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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 8 4044-4052
Copyright © 2004 by The Endocrine Society

Genome-Wide Linkage Analysis Reveals Evidence for Four New Susceptibility Loci for Familial Euthyroid Goiter

Yvonne Bayer, Susanne Neumann, Birgit Meyer, Franz Rüschendorf, Andreas Reske, Thomas Brix, Laszlo Hegedüs, Pavel Langer, Peter Nürnberg and Ralf Paschke

III. Medical Department (Y.B., S.N., A.R., R.P.), University of Leipzig, 04103 Leipzig, Germany; Gene Mapping Center (B.M., F.R., P.N.), Max Delbrück Center for Molecular Medicine, 13092 Berlin, Germany; Department of Endocrinology (T.B., L.H.), Odense University Hospital, 5000 Odense, Denmark; Department of Internal Medicine (P.L.), Faculty of Medicine, P. J. Safárik University, 040 60 Kosice, Slovakia; and Institute of Medical Genetics (P.N.), Charité University Hospital, Humboldt University, 10099 Berlin, Germany

Address all correspondence and requests for reprints to: Ralf Paschke, M.D., III. Medical Department, University of Leipzig, Ph.-Rosenthal-Straße 27, 04103 Leipzig, Germany. E-mail: pasr{at}medizin.uni-leipzig.de.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Euthyroid goiter is characterized by diffuse or nodular enlargement of the thyroid gland. Iodine deficiency and cigarette smoking have been identified as important environmental factors. However, family and twin pair studies suggest a strong genetic predisposition. Therefore, we performed the first extended genome-wide scan to identify susceptibility loci that predispose for euthyroid goiter using 450 microsatellite markers in 18 extended Danish, German, and Slovakian families. Parametric and nonparametric multipoint linkage analyses were performed. The highest nonparametric LOD scores were obtained for chromosomes 2q and 3p with values of 2.54 at D2S1363 and 2.25 at D3S3038, respectively. Assuming heterogeneity and dominant inheritance, heterogeneity LOD scores (HLOD) of 2.71 and 1.94 were calculated for 2q and 3p, respectively. Furthermore, nonparametric LOD scores of 1.87 (HLOD 1.39) at D7S1808 on 7q and 1.79 (HLOD 1.80) at D8S264 on 8p were obtained. Haplotyping of families contributing to the linkage signals revealed four families compatible with a putative locus on 3p and one family each showing strict cosegregation with the loci on 2q, 7q, and 8p. The four novel candidate loci corroborate the assumed heterogeneity in the etiology of euthyroid familial goiter. For the first time, a more prevalent putative locus, present in 20% of the families investigated, was identified.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
EUTHYROID MULTINODULAR GOITER [Online Mendelian Inheritance in Man 138800] is a common thyroid disease characterized by nodular enlargement of the thyroid gland. Euthyroid goiter occurs both endemically and sporadically. In regions with mild iodine deficiency, the prevalence of endemic goiter amounts to more than 10% within a population (1, 2, 3) and is even higher for schoolchildren (4), whereas the prevalence for sporadic euthyroid goiter is 5% or less in nonendemic regions (3). However, not all individuals in the same iodine deficiency region develop a goiter, and iodine supplementation does not prevent goiter development in all treated subjects or individuals. The etiology of euthyroid goiter is therefore still incompletely understood. It is assumed that the development of goiter depends on various interactions between genetic and environmental factors (5). The major environmental influence is attributable to iodine intake. Moreover, other factors such as age, sex, and cigarette smoking are known to influence the development of goiter. However, family and twin pair studies in endemic and nonendemic areas clearly demonstrated a genetic predisposition for goiter development. The importance of genetic factors is also evident from clustering of goiters within families (6, 7, 8, 9) and from a higher concordance rate for goiter in monozygotic than in dizygotic twins (10). From the latter it was suggested that the heritability of the liability to the development of simple goiter in women is approximately 82% in a Danish population (5, 10). Children of parents with goiters have a significantly higher prevalence of goiter compared with children of nongoitrous parents (11). Therefore, genetic factors most likely predispose to the development of euthyroid goiters.

The genes coding for proteins involved in thyroid hormone synthesis such as thyroglobulin (TG), thyroid peroxidase (TPO), sodium iodide symporter, pendrin (PDS), and the TSH receptor (TSHR) are obvious candidate genes, which are thought to be involved in the molecular etiology of familial euthyroid goiter. A linkage study (6) performed in a large Canadian family with 18 cases of nontoxic multinodular goiter (MNG) identified a locus on chromosome 14q, called MNG-1. We have confirmed this locus in a German family (8). In this family, haplotypes of the MNG-1 locus and the TSHR, close to MNG-1, cosegregate with familial euthyroid goiter. In contrast, TSHR was clearly excluded as a candidate gene in the Canadian family (6). A further indication for a genetic predisposition for euthyroid familial goiter came from the analysis of an Italian pedigree with an X-linked autosomal pattern of inheritance of MNG, including 10 affected females and two affected males (7). A significant LOD score of 4.73 was calculated, which confirmed the conclusion that defects in the Xp22 region could cause the euthyroid goiters in this family. These candidate loci were investigated in four German and one Slovakian family (9). However, only one German family had a confirmed linkage to the locus MNG-1 (8). It is not clear how strong the contribution of the locus to the phenotype is. The absence of significant LOD scores in all investigated families did not allow confirmation or exclusion of the candidate loci MNG-1 or Xp22 and furthermore suggested genetic heterogeneity in the etiology of euthyroid goiter. Only weak indications for linkage to Xp22 and PDS were obtained in one family for each locus.

Thus, no reproducible common candidate region for euthyroid familial goiter has so far been identified. Therefore, we carried out the first genome-wide linkage analysis to identify new and more common susceptibility loci that predispose to euthyroid goiter using 450 microsatellite markers in 18 extended Danish, German, and Slovakian families. Slovakia is an iodine-sufficient region with a mean urinary excretion of 36–144 µg/liter (12). In Germany, iodine intake has increased over the last years, currently resulting in a moderate to mild iodine deficiency (mean urinary iodine excretion, 88 µg/d) (13, 14). Denmark is a country with borderline iodine deficiency (mean urinary iodine excretion, ~60 µg/liter) (15).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Families

Families with euthyroid goiters were recruited by the European Thyroid Association Working Group on "Genetics of euthyroid goiter" [www.eurothyroid.com]. Inclusion criteria were as follows: TSH, free T3 (fT3), fT4, and TG, TPO, and TSHR antibodies were in the normal range. Autoantibodies in serum were measured by the Brahms immunoassay (Brahms Diagnostica, Berlin, Germany). Laboratory tests for TSH, fT3, and fT4 were performed using Roche Diagnostics tools (Mannheim, Germany) using standard procedures. Goiter was documented in at least two generations. Goiter was defined as a previous thyroidectomy for benign goiter, as a clinically enlarged thyroid gland, or as a thyroid volume (determined by ultrasound) of more than 18 ml in females and more than 25 ml in males (16). Furthermore, we also included individuals with nodular goiters in our study. However, we did not perform fine-needle aspirations of the nodular goiters. A total of 18 pedigrees, comprising 79 affected and 68 unaffected individuals, were investigated in the genome scan. They included 11 families from Denmark, six extended German families, and one extended Slovakian family. Some German families were also included in our recent linkage studies. The families with the number G1004 were investigated by Neumann et al. (8), and the families with the numbers G1001, G1002, G1004, G1005, and S1006 were investigated in our previous linkage study (9). The study was approved by the ethical committees of all three participating university hospitals. Data of the clinical characterization of all families such as number of affected patients per family, number of affected/unaffected individuals, age of onset, and smoking behavior are summarized in Table 1Go.


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  		TABLE 1. Clinical characteristics of the euthyroid goiter families

 
Genotyping

DNA was extracted from leukocytes using QIAamp DNA blood midi kit (QIAGEN GmbH, Hilden, Germany). A genome-wide scan for linkage was performed using 450 evenly spaced microsatellite markers with an average spacing of 11 cM, from a modified Marshfield Weber 9 Linkage mapping set (Center for Medical Genetics, Marshfield, WI) covering all 22 autosomes and the X chromosome. Six nanograms of genomic DNA were amplified for each marker in a 10-µl reaction volume on an MJ PTC 225 Tetrad Cycler (MJ Research, Inc., Waltham, MA). The PCR mix contained 0.53 µM each primer, 0.1 µM each deoxynucleotide triphosphate, 0.5 U Taq polymerase, and 1x reaction buffer with 1.5 mM MgCl2. The forward primers were labeled at their 5' ends with a fluorescence dye such as FAM, HEX, NED, or TET. Equal aliquots of PCR products were pooled and 3 µl was mixed with an equal volume of MegaBace ET400-R size standard (Amersham Biosciences AB, Uppsala, Sweden). Genotypes were determined on a MegaBACE 1000 automated sequencer (Amersham Biosciences). For allele calling, the Genetic Profiler 1.0 software (Molecular Dynamics, Inc., Sunnyvale, CA) was used. Mendelian inheritance of marker alleles was tested with the PedCheck program (17).

Statistical analysis

A dominant model with reduced penetrance was assumed using a penetrance vector of [0.01; 0.9; 0.9]. Allele frequencies of 0.99 for the wild-type allele and 0.01 for the mutant allele were assumed. Two point LOD scores were computed using the MLINK program of LINKage 4.0 package (18). For each pedigree, multipoint nonparametric LOD [NPL/Statistic E (StatE)] scores and the corresponding P values were calculated using GeneHunter version 2.1 (19) and SIMWalk version 2.82 (20). GeneHunter uses the nonparametric Zall statistic to estimate the significance of allele sharing only among all affected individuals. The SIMWalk program uses the identity by descent measurements at the marker loci for the allele-sharing test. The program reports five statistics and their empirical P values, which measure the degree of clustering and its significance. The fifth statistic (StatE) is the Zall statistic implemented in GeneHunter. The NPL/StatE scores were summarized over all pedigrees. Multipoint parametric LOD scores were calculated for each pedigree using the programs described above and summarized over all pedigrees. LOD scores calculated under the assumption of heterogeneity (HLOD) were estimated by varying values of {alpha} (proportion of linked families) and {theta} until the HLOD scores were maximized.

Direct sequencing of the candidate genes thyroid hormone receptor interactor 12 (TRIP12) (2q), THRB (3p), and TRIP6 (7q)

For mutation analysis, all exons of THRB1, TRIP12, and TRIP6 were amplified including their flanking splice sites. All used primers for mutation analysis were chosen from the DNA sequence by hand or by the program Primer3. We used the following PCR program: 95 C initial denaturing for 5 min, 95 C denaturing for 30 sec, 55–60 C annealing for 30–45 sec (optimized for each primer pair), and 70 C elongation for 30 sec. Additionally, at the end of each PCR reaction we used 70 C for 10 min. For further optimizing we used the touchdown strategy. PCR fragments were purified with polyethyleneglycol precipitation (13% polyethyleneglycol 8000, 10 mM MgCl2) at room temperature. Purified PCR products were sequenced using the Big Dye terminator sequencing chemistry (Applied Biosystems, Foster City, CA). Sequencing reactions were analyzed on a Genetic Analyzer ABI 377 (Applied Biosystems).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Each of the 450 microsatellite markers typed was analyzed using multipoint nonparametric and parametric allele-sharing tests in GeneHunter and SIMWalk for a dominant pattern of inheritance in all 18 euthyroid goiter families. Figure 1AGo shows the genome-wide multipoint NPL curves over all investigated chromosomes summarized over all pedigrees. Two regions, on chromosomes 2 and 3, showed the highest peak NPL scores. The NPL scores of 2.54 at marker D2S1363 on chromosome 2q and 2.25 at marker D3S3038 on chromosome 3p were calculated (Fig. 1AGo and Table 2Go). Also, more suggestive NPL scores of 1.87 for chromosome 7q at D7S1808 and 1.79 at D8S264 on chromosome 8p were obtained (Table 2Go). Under the assumption of a dominant model and genetic heterogeneity, suggestive HLOD scores of 2.71 for chromosome 2q and 1.94 for chromosome 3p were calculated (Fig. 1BGo and Table 2Go). In addition, for the two regions on chromosome 7q and 8p, HLOD scores of 1.39 as well as 1.80, respectively, were obtained (Fig. 1BGo and Table 2Go). The haplotype segregation analyses performed in each pedigree confirmed indications for linkage to chromosome 2q in one family (G1001) and to chromosome 3p in a subset of four families (G1003, G1004, D2018, D2030) of the initial 18 families. Furthermore, haplotype analysis on 7q (G1002) and 8p (S1006) revealed cosegregation in one family for each locus. The smallest common haplotype for the disease intervals was determined by the proximal and distal boundaries and defined a 46-cM interval on 2q between D2S434 and D2S125 as well as a 16-cM interval between D3S1259 and D3S1266 on chromosome 3p (Fig. 2GoGoGo, A and D). Moreover, haplotyping revealed a 32-cM interval on 7q between the markers D7S820 and D7S3061 (Fig. 2BGoGoGo) and a further candidate region on 8p of 34 cM between D8S1119 and D8S1179 (Fig. 2CGoGoGo). These four novel candidate loci, which predispose to euthyroid goiter in several of the 18 families, confirm our previous hypothesis of genetic heterogeneity in the etiology of euthyroid goiter (9).



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  		FIG. 1. Summary of the genome scan for the euthyroid goiter susceptibility loci in 18 extended families. A, The nonparametric multipoint SIMWalk score is shown, across all autosomes and the X chromosome, from pter to qter. B, LOD scores were calculated using SIMWalk H assuming genetic heterogeneity and a dominant model of inheritance. The genetic distances are presented in centiMorgans (cM) over the entire genome. The black arrows indicate the four novel candidate regions on chromosomes 2q, 3p, 7q, and 8p.

 

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  		TABLE 2. Nonparametric LOD scores (StatE) and parametric HLOD scores for the four novel susceptibility loci on 2q, 3p, 7q, and 8p summarized for all families

 


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  		FIG. 2. Haplotype analysis in several families with contribution to one of the four novel loci. Haplotypes were constructed using the GeneHunter package. Blackened circles represent affected females; blackened squares affected males. Question marksrepresent unknown diagnosis, and white symbolsdescribe unaffected individuals. The haplotype cosegregating in the families is shown by filled frames. The proximal and distal boundaries define the smallest common haplotype of the familial euthyroid goiter locus in each family. A, Pedigree of the family linked to the locus on 2q. The critical interval is defined by the markers D2S434 and D2S125. B, Pedigree of the family linked to the locus on 7q. The disease region is defined by the markers D7S820 and D7S3061. C, Pedigree of the family linked to the locus on 8p. The critical interval is defined by the markers D8S1119 and D8S1179. D, Pedigrees of the families linked to the locus on 3p. The haplotype cosegregating in the individual families is shown by filled frames. The smallest common haplotype of the familial euthyroid goiter locus in each family was determined. The overlapping haplotypes place the disease interval between the markers D3S1259 and D3S1266.

 


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  		FIG. 2A. Continued

 


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  		FIG. 2B. Continued

 
To further examine the potential role of the novel euthyroid goiter susceptibility loci on 2q, 3p, 7q, and 8p, we looked for candidate genes in the regions of interest. The analysis of the candidate region on 2q offered 170 known genes (Ensembl database: www.ensembl.org) and comprises an entire physical region of 23 Mb. Within the candidate region, we identified the plausible candidate gene for thyroid hormone receptor interactor 12 (TRIP12), whose gene product potentially interacts with the thyroid hormone receptor ß (THRB). However, direct sequencing of TRIP12 in family G1001 showed no sequence variations, which could explain the obtained haplotype cosegregation with the phenotype.

The second locus on 3p (physical area of 15 Mb) includes 67 known genes on the Ensembl database within a critical 16-cM region. Within this region, the gene for the THRB maps on 3p24.3. THRB generates two isoforms by alternative splicing. The THRB1 variant is most abundant in kidney and liver, and its level of expression is unaffected by T3, whereas THRB2 is found exclusively in the pituitary and hypothalamus. The mRNA encoding this receptor is down-regulated by T3. Mutations in the THRB gene result in thyroid hormone resistance (RTH), which is characterized by the development of goiter as a hallmark. Therefore, THRB was chosen for mutation screening. To date, all identified mutations are located in three amino acids clusters in exons 5–8 of the ligand-binding domain of THRB (cluster 1, THRB1 234–282; cluster 2, THRB1 310–383; cluster 3, THRB1 429–460). Thus, all six common exons [numbers 3–8, according to the nomenclature of Beck-Peccoz et al. (21)]of the two alternative splice products and all other coding and noncoding exons of the THRB1 gene were sequenced in two affected individuals in each of the families with observed segregation of the haplotypes. However, the absence of germline mutations in these patients reduces the probability that THRB is one of the causative genes for euthyroid familial goiter.

Furthermore, in one extended German family (G1002) cosegregation of a tentative disease haplotype spanning an interval of 32 cM was observed on chromosome 7q (Fig. 2BGoGoGo). The cosegregating chromosomal section includes 261 known genes (Ensembl database) in a physical region of 39 Mb. Furthermore, this candidate locus contains the PDS gene (PDS) on 7q31. This result confirms our previous investigation (9) in the same family (G1002), in which we obtained weak indication for linkage to PDS located in the identified chromosomal region. However, no mutations were identified in PDS in this family in the previous investigation (9). Thus, our previous results suggest that the causative gene for euthyroid goiter in this family is close to the PDS gene. The defined region of interest on chromosome 7q comprises the TRIP6 gene at a distance of 6 Mb to the PDS gene. However, direct sequencing revealed no mutations in TRIP6. Therefore, the causative gene for euthyroid goiter in this family is still unknown.

The disease interval on chromosome 8p defined by one Slovakian family (S1006) comprises 155 known genes in a physical region of 39 Mb. However, our analysis did not reveal any plausible functional first-line candidate genes within this locus.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
A number of investigations have tried to identify susceptibility genes for euthyroid goiter (6, 7, 8, 9, 10, 22, 23, 24, 25). Family and twin pair studies have clearly demonstrated a genetic predisposition for euthyroid goiter (10, 26, 27, 28, 29). A family history positive for euthyroid goiter over several generations and an early onset of disease (Table 1Go) are strong indications for a genetic cause in the etiology of euthyroid familial goiter. Linkage studies have identified candidate genes and loci such as MNG-1 on 14q31 (6, 8), TSHR on 14q31 close to MNG-1 (8), and the Xp22 locus on the X chromosome (7). However, only one German family had a confirmed linkage to the locus MNG-1 (8). It is not clear how strong the contribution of the locus to the phenotype is. Therefore, it might be impossible to detect a clear evidence for linkage. All of these linkage studies investigated only one family, and none of these candidate genes in single families has been reported to have a more general significance. Moreover, in our previous study (9), we investigated five euthyroid goiter families for the described loci and for thyroid candidate genes such as TG, TPO, sodium iodide symporter, and revealed indications for genetic heterogeneity. Furthermore, genetic heterogeneity is present in other thyroid diseases such as Graves’ disease. Linkage analyses revealed different candidate regions that contribute to Graves’ disease (30, 31, 32, 33, 34, 35, 36, 37). As a consequence, we performed the first extended genome-wide linkage analysis in 18 families with euthyroid familial goiter to identify new and more general susceptibility loci. Using a dominant model, we obtained suggestive indications of linkage to four novel susceptibility loci with maximum parametric HLOD scores on 2q at D2S1363, on 3p at D3S3038, on 7q at D7S1808, and on 8p at D8S264. These results prompted us to perform haplotype analyses and define critical intervals of interest. For one German family (G1001), we observed a 46-cM interval on 2q between the markers D2S434 and D2S125, as well as in four families on 3p of 16 cM between D3S1259 and D3S1266 (Fig. 2GoGoGo, A and D). Additionally, in one family a weak indication of linkage to an interval between the markers D7S820 and D7S3061 on 7q was revealed. Moreover, in another family a cosegregation haplotype was delimited on 8p in a region of 34 cM. These results represent suggestive evidence that genetic factors on chromosomes 2q, 3p, 7q, and 8p confer susceptibility for euthyroid goiter in some families. The nonsignificant, low HLOD scores suggest the presence of multiple loci or genes that contribute to the phenotype of euthyroid goiter. Furthermore, it is possible that the genetic variants responsible for euthyroid familial goiter have not yet been identified by the various linkage studies, because it could be that several susceptibility loci with individually nonsignificant impacts are responsible for the etiology of euthyroid multinodular goiter, which can most likely be characterized as a complex genetic disease. Moreover, it is possible that different candidate genes or loci cause euthyroid goiter in different families. Furthermore, only seven of 18 families showed suggestive indications for linkage to the four novel candidate regions on 2q, 3p, 7q, and 8p. This fact also suggests not yet identified genetic loci or association mechanisms with minor effects for euthyroid goiter in the remaining 11 families. Furthermore, in our present study we could not identify any suggestive or evident indications for linkage to the previously identified loci on 14q (MNG-1) (8) and Xp22 (7). Because of the nonsuggestive and nonsignificant HLOD scores for both loci, in the present study, a clear conclusion as to whether these loci contribute or not to the phenotype of euthyroid goiter is not possible.

To further investigate the regions of interest, we performed a mutation screening for functional candidate genes that contribute to euthyroid goiter. TRIP12, which is located in the candidate region on chromosome 3p, is a plausible candidate gene because of the molecular interaction of its gene product with that of THRB. Therefore, a mutation screening of TRIP12 in one affected family member from the German family (G1001) that contributes to the HLOD score of 2.71 was performed. However, no sequence variation could be observed. Consequently, it is unlikely that TRIP12 is a causative genetic factor contributing to the observed goiter phenotype in family G1001.

The candidate region on chromosome 3p comprises the candidate gene THRB that encodes for the THRB. THRB constitutes a logical candidate gene, because nonsense mutations in this gene are the cause of RTH for which goiter constitutes a hallmark (38, 39, 40). The syndrome of RTH is characterized by persistent elevation of serum fT3 and fT4 and nonsuppressed TSH. The elevated fT3 and fT4 levels compensate for the impaired THRB. Hence, most of the RTH patients are clinically euthyroid and develop a goiter. Increased fT3 or fT4 values have not been observed in any of our investigated families. There is a possibility of unknown mutations in THRB, outside the described hot spot regions. However, the screening of the plausible candidate gene THRB on 3p24 revealed no germline mutations in any of 10 exons in selected family members. Therefore, the causative gene on 3p is still unknown for these families.

In agreement with our recent study (9), the chromosomal section on 7q, which we identified as a third candidate region for euthyroid goiter in this investigation, includes the PDS gene. In our previous study we also obtained indications for linkage to PDS by LOD scores and haplotype cosegregation. However, we found no mutations in the PDS gene, which cosegregates with the disease phenotype, and we postulated a candidate gene close to PDS in this family. The HLOD score of 1.39 is very suggestive and confirms our previous results. There is also the gene for TRIP6 within the candidate region on 7q. Sequencing of TRIP6 in two affected individuals of this family (G1002) revealed no variations in this gene. Interestingly, in this context, the PDS gene was recently identified as a new susceptibility gene for autoimmune thyroid diseases (41). None of the sequenced candidate genes harbored any germline mutations and can thus be excluded as general candidates for euthyroid goiter in the reported families. There are no obvious first-line candidate genes in the identified locus on chromosome 8p. In conclusion, our study reveals novel susceptibility loci. However, it is unclear whether other previously identified loci such as MNG-1 and Xp22 predispose partially to euthyroid goiter. Some studies have investigated one large multigenerational pedigree (6, 7). However, in subsequent studies it was impossible to confirm these loci in additional families.

In addition to genetic factors, environmental influences are of importance in the etiology of nontoxic goiter. Iodine deficiency and cigarette smoking are the most important environmental risk factors (42, 43). Therefore, the analysis of smoking habits was included in the clinical characterization of families in this study (Table 1Go). In the most extended families, including most of the affected individuals, there was only a limited number of smokers among the affected family members, strengthening the finding of a major genetic contribution.

In summary, this study confirms the genetic heterogeneity of euthyroid familial goiter and identifies the first candidate region with a prevalence of 20% in the families investigated. Additional studies will have to confirm the newly identified loci on 2q, 3p, 7q, and 8p in more families. It will also be necessary to narrow down the candidate regions by further microsatellite markers and to perform association studies with single nucleotide polymorphism markers in further families and especially in case-control individuals to reveal reliable results regarding possible genetic associations between single nucleotide polymorphisms and the phenotype of euthyroid goiter.


   Acknowledgments
 
We thank the families for their participation in this study. We thank Elisabeth Kirst, Elisabeth Peter, and Beate Jeßnitzer for their excellent technical assistance. We thank Dagmar Führer for her help with the clinical investigations, including the ultrasonography in all the German families. Furthermore, we thank Gudrun Nürnberg for her assistance with the preparation of the figures. Finally, we thank all families for their continuous support.


   Footnotes
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft to S.N. (DFG/NE 844/1-1), the Agnes and Knut Mörk Foundation, and the German Federal Department of Education and Research to P.N. (01KW9967).

Abbreviations: fT3, Free T3; fT4, free T4; HLOD, heterogeneity LOD scores; MNG, multinodular goiter; NPL, nonparametric LOD; PDS, pendrin; RTH, thyroid hormone resistance; StatE, Statistic E; TG, thyroglobulin; THRB, thyroid hormone receptor ß; TPO, thyroid peroxidase; TRIP6, thyroid hormone receptor interactor 6; TSHR, TSH receptor.

Received December 5, 2003.

Accepted April 25, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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