Detection of the Thyroxine-Binding Globulin (TBG) Gene in Six Unrelated Families With Complete TBG Deficiency*

Abstract

T4-binding globulin (TBG) is a glycoprotein of hepatic origin which transports thyroid hormone in serum. Inherited TBG defects in man are X-chromosome linked and are expressed in hemizygotes as complete deficiency, partial deficiency, or excess. Since TBG is not necessary for thyroid hormone action, affected subjects are healthy. Using DNA probes for human TBG, we searched for restriction fragment length polymorphisms in six affected males belonging to 6 unrelated families with inherited complete TBG deficiency and an equal number of normal males. TBG could not be detected in the serum of any of the TBG-deficient males by a specific and sensitive RIA capable of detecting as little as 5 micrograms TBG/L or 0.031% of the average normal serum TBG concentration. DNA isolated from white blood cells was digested with 11 restriction endonucleases, and the digests were submitted to DNA blot analysis using two cloned TBG-DNA probes which together covered the entire protein coding and the 5'-flanking sequences of the TBG gene. A total of 26 different bands were detected on DNA blots, identifying 18 restriction sites located within the 4.2-kilobase TBG gene, which includes intronic, exonic, and 5'-flanking sequences. This analysis, which sampled 2.3% of the total TBG genome, failed to reveal differences in fragment size among the 6 TBG-deficient and 6 normal males examined. One restriction endonuclease (NcoI) identified normal sequences at the putative promoter region of the gene, and four other endonucleases (TaqI, SstII, MspI, and HpaII) recognized the cytosine-guanine dinucleotide phosphate sequences representing potential mutation hot spots. Although C was methylated at these sites, no C to T (thymidine) transitions were found. These data suggest that large deletions, insertions, or rearrangements of the TBG gene, or mutations at sites of methylated cytosine-guanine dinucleotide phosphate dimers are not common mechanisms for inherited complete TBG deficiency in man.

Screening of nineteen unrelated families with generalized resistance to thyroid hormone for known point mutations in the thyroid hormone receptor beta gene and the detection of a new mutation.

“…DNA was digested to completion with Eco RI (New England Biolabs, Beverly, MA), then subjected to 1.0% agarose gel electrophoresis, and following denaturation was transferred onto nitrocellulose paper (Southern blot). Hybridization was carried out under conditions previously described (43), using the hTR(B, cDNA probe pheA4 (14) which is complementary to all exons (two 5'-noncoding and eight coding) (27). The probe covers 80 nucleotides (30%) of the last coding exon (exon 8).…”

Replacement of Leu227by Pro in Thyroxine-Binding Globulin (TBG) Is Associated with Complete TBG Deficiency in Three of Eight Families with This Inherited Defect*

“…In a study of six unrelated males with TBG-CD, we noted no differences in the number or pattern of TBG gene fragments generated with 11 restriction endonucleases and analyzed using both a TBG cDNA and a TBG genomic DNA (gDNA) probe (11). These data indicated that TBG-CD is not usually due to deletions or rearrangements of the TBG gene.…”

“…Nineteen were French Canadians, 8 were Caucasians of European but not French origin, 3 were Mexican, 3 were Australian Aborigines, and 2 were Japanese. Three of the French Canadian subjects with TBG-C were each unaffected relatives of families with TBG-CD (CD1, CD2, and CD3) (11). Data on the inheritance pattern of TBG-CD in the 7 families of French Canadian origin has been previously reported (12).…”

“…We now report the results of cloning and sequencing of the TBG-coding regions adjacent introns and the promoter region of the gene of one male with inherited TBG-CD (CD5) (11,12). The sequence differed in two sites: codon 227 CCA (proline) rather than CTA (leucine) and codon 283 TTT (phenylalanine) in place of TTG (leucine).…”

Sequence of a Variant Thyroxine-Binding Globulin(TBG) in a Family with Partial TBG Deficiency in Japanese(TBG-PDJ).

“…Serum total T4 (TT4), total T3 (TT3), free T4 (FT4) and TSH were measured with commercial kits. DNA amplificationand sequencing of TBG gene Genomic DNA was obtained by extraction from white blood cells, as previously described [15]. DNA from the propositus served as a template to amplify the coding regions (Exon 0 through Exon 4) and the adjacent exon/intron junctions of the TBG gene by the polymerase chain reaction (PCR) with Taq DNA polymerase (Perkin-Elmer Cetiis Corp., Norwalk, CT), using Parkin-Elmer Cetus Thermal Cycler (model PJ 1000), under the conditions and with the oligonucleotide primers described previously [1 1 ].…”