Abstract.Metabolic abnormalities in thyroid hormonogenesis cause congenital goiter. Here we studied a case of mild hypothyroidism caused by a novel missense mutation in the thyroglobulin (TG) gene.

Sequences of the full genomes of 259 clinical isolates of Mycobacterium tuberculosis, obtained from foreign-born and Japan-born patients in Tokyo, Japan, were determined, and a phylogenetic tree constructed by concatenated single-nucleotide polymorphism (SNP) sequences. The 259 isolates were clustered into four clades: Lineage 2 (East Asian or "Beijing" genotype; n = 182, 70.3%), Lineage 4 (Euro-American, n = 46, 17.8%), Lineage 1 (Indo-Oceanic, n = 23, 8.9%), and Lineage 3 (East African-Indian, n = 8, 3.1%). Of the 259, 36 (13.9%) were resistant to at least one drug. There was no multi-drug-resistant isolate. Drug resistance was greater for the strains in Lineage 2 than the non-Lineage 2. The proportion of Lineage 2 isolates was significantly smaller in foreign-born (n = 43/91, 47.3%) than in Japan-born (n = 139/168, 82.7%) patients, whereas the proportion of Lineage 1 isolates was significantly larger in foreign-born (n = 19/91, 20.9%) than in Japan-born (n = 4/168, 2.4%) patients. We also found eight SNPs specific to the typical Beijing sub-genotype in Lineage 2, including 4 non-synonymous SNPs. Of the 259 isolates, 244 had strain-specific SNP(s) and small (1-30-bp) insertions and deletions (indels). The numbers of strain-specific SNPs and indels per isolate were significantly larger from foreign-born (median 89, range 0-520) than from Japan-born (median 23, range 0-415) (p 3.66E-15) patients. These results suggested that M. tuberculosis isolates from foreign-born patients had more genetic diversity than those from Japan-born patients.

We analyzed the thyroglobulin (Tg) gene of 2 unrelated patients with congenital goiter and the Tg gene of 2 siblings with the variant type of adenomatous goiter. The clinical characteristics of the patients with congenital goiter and the variant type of adenomatous goiter were very similar, except for serum Tg levels, which were less than 15 pmol/L in the patients with congenital goiter, but 117-181 pmol/L in the patients with the variant type of adenomatous goiter (normal, 15-50 pmol/L). The tissue content of Tg in the thyroid glands of all 4 patients was reduced at 0.9-3.8% of total protein (normal, 19-40%). The missense mutation C1263R was detected in the 2 unrelated patients with congenital goiter; the pedigree study showed an autosomal recessive pattern of inheritance. In the 2 siblings with the variant type of adenomatous goiter, the missense mutation C1995S was homozygously detected. In the Tg complementary DNA of 110 normal subjects, the allelic frequencies of the C1263R and C1995S mutations were each less than 0.5%. Also in the normal subjects were detected 35 nucleotide polymorphisms, the insertion of 3 nucleotides, and 1 alternative splicing, each of which was not associated with any specific thyroid disease. From these data, the molecular mechanism of the C1263R and C1995S mutations was elucidated. We first analyzed the carbohydrate residues of C1263R Tg and C1995S Tg. Sensitivity to treatment by endoglycosidase H suggests that C1263R Tg and C1995S Tg were retained in the endoplasmic reticulum (ER). Also, the presence of endoglycosidase H-resistant Tg as well as endoglycosidase H-sensitive Tg in the patients with the variant type of adenomatous goiter suggests that a fraction of C1995S Tg was transported to the Golgi and associated with the mildly increased serum Tg levels. Native PAGE and Western blot analysis with anti-Tg antibody showed that C1263R Tg and C1995S Tg form high mol wt aggregates in the ER. Our results suggest that missense mutations that replace cysteine with either arginine or serine cause an abnormal three-dimensional structure of Tg. Such misfolded Tg polypeptides are retained in the ER as high mol wt aggregates.

Abstract. Congenital hypothyroidism is caused by several mechanisms.The most common cause worldwide is iodine deficiency, but in iodine-sufficient regions thyroid dysgenesis is the most common cause of congenital hypothyroidism.In the present study we analyzed the thyroid transcription factor-1 (TTF-1) gene in patients with congenital hypothyroidism due to thyroid dysgenesis: three patients with athyrosis, five with ectopy, and one with hypoplasia.Genomic DNA was isolated from peripheral leukocytes, and the TTF-1 gene, including a 5' flanking region, two exons and one intron was amplified by polymerase chain reaction (PCR) with 4 pairs of primers. The PCR products were directly sequenced by the Dye Terminator Cycle Sequencing method. We could not find any mutations specific for the thyroid dysgenesis in the 5' flanking region, two exons and one intron in the TTF-1 gene, but two heterozygous nucleotide substitutions were detected in the intron: a G to A transition at nucleotide 469 (G469A) and a C to A transversion at nucleotide 866 (C866A). The same nucleotide changes were detected in some normal subjects. Allelic frequencies of the polymorphisms G469A and C866A were 23% and 10%, respectively.Another normal polymorphism in the 5' flanking region was a G to T transversion at nucleotide -845 from the transcription start site (G-845T). The allelic frequency of the polymorphism G-845T was 28%. We also found 12 polymorphisms in the 5' flanking region, two in the intron and one in the 3' untranslated region. These polymorphisms were detected in 100% chromosomes. These results suggest that congenital hypothyroidism associated with thyroid dysgenesis is unlikely to be caused by mutations in the TTF-1 gene in which, however, were detected normal polymorphisms in the 5' flanking region, intron and 3' untranslated region.

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