Pohlenz
et al. reported a homozygous nonsense mutation C272X
(Cys272stop) in a Brazilian kindred (16) and a compound
heterozygous mutation of Q267E (Gln267Glu)/deletion (67
bp) in a patient of Mexican origin (18). Thus, NIS mutations have
been detected in only two patients with ITD outside Japan; the clinical
features of such ITD patients with NIS mutation have not been well
characterized.
We reported nine patients with congenital hypothyroidism in a Hutterite family living in central Canada (9). Five of these patients were confirmed to have complete ITD. Since this previous report (9), we have found nine additional infants with congenital hypothyroidism in the family by neonatal screening. In the present study, we identified a novel, missense, and loss of function germline mutation of the NIS gene in the patients from this largest ITD family reported to date. We also describe here the clinical features of the patients and discuss the genotype-phenotype relationship and clinical significance of gene diagnostics in this family.
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Subjects and Methods |
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The pedigree of the Hutterite family with ITD is shown in Fig. 1
. We constructed the pedigree by direct
interviews in the present study, and it includes a part of the pedigree
from the previous report (9). The generation of the patients is
designated VI, which corresponded to generation IX in the previous
pedigree (9). Individuals with congenital hypothyroidism are shown by
closed symbols. The geographic and cultural isolation of
this religious group living in rural colonies has led to a remarkable
degree of consanguinity. Most of the patients were born from a
consanguineous marriage. The clinical features and biochemical results
of neonatal screening of the patients in this family are summarized in
Table 1. Some of the data from the seven
patients described in the previous report (9) are duplicated for ease
of comparison and comprehension.
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Genomic DNA was extracted as previously described (17, 20) from peripheral blood cells of the patients and their family members with their informed consents. Each exon was amplified by PCR with a pair of primers derived from the flanking introns. Exons 3 and 4, 6 and 7, 9 and 10, and 11 and 12 were coamplified with intervening introns. The locations of all intronic primers were at least 22 nucleotides distant from the exon(s) to be amplified. Nucleotide sequences of all exons, from nucleotide -37 to +1952 in NIS complimentary DNA (cDNA) covering the full-length coding region, and those of all exon-intron boundaries containing at least 15 nucleotides in introns (GenBank accession no. AF049198-AF049220), were determined in both orientations by direct sequencing with a GeneScan DNA sequencer 373A (Perkin Elmer Corp., Foster City, CA) (19).
Detection of the G395R (Gly395Arg;
GGAAGA) mutation by AlwI digestion
Exons 9 and 10 were coamplified with the intervening intron 9
using primers derived from intron 8 (5'-GATGGTGTGGACGGTCTCTCCAT-3') and
intron 10 (5'-AAGGTGCCCCCACCTCTCAGGA-3'). In the wild-type (WT) allele,
there is an AlwI site (GGATCN4
) in exon 10,
which produces fragments of 267 and 103 bp. When the G395R mutation
(GGATCAGATC) exists, the 370-bp PCR product remains undigested.
Construction of expression vectors, transfection, and iodide uptake assay
WT human NIS cDNA construct was obtained by TA cloning of the
full-length (nucleotide -59 to +1975) human NIS cDNA in the pCR3.1
vector (Invitrogen, San Diego, CA) under control of the
cytomegalovirus promoter (17, 19). Mutant construct G395R was generated
by site-directed mutagenesis. COS-7 cells were transfected with 25 µg
mutant or WT NIS DNA or control vector DNA (pCR3-CAT,
Invitrogen) by electroporation. To mimic the family
members who had heterozygous G395R mutation, 12.5 µg each of two
kinds of constructs were used to transfect COS-7 cells. To monitor
transfection efficiencies, 0.1 µg pSVGH was cotransfected with mutant
or WT NIS plasmid cDNA or control vector. Cells were aliquoted into
24-well plates (
105 cells/well). Forty-eight hours after
transfection, the medium was taken for RIA of human GH concentration,
and assays of iodide uptake were performed as previously described (17, 19). Iodide uptake was determined by incubating cells with 500 µL
HBSS incubation buffer (HBSS containing 0.5% BSA and 10 mmol/L
HEPES-NaOH, pH 7.4) with about 0.1 µCi carrier-free
Na125I and 10 µmol/L NaI to give a specific activity of
approximately 20 mCi/mmol at 37 C for 2 min. After finishing the
incubation, cells were washed twice on wet ice with ice-cold 2 mL HBSS
incubation buffer as quickly as possible (<15 s). Cells were
solubilized with 1 mL 0.1 mol/L NaOH, 0.1% (wt/v) SDS, and 2%
Na2CO3 and subjected to protein concentration
determination by the Bradford method (22) using BSA as standard and to
counting of radioactivity by a 
-counter. Some wells of cells were
trypsinized for cell number counting. Data for iodide uptake are
expressed as picomoles per min/mg cell protein.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Previously, we described nine children with congenital
hypothyroidism in a Hutterite family with extensive consanguinity
living in central Canada (9). Five of the patients who showed
negligible 123I uptake and low saliva/serum iodide and
technetium ratios were diagnosed as having definite and complete ITD
(Table 1
) (9).
Since then, we have identified 9 additional children with congenital
hypothyroidism by neonatal screening in this family, bringing the total
number of patients to 18. The pedigree shown in Fig. 1 indicates only
relationships that were confirmed by direct interviews in the present
study. Upper generations that were not directly confirmed this time but
were described in the figure of the 1985 paper (9) were eliminated from
Fig. 1. The 2 patients included in the previous pedigree (9) who were
not available for further characterization even in the original study
(VIII-27 and VIII-29) were excluded from Fig. 1.
No patients showed clinical hypothyroidism at the time of diagnosis. T4 treatment for most of patients was started in the neonatal period and successfully continued; we had no chance to administrate large amounts of iodide to these patients.
NIS mutation
Initially, we sequenced all exons and flanking introns of NIS
genomic DNA in patients VI-27 and VI-31 who had been characterized most
extensively and were diagnosed definitively as having ITD. In both of
these patients, a novel and homozygous nucleotide change of GA at
nucleotide +1530 in exon 10 resulting in a change of Gly395
to Arg (GGAAGA) in the 10th transmembrane domain was identified
(Fig. 2). No other nucleotide changes
were found in the coding region or exon-intron boundaries. This
mutation resulted in disappearance of the AlwI site
(GGATCN4) between nucleotides +1538 and +1539. In the WT
allele, Alw I digestion produced fragments of 267 and 103 bp
from the PCR product containing exons 9 and 10. When the G395R mutation
(GGATCAGATC) was present, the 370-bp PCR product remained
undigested. AlwI digestion confirmed the G395R mutation and
revealed the presence of the G395R homozygous mutation in the 10
patients (Fig. 3): VI-10, VI-16, VI-27,
VI-28, VI-29, VI-31, VI-32, VI-53, VI-54 and VI-56. The parents of
patients VI-10 (V-6 and V-30); VI-28, VI-29, and VI-31 (V-14 and
IV-24); VI-32 (V-11 and V-31); and VI-53 and VI-54 (IV-23 and V-53)
were heterozygous, confirming that these patients were homozygous for
G395R. Mothers of patients VI-16 (V-8), VI-27 (V-13), and VI-56 (V-58)
were heterozygous for G395R, but no blood samples were obtained from
their fathers.
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Brothers and sisters of patients with homozygous G395R mutation, i.e. the brother of patients VI-53 and VI-54 (VI-51), the brother of patient VI-32 (VI-33), the sister of patient VI-10 (VI-8), and the sister of patient VI-59 (VI-58), were shown to be heterozygous for the G395R mutation. None of the individuals heterozygous for the G395R mutation had thyroid disorders.
No blood samples were obtained from the families of patients VI-36, VI-38, and VI-39 or from patient VI-57, who was very young.
Patient VI-40, who was described as IX-24 in the original report (9),
did not have the G395R mutation. Full-length sequencing was performed,
but no NIS mutation was found in the coding region or exon-intron
boundaries. The saliva/serum
99mTcO4- ratio (normal, 20 or
higher) of this patient was 14.0, which was much higher than those of
the other patients with ratios lower than 1.0 (Table 1). Further, he
showed a small amount of early uptake of99mTcO4- by the salivary glands, which
disappeared by 30 min. Therefore, in the original study, he was
considered to have a less severe or partial defect (9).
Including the finding of the NIS mutation in the 10 patients in this family, the total number of ITD patients with identified NIS mutation(s) worldwide has become 24. NIS mutations of the 20 cases of the 24 were identified in our laboratory.
Expression experiments
Expression experiments by transfection of the mutant (G395R) NIS
cDNA into COS-7 cells showed no perchlorate-sensitive iodide uptake
(Fig. 4), confirming that the mutation is
the direct cause of the ITD in these patients. Cells cotransfected with
WT/G395R, mimicking the heterozygous state in unaffected family
members, had approximately half the level of iodide uptake activity as
that observed in cells transfected with 25 µg WT NIS DNA, but a level
similar to that in cells transfected with 12.5 µg each of WT NIS and
control vector, pCR3-CAT DNAs. These results suggested that the G395R
mutant NIS protein does not interfere with the function of WT NIS (no
dominant negative effect) similar to other NIS mutants, G93R, T354P,
and G543E, and iodide uptake activity in transfected cells is
correlated with the level of WT NIS expression. Cotransfection with
pSVGH and measurement of the GH concentration in the culture
medium showed no differences in transfection efficiencies among
the transfectants.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Patients VI-16, VI-27, VI-28, VI-29, and VI-31, who only had alleles with the G395R mutation, in whom 123I thyroidal uptake, and 123I and 99mTcO4- saliva/serum ratios were very low, were definitively diagnosed as ITD. Patient VI-10, who had no 99mTcO4- uptake in the thyroid or salivary glands, and patients VI-32, VI-53, VI-54, and VI-56, who only had G395R mutant NIS alleles but who had never undergone thyroidal scanning or further clinical studies, were presumed to have ITD. Further, the congenitally hypothyroid patient VI-59, whose genotype was unknown but whose parents were heterozygous for G395R, was likely to have ITD caused by homozygous G395R NIS mutation.
The hypothyroidism in patient VI-40 without a NIS mutation was considered to be due to a cause other than partial or less severe ITD. However, the limited availability of clinical data for the patient did not allow us to reach any definite conclusion.
It was notable that ITD patients in this family were predominantly
female; the male/female ratio of the patients shown in Fig. 1 was 4:12.
Further, of the 10 patients with homozygous G395R mutation, 9 were
female. In ITD patients with another NIS mutation(s), the ratio was 7:7
(15, 16, 17, 18, 19, 20, 21). Whether the female predominance in the family was due to
chance or to a specific cause(s) is unknown.
Previously, NIS was suggested to be composed of 12 transmembrane domains with both amino- and carboxyl-termini inside the plasma membrane (22, 23). However, by characterization of potential asparagine-linked glycosylation sites, NIS protein is now considered to have 13 transmembrane helices (24). The highly hydrophobic region comprising amino acid residues 389–410 located between the previously proposed transmembrane domains 9 and 10 forms a new transmembrane helix (24). Therefore, G395R involves a residue located in the 10th transmembrane domain of NIS.
Gly395 is conserved between human and rat NIS, and amino acids in the 10th transmembrane helix (residues 389–410) are identical in 21 of 22 between the two homologues (22, 23). This Gly is conserved in sodium/glucose cotransporter 1 from various species, but not in human sodium/myo-inositol cotransporter, rabbit sodium/nucleoside cotransporter, or pig sodium/neutral amino acid cotransporter (Swiss-Prot).
Four kinds of NIS missense mutations causing ITD, G93R, T354P, G395R,
and G543E, all of which were identified in our laboratory, are located
in the transmembrane domains. Three of these mutations involve a
conserved Gly residue that is a destabilizer in an 
-helix,
suggesting possible structural and/or functional importance. However,
we must await detailed three-dimensional characterization by
mutagenesis, modeling, and purification before discussing the roles of
the involved residues.
It is noteworthy that no ITD patients in this Hutterite family had or developed goiter, in contrast to many previously reported patients with ITD who had diffuse (sometimes huge) or nodular goiter. This might be a specific characteristics of the G395R mutation. A more likely explanation is the early diagnosis. Most of these patients were diagnosed by neonatal screening and were treated with T4 at 3–14 days of age. For the older patients, treatment was started at 1–4 months of age, much earlier than that in other patients with ITD. We speculate that T4 treatment from the early neonatal period may prevent the development not only of cretinism but also of goiter. There have been no other reports of patients with ITD diagnosed as having congenital hypothyroidism by neonatal screening and treated from the neonatal period. Neonatal screening for hypothyroidism was started in 1977 in Manitoba as the first provincewide screening program in Canada. The average age of treatment of patients with congenital hypothyroidism in Manitoba is 10 days.
The description by Fujiwara et al. (21) that ITD is usually found by neonatal screening as primary hypothyroidism seems incorrect. With the exception of ITD patients in this Hutterite family, few reported ITD patients have undergone neonatal screening for cretinism. Most were diagnosed as having transient hyperthyrotropinemia (case b2 in Ref. 19) or as normal (case 6 in Ref. 20 and case b1 in Ref. 19). Although only case 1 in Ref. 21 among the ITD cases reported by Fujiwara et al. showed high TSH value by neonatal screening, her thyroid function normalized at 4 weeks of age, and T4 treatment was started at 3 months of age. The reason why Japanese ITD patients do not show very high TSH levels in the neonatal period may be because of high iodide intake.
It is interesting that Fujiwara et al. (21) speculated that the thyroid-stimulating effect of TSH may be enhanced by low intrathyroidal iodide concentration. Extremely high TSH for a short period of time in early life might greatly enhance the initiation of somatic mutations or focal hyperplasia in multiple thyroid follicular cells and result in multinodular goiter at a young age.
It is now possible to use gene diagnostics rather than uptake of isotopes in the thyroid and salivary glands and saliva/serum iodide or technetium ratios to identify this unique NIS mutation in infants born with congenital hypothyroidism in this family. Gene diagnostics can also be used to determine the carrier state of potential parents for genetic counseling and to arrange rapid and early diagnosis by selective cord blood TSH screening and/or direct analysis of G395R mutation by AlwI digestion, which can be performed within 3 h.
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Acknowledgments |
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Footnotes |
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Received March 30, 1999.
Revised May 27, 1999.
Accepted May 27, 1999.
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References |
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