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Original Studies |
Third Medical Department, University of Leipzig (S.N., F.A., A.R., R.P.), D-04103 Leipzig; the Department of Pediatrics, University of Leipzig (H.W.), D-04317 Leipzig; and the Microsatellite Center, Max Delbrück Center for Molecular Medicine (M.J., A.R.), D-13125 Berlin, Germany
Address all correspondence and requests for reprints to: Dr. Ralf Paschke, Third Medical Department, University of Leipzig, Ph. Rosenthal Strasse 27, 04103 Leipzig, Germany. E-mail: pasr{at}server3.medizin.uni-leipzig.de
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In hypothyroid goiters several molecular defects in the thyroglobulin (TG) and thyroperoxidase (TPO) genes have been identified. The extent of TG messenger ribonucleic acid (mRNA) expression induced by the different mutations was related to the degree of hypothyroidism (7, 8, 9, 10, 11). Recently, in patients whose thyroid function ranged from mildly hypothyroid to euthyroid, two different missense mutations in the TG gene have been detected, causing an abnormal three-dimensional structure of TG (12). In a previous study, abnormalities in the degree of glycosylation and the iodine content of TG were described in patients with nonendemic simple goiter (13). Some of these cases were associated with a point mutation in exon 10 of the TG gene (14). The same mutation was only observed in 1 of 36 patients with endemic goiter (15). Mutations responsible for dyshormongenesis have also been described in the TPO gene. Abnormal TPO synthesis is caused by a heterogeneous spectrum of TPO mutations (16, 17, 18, 19, 20, 21, 22) that leads to a complete loss or a decrease in TPO activity. As the cloning and molecular characterization of the human Na+/I- symporter gene (NIS) (23) several defects in this gene have been detected in patients with different phenotypes of thyroid diseases (24- 28). In contrast to hypothyroidism, to date only 1 mutation in the NIS gene has been identified in a patient with euthyroid goiter (25).
The TSH receptor (TSHR), in accordance with its central role in thyroid function and growth, was excluded as a candidate gene in a family with nontoxic multinodular goiter (MNG) (29). However, MNG-1 on chromosome 14q outside the TSHR locus was established as a susceptibility locus in the same family. Mutations in the TSHR have been found in rare cases of congenital hypothyroidism (30, 31). However, the TSHR was excluded as a candidate gene for familial congenital hypothyroidism (32).
Recurrent or relapsing goiter occurring in families represents a phenotype strongly suggesting a genetic etiology. However, few studies have addressed the possible molecular defects. We, therefore, investigated a proposita with a euthyroid relapsing goiter and her family, in which several members had goiters. Sequencing results for the cDNAs and linkage analysis with microsatellites of the possible candidate genes TG, TPO, and NIS, indicate that they are not responsible for the euthyroid goiters in this family. However, we provide first confirmation for the recently identified MNG-1 locus (29) as a locus for nontoxic goiter.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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)Patient I-2 (74 yr old). A euthyroid goiter was identified at the age of 22 yr in this subject. The patient was treated with 100 µg T4 after thyroid resection. Thyroid hormone levels during T4 treatment were within the normal range (TSH, 0.44 mU/L). There were no thyroid autoantibodies detectable.
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Patient II-3 (48 yr old). At the age of 19 yr, euthyroid goiter was diagnosed. As treatment with L-T4 did not lead to volume reduction, the patient underwent subtotal thyroidectomy at the age of 22 yr. During postoperative thyroid hormone substitution the goiter recurred. She therefore underwent total thyroid resection. At present the patient is euthyroid while taking 100 µg T4/day.
Patient II-5 (37 yr). An enlarged thyroid gland was found at the age of 15 yr in this patient. Because of unsuccessful therapy with T4, the patient underwent resection of the thyroid gland at the age of 16 yr. During T4 substitution she developed goiter relapse. A second thyroidectomy was performed. At present she receives 100 µg T4/day. There is no evidence of thyroid enlargement.
Patient III-1 (18 yr old, proposita). A euthyroid goiter was identified at the age of 6 yr. T4 treatment was started with a dose of 100 µg T4/day. During the next year she presented with increased thyroid volume. She underwent subtotal resection of the thyroid gland followed by therapy with 100 µg T4 and 200 µg iodine/day. Five years later the subtotal resection was repeated because of recurrent goiter (14 mL). A second goiter relapse occurred at the age of 15 yr. At present she receives 125 µg T4/day.
Patients III-3 (23 yr old) and III-4 (28 yr old). Both individuals presented with mild thyroid enlargement and euthyroidism. Substitution with T4 and iodine was started with 100 µg T4 and 200 µg iodine/day.
Family members I-1, II-4, and III-2 showed normal thyroid hormone levels without signs of thyroid enlargement. Individuals I-3, III-5, and IV-1 could not be investigated.
RNA and DNA extraction
Thyroid tissue removed at surgery was immediately frozen and kept at -80 C. Total RNA was extracted using InViSorb RNA kit II (InViTek GmbH, Berlin, Germany), and mRNA was extracted using the mRNA DIRECT kit (DynAl, Oslo, Norway). RNA samples were stored in diethylpyrocarbonate-treated water at -20 C for later preparation of cDNA. Genomic DNA was extracted from peripheral blood lymphocytes using the QIAamp Blood Kit (Qiagen, Hilden, Germany).
Preparation of cDNA, RT-PCR, and direct DNA sequencing
The Ready To Go T-Primed First Strand Kit from Pharmacia Biotech (Uppsala, Sweden) was used to prepare the first strand of cDNA from thyroid tissue total RNA, according to the manufacturer’s instructions. A complete list of primers used for amplification of the entire TG, TPO, and NIS genes by PCR and for DNA sequencing will be provided by the authors upon request. The PCR reactions were performed in an MJ Research, Inc. thermocycler PTC 200 (Biozym, Oldendorf, Germany). Amplification of TG, TPO, and NIS cDNA fragments was performed in a 50-µL reaction mixture containing 0.5 µg cDNA as template, 10 µmol/L of each primer, 10 mmol/L of each deoxy-NTP, 1 U InViTAQ DNA polymerase (InViTek GmbH, Berlin, Germany), and the appropriate 10-fold concentrated incubation buffer with 1.5 mmol/L MgCl2. The PCR conditions were as follows: 30–35 cycles consisting of denaturation for 30 s at 94 C, primer annealing for 30 s at temperatures depending on the primer used, and extension for 30 s to 1 min at 72 C.
For direct sequencing, PCR fragments were purified with polyethylene glycol precipitation (13% polyethylene glycol 8000, 10 mmol/L MgCl2) at room temperature. Purified PCR fragments were sequenced using dRhodamine Terminator Cycle Sequencing chemistry (ABI). Sequencing reactions were analyzed on a Genetic Analyzer ABI 310 (ABI, Perkin Elmer Corp., Foster City, CA).
Amplification and sequencing of the TSHR gene
Genomic DNA extracted from blood lymphocytes was amplified by PCR. Exons 1–10 of the TSHR were amplified using the PCR primers previously published by De Roux et al. (33) and modified PCR conditions as described by Führer et al. (34). PCR products were purified and sequenced as described above.
Digoxygenin (DIG)-labeled RNA probes for Northern analysis
The probes pHTgM1, pHTgM3, and pHTgB2 (35, 36) mapping at positions 150-1539, 3185–5125, and 4874–5852 bp in the TG cDNA were used for Northern analysis. These probes were digested with the restriction enzyme PstI (MBI Fermentas, Vilnius, Lithuania), and the resulting 0.79-kb pHTgM1, 0.9-kb pHTgM3, and 0.96-kb pHTgB2 fragments were cloned in the PstI-digested vector pBSK+. For examination of TPO mRNA, a 1.02-kb PstI fragment from human TPO cDNA was used as probe (37). The integrity and orientation of the insert were confirmed by sequencing.
To produce DIG-labeled RNA probes, cloned DNA was transcribed in vitro with T3 or SP6 RNA polymerases in the presence of DIG-UTP as described by the manufacturer (Roche Molecular Biochemicals, Mannheim, Germany). DIG-labeled RNA probes were used for hybridization of the Northern blots. The control probe was a 1.2-kb cDNA fragment encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Northern blot analysis
Ten micrograms of formaldehyde-denatured mRNA from goiter and control tissue (normal thyroid tissue surrounding a cold thyroid nodule) were analyzed by electrophoreses on a horizontal 1% agarose gel in 0.1 mol/L MOPS [3-(N-morpholino)propanesulfonic acid; 40 mmol/L sodium acetate and 5 mmol/L ethylenediamine tetraacetate buffer, pH 7.0] and transferred to a positively charged nylon membrane (Boehringer Mannheim, Mannheim, Germany) using 20 x SSC as previously described (38). The membrane was baked for 30 min at 120 C. Prehybridization and hybridization were carried out at 68 C in DIG Easy Hyb solution (Boehringer Mannheim). The detection of DIG-labeled nucleic acids was performed according to the protocol of the CDP-Star kit (Boehringer Mannheim). The membranes were exposed for 30 s to 10 min at room temperature to Hyperfilm ECL (Amersham Pharmacia Biotech, Aylesbury, UK).
Generation of RNA transcription templates for ribonuclease protection assay (RPA)
Total RNA isolated from normal thyroid tissue was used to generate single stranded cDNA (Amersham Pharmacia Biotech, Braunschweig, Germany), followed by amplification of a 568-bp NIS cDNA fragment. This fragment was cloned into pGEM-T vector (Promega Corp., Madison, WI). The resulting plasmid was digested with SmaI and SalI (MBI Fermentas), releasing a 160-bp fragment consisting of 130 bp of NIS cDNA and 30 bp of pGEM-T multiple cloning sequence at the 3'-end. The 5'-overhang generated by SalI was filled up by T4 DNA polymerase, and the product was then cloned into the EcoRV-site of pBluescript II SK vector (Stratagene, Heidelberg, Germany). The plasmid was digested with SpeI, and the resulting 130-bp NIS cDNA fragment of interest was subcloned into the SpeI site of the pBluescript-II SK vector. To terminate the in vitro transcription, this plasmid was digested with NotI (MBI Fermentas).
To obtain the 75-bp GAPDH probe, a pGEMGAPDH plasmid (pGEM-Vector containing a 548-bp human GAPDH cDNA fragment; a gift from Dr. E. Ueberham, Institute of Biochemistry, Leipzig, Germany) was transcribed in vitro after digestion with DdeI (New England Biolabs, Inc., Schwalbach/Taunus, Germany).
RPA
The RPA was performed using the Riboquant kit from
PharMingen (San Diego, CA). Transcription templates (see
above) were labeled with [
-32P]UTP by
in vitro transcription using T7 RNA polymerase. The
[-32P]UTP-labeled probes were hybridized for
12–16 h at 56 C in solution in excess to three different target RNA
samples extracted from normal thyroid tissue, the proposita’s thyroid
tissue, and tissue obtained from patients with Graves’ disease.
The remaining free probe and single stranded RNA were digested with RNases (Riboquant kit from PharMingen). The RNase-protected probes were purified by phenol/chloroform extraction and ethanol precipitation, separated on a 6% polyacrylamide gel, and quantified by phosphorimaging on the Molecular Imager System GS-525 with Multi Analyst software (Bio-Rad Laboratories, Inc., Munich, Germany) and autoradiography on Kodak Biomax Film (Kodak, Deisenhofen, Germany). The ratio of NIS/GAPDH was subsequently calculated.
Allele determination and genotyping of the affected family
Two or three microsatellite markers for each gene were used to
genotype the family (Table 2). Genotyping was performed by PCR using
one fluorescent dye-labeled primer and genomic DNA extracted from
peripheral blood lymphocytes as template. Amplification reactions were
performed in a total volume of 20 µL containing 50 ng DNA, 10 pmol of
each primer, 10 mmol/L deoxy-NTPs, 0.5 U InViTAQ DNA polymerase, and
the appropriate 10 x incubation buffer with 1.5 mmol/L
MgCl2. The PCR reactions were denatured at 94 C
for 5 min, followed by 30 cycles at 94 C for 30 s and touchdown
annealing with 2 cycles at 61 C, 2 cycles at 59 C, 2 cycles at 57 C,
following 24 cycles at 55 C for 30 s, and 72 C for 30 s. The
PCR products were separated in 6% polyacrylamide denaturing gels on a
373A ABI automated sequencer (ABI Perkin Elmer Corp.,
Foster City, CA) and analyzed with Genescan software 2.2. Allele typing
was performed using Genotyper software 2.0 (ABI Perkin Elmer Corp.).
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Linkage analysis was performed using logarithm of odds
(LOD) score determination. LOD scores were calculated with
two-point analysis using the MLINK program (39) with the assumption of
different recombination fractions (
). In addition, a multipoint
analysis was performed using GENEHUNTER software (40, 41). This
software package allows extraction of complete multipoint inheritance
information from a pedigree. Calculations for multipoint LOD scores
involved all screened markers for the respective candidate gene. The
multipoint inheritance information allows the reconstruction of maximum
likelihood haplotypes for all individuals in the pedigree.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The presence and the quantity of TG and TPO gene transcripts were
studied by Northern blot. Analysis of the index patient’s mRNA showed
a clear hybridization signal of 8.4 kb with three different TG probes,
pHTgM1, pHTgM3, and pHTgB2 (data not shown), corresponding to the
expected TG mRNA size. Hybridization of the patient’s thyroid RNA with
a 1-kb TPO cDNA probe detected a 3-kb TPO mRNA transcript (data not
shown). No difference in the size or intensity of TG and TPO mRNA in
comparison to RNA from normal thyroid tissue could be observed. A GAPDH
probe was used to estimate the mRNA quantity. NIS mRNA transcripts were
only weakly detectable by Northern analysis in the patient’s goiter
tissue and in control tissue. Therefore, we examined the NIS mRNA
expression by RNase protection assays. In the goiter tissue, a 130-bp
NIS mRNA fragment hybridizing with the
32P-labeled NIS in vitro RNA
transcript probe was identified. The expression of NIS mRNA in the
proposita’s goiter tissue was significantly reduced. Quantitative
densitometry and normalization of the amount of NIS mRNA relative to
the amount of GAPDH mRNA revealed a decrease in NIS expression in the
proposita’s goiter tissue to 42 ± 2.4% of that in normal
thyroid tissue (set at 100%). In contrast, in Graves’ disease tissue
a higher level of NIS expression compared to that in normal thyroid
tissue was observed (1093 ± 91.5%; Fig. 2).
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RNA extracted from the second goiter of the index patient was
analyzed by RT-PCR. As controls, normal thyroid tissue and tissue from
a patient with Graves’ disease were used. The TG-coding sequence was
amplified from cDNA position 57–8383 in 20 overlapping fragments. The
TPO cDNA was amplified from position 18–3024 in 8 overlapping
fragments. The NIS cDNA sequence was divided into 5 overlapping
portions from position 227-2456. The size of the PCR fragments
investigated by agarose gel electrophoresis was not different for cDNA
prepared from goiter, normal thyroid, and Graves’ thyroid tissue. All
RT-PCR-generated products were directly sequenced using the PCR
reaction primers. Five single nucleotide substitutions in the TPO gene
were detected by comparison to the published sequence (42). Sequence
analysis of the TPO cDNA revealed amino acid substitutions at positions
859 in exon 7, 1207 and 1283 in exon 8, 2263 in exon 12, and 2630 in
exon 15 (Table 1). These identified
variations are polymorphisms previously described by Abramowicz
et al. (16) and Bikker et al. (18).
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Table 2
summarizes all markers used
as well as the maximum and minimum LOD scores calculated assuming a
dominant mode of inheritance and a penetrance of 1. The LOD scores were
calculated assuming different recombination fractions () for the
microsatellite markers in the two-point analysis. LOD scores of -2 and
less indicate linkage exclusion. Two-point analysis of the markers
D8S263 and D8S272 flanking the TG gene gave LOD scores of -3.88 and
0.13, respectively, at = 0. For the NIS gene, linkage was
clearly excluded by LOD scores of -4.76 for the marker D19S226 at
= 0.001 and -2.8 at = 0 for the marker D19S414.
Likewise, linkage was excluded for the TPO gene with both gene flanking
markers by negative LOD scores of -2.7 for the marker D2S2268 and
-2.4 for the marker D2S319 at = 0.01. With the additional
microsatellite marker sRA, located in intron 10 of the TPO gene,
similar linkage results were obtained. A two-point LOD score of -2.1
at = 0.001 was noted for sRA. None of these markers showed a
maximum LOD score of more than 1 for different recombination fractions.
The multipoint analysis confirmed these results. Using the two markers
for TG D8S263 and D8S272 gave a negative LODmax of -0.25. The LODmax
was -2.3 for D2S2268, D2S319, and sRA. Multipoint analysis computed
for the markers D19S226 and D19S414 gave a LODmax of -1.41.
The TSHR (mapped on 14q31) was analyzed with two microsatellites, which
are located in introns 2 and 7 of the TSHR gene (33). For the markers
(AT)TSHR and (CT)TSHR, positive two-point LOD scores of 1.5 and 1.2,
respectively at = 0 were obtained. The markers D14S1030 and
D14S1054 were chosen to study the candidate locus MNG-1 on chromosome
14q. A maximum two-point LOD score of 1.5 at = 0 was obtained
for D14S1030. D14S1054 also gave a positive LOD scores of 1.2 at
= 0. A multipoint LODmax score of 1.5 was achieved by
inclusion of the markers (AT)TSHR, CT(TSHR), D14S1030, and D14S1054.
The genetic distance between the TSHR locus and the MNG-1 locus is
approximately 22 centimorgans. The cosegregation of the
corresponding haplotypes supports a linkage of the region between TSHR
and MNG-1 on chromosome 14q with euthyroid goiter in this family (Fig. 1).
Sequencing of the entire coding region of the TSHR gene
As positive LOD scores were obtained with both microsatellite markers within the TSHR gene, and all affected individuals carry the same haplotype, the proposita’s genomic DNA was screened for germline mutations by sequencing of all 10 TSHR exons. However, apart from a previously described polymorphism in codon 87 of TSHR exon 3 (43) resulting in an amino acid substitution of leucine to valine, no other mutations in the TSHR could be detected.
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Discussion |
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In our proposita we identified several polymorphisms. However, their possible functional importance was unclear. To gain further insights into the familial nature of the disease and to examine the meaning of the identified nucleotide variations, members of the proposita’s family were carefully characterized. On collection of clinical data from the family, it was apparent that males were more often affected than females. As women are more often affected, this presented an unusual finding. This observation together with the family history and the goiter relapses in the proposita suggested the existence of molecular alterations. We therefore investigated the mRNA transcripts of the TG and TPO genes from the proposita’s goiter tissue by Northern blot analysis, but could not detect a difference in size and quantity in comparison to control thyroid tissue. Due to the inherent lower sensitivity of the method, the detection of mRNA transcripts of the NIS gene with Northern blot was not possible. Therefore, we examined the NIS mRNA transcript by RNase protection assay, which is more sensitive than Northern analysis. In contrast to the TG and TPO genes, the amount of the NIS mRNA transcript was markedly decreased in the goiter tissue compared to that in normal thyroid tissue. As no splice variants by RT-PCR and no nucleotide changes could be detected in the NIS cDNA, and as the NIS gene was excluded as a candidate gene by linkage analysis, the decreased NIS expression is most likely a secondary event. The regulation of the human NIS gene is largely unknown at present. It is conceivable that the down-regulation of NIS gene expression in the large goiter maintains euthyroidism in the proposita via decreased I- uptake activity in the goiter tissue with normal TG and TPO gene expression despite thyroid growth. A distinctly higher NIS mRNA transcript level was observed in thyroid tissue from a patient with Graves’ disease, which was also used as a control. This observation confirms the findings of Saito et al. (46), which showed that the expression of human NIS is increased in Graves’ thyroid tissue compared to that in normal thyroid tissue.
As the examination of TG and TPO mRNA expression did not indicate a functionally important genetic alteration, further studies had to be carried out to investigate a possible pathogenetic role of TG or TPO alterations in the goiter genesis in this family. In the absence of suitable assay systems for TG and TPO, we therefore opted for linkage analysis with microsatellite markers to test the hypothesis that the candidate genes, TG, TPO, NIS, and TSHR, contribute to the euthyroid goiter disease in this family. The polymorphic microsatellite markers for the genes TG, TPO, and NIS gave two-point and multipoint LOD scores in the linkage analysis, which were negative or below 1 for all assumed recombination fractions. As no significant evidence for linkage was found on the assumption of either homogeneity or heterogeneity, we conclude, that these candidate genes can be excluded as a major cause of the euthyroid goiter disease in this family. In contrast, we have found evidence for linkage of familial euthyroid goiter to a locus on chromosome 14q, which was first described by Bignell et al. (29) as a locus for nontoxic multinodular goiter (MNG-1) in a large Canadian family. The haplotype clearly cosegregates with the euthyroid goiter in our family.To our knowledge, the present results are the first confirmation for the MNG-1 locus as a susceptibility gene locus for nontoxic goiter on chromosome 14q. Using linkage analysis, Tomer et al. (47) identified the GD-1 locus on chromosome 14q31 that was linked to Graves’ disease. GD-1 was localized within 2 centimorgans of the MNG-1 locus for nontoxic multinodular goiter. Consequently, our data also support the hypothesis (48) postulating the presence of a thyroid disease gene complex on chromosome 14q, etiologically related to thyroid diseases in general.
The TSHR is coupled mainly to the cAMP cascade. The cAMP pathway exerts a dual function, as it regulates both thyroid hormone production and proliferation of thyroid epithelial cells. Therefore, our study included analysis of the TSHR gene. In previous studies the TSHR has been excluded as a candidate gene for familial congenital hypothyroidism (32), multinodular nontoxic goiter (29), and Graves’ disease (47) by linkage analysis. However, in our family the TSHR gene could not be excluded as candidate locus by linkage analysis, in particular because for both TSHR microsatellites the haplotype cosegregates with familial euthyroid goiter. We therefore sequenced the entire TSHR-coding region. However, except for a polymorphism, no genetic alteration could be detected.
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Acknowledgments |
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Footnotes |
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Received December 9, 1998.
Revised June 7, 1999.
Accepted June 25, 1999.
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