The screening for mutations in the thyroglobulin cDNA from six patients with congenital hypothyroidism.

The six patients described in this study were clinically diagnosed with congenital hypothyroidism. Based on clinical and pathophysiological parameters, the cause of the thyroid dyshormonogenesis was suspected to be a defect in the synthesis of thyroglobulin, the matrix protein for thyroid hormone synthesis in the thyroid gland. After RNA isolation from six goitrous tissues and control thyroid tissues, RT-PCR was used to amplify 20 overlapping thyroglobulin (TG) cDNA fragments. Two alternative splice transcripts were identified: a transcript with a deletion of nucleotides 177-274 and a transcript with a deletion of nucleotides 3430-3736 that result in frame shifts and the introduction of premature stop codons. Two alternatively spliced transcripts not changing the reading frame were also identified: a transcript containing a deletion of nucleotides 4529-4699 and a transcript with a deletion of nucleotides 7301-7561. All these transcripts were expressed in thyroid tissue of both patients and controls. Nucleotide sequence analysis and comparison to the revised TG sequence (1997) revealed one revision and eight polymorphisms that did not result in amino acid changes and four polymorphisms that did change amino acid codons. In three patients a homozygous mutation was present at nucleotide position 229, causing a glycine to serine amino acid substitution. The clinical description 'thyroglobulin synthesis defect' in this population cannot be explained by major mutations in the coding region of the TG gene. Furthermore, the presence and level of expression of the alternatively spliced transcripts do not co-segregate with thyroid dyshormonogenesis, since in normal thyroid tissue the same alternatively spliced transcripts are present.

Caprine homologue of rodent 5'-AMP-activated protein kinase subunit and yeast SNF4/CAT3 is down-regulated by thyroid hormone.

We isolated a cDNA, B12, that was down-regulated by thyroid hormone (TH) in the goat cerebellum, using a polymerase chain reaction (PCR)-based subtractive hybridization and differential screening procedure. Northern blot analysis of RNA from cerebellum of T4-treated and untreated hypothyroid goats confirmed that clone B12 was TH-regulated with an average reduction in expression of 21% after 4 days of T4 supplementation. Other tissues from a T4-treated and an untreated hypothyroid goat also revealed down-regulation of B12, with the highest reduction in expression found in the thyroid gland (38%). Steady-state levels of the approximately 1.8 kb B12 mRNA were higher in brain than in peripheral tissues. In situ hybridization showed that B12 mRNA in the brain is mainly present in various layers of the cerebellum and the cerebral cortex, the hippocampus, and the olfactory tubercle and is predominantly expressed in neurons. Sequence analysis of the caprine B12 cDNA clone, and the murine homologue, revealed 61% similarity to SNF4/CAT3, a regulator involved in the transcriptional control of glucose-repressible genes in yeast, and 99% identity to a rat 5'-AMP-activated protein kinase subunit, which is involved in the regulation of fatty acid, glycogen and isoprenoid metabolism. In view of these homologies, B12 might encode a regulator involved in distinct metabolic pathways and therefore, TH might also affect gene expression indirectly by down-regulation of regulators like B12.

RC3/neurogranin structure and expression in the caprine brain in relation to congenital hypothyroidism.

In view of the profound effects of thyroid hormone deficiency on the central nervous system (CNS), neuronal genes regulated by thyroid hormone could potentially be involved in the development of the CNS. Expression of the neuronal gene RC3/neurogranin was shown to be induced by thyroid hormone in the rat. No data are available on RC3 expression in mammals with prenatal brain development, like humans. To study RC3 mRNA expression in a genetic in vivo model of congenital hypothyroidism, which also resembles the human situation in the timing of brain development relative to birth, we used an inbred strain of congenitally hypothyroid goats. We isolated a cDNA for the caprine RC3 homolog. The deduced amino acid sequence had 99% similarity with the rat and bovine protein sequence. An analysis of the developmental expression of RC3 mRNA levels showed a 3-fold increase between E90 and P0. In situ hybridization analysis showed that in euthyroid goats, the RC3 expression pattern was region-specific and resembled that in rats. However, in contrast to rats, hypothyroid goats showed only a reduced RC3 mRNA expression in the striatum. Hypothyroidism had no effect on RC3 mRNA expression in all other brain regions. T4-treatment of the hypothyroid fetus increased RC3 mRNA expression in the striatum to euthyroid control levels. These data suggest that thyroid hormone is a regulator of RC3 gene expression in the caprine brain, and that the striatum is highly sensitive to thyroid hormone deficiency.

Molecular basis of an adult form of Sandhoff disease: substitution of glutamine for arginine at position 505 of the beta-chain of beta-hexosaminidase results in a labile enzyme.

Sandhoff disease is a lysosomal storage disorder characterized by accumulation of GM2 ganglioside due to mutations in the beta-chain of beta-hexosaminidase. Hexosaminidase activity is negligible in infantile Sandhoff disease whereas residual activity is present in juvenile and adult forms. Here we report the molecular basis of the first described adult form of Sandhoff disease. Southern analysis of chromosomal DNA indicated the absence of chromosomal deletions in the gene encoding the beta-chain. Northern analysis of RNA from cultured fibroblasts demonstrated that at least one of the beta-chain alleles was transcribed into normal-length mRNA. Sequence analysis of the entire cDNA prepared from poly-adenylated RNA showed that only one point mutation was present, consisting of a G-->A transition at nucleotide position 1514. This mutation changes the electric charge at amino acid position 505 by substitution of glutamine for arginine in a highly conserved part of the beta-chain, present even in the slime mold Dictyostelium discoideum. The nucleotide transition generated a new restriction site for DdeI, which was present in only one of the alleles of the patient. Reverse transcription of mRNA followed by restriction with DdeI resulted in complete digestion at the mutation site, demonstrating that the second allele was of an mRNA-negative type. Transfection of COS cells with a cDNA construct containing the mutation but otherwise the normal sequence resulted in the expression of a labile form of beta-hexosaminidase. These results show that the patient's is a genetic compound, and that the lability of beta-hexosaminidase found in this form of Sandhoff disease is based on a single nucleotide transition.

Isolation of cDNAs encoding subunit VIb of cytochrome c oxidase and steady-state levels of coxVIb mRNA in different tissues.

A full-length cDNA clone specifying the nuclear-encoded subunit VIb of human cytochrome c oxidase (COX) was isolated from a human skeletal muscle cDNA expression library. This was done with antiserum directed against the group of subunits VIa, b and c of bovine heart COX. A potential ribosome-binding site was located immediately upstream from the initiation codon. The predicted amino acid sequence revealed 85% similarity with the corresponding subunit of bovine heart COX. Subunit VIb lacks a cleavable presequence for mitochondrial addressing. We assume that there are no tissue-specific isoforms of subunit VIb, since (i) in a Northern blot experiment a single hybridizing band of approx. 500 nucleotides was demonstrated in RNA from liver, skeletal muscle, MOLT-4 cells and fibroblasts and (ii) a full-length cDNA clone with an identical sequence was isolated from a human liver cDNA library. Steady-state levels of the coxVIb transcript were different in the tissues examined.

Rapid shift in genotype of human mitochondrial DNA in a family with Leber's hereditary optic neuropathy.

Mitochondrial DNA isolated from white blood cells was investigated in families suffering from Leber's hereditary optic neuropathy. A recently described mutation at nucleotide position 11778 was present in 5 out of 12 families and heteroplasmic mitochondrial DNA was observed in 2 of these 5 families. A rapid shift in genotype was found in one of the families with heteroplasmy: the grandmother had 60 percent mitochondrial DNA mutated at nucleotide position 11778, the mother 55 percent, and the two sons at least 95 percent. These data indicate that the number of mitochondrial DNA molecules transmitted to the progeny passes a developmental bottleneck, as previously proposed to occur in bovine oogenesis.

Free-energy carriers in human cultured muscle cells.

Creatine phosphate (CrP), adenosine triphosphate (ATP), creatine kinase (CK), adenylate kinase (AK), protein, and DNA were quantified in human muscle cell cultures undergoing transition from dividing myoblasts to multinucleate myotubes. CrP is negligible in cultures grown in commonly applied media but increases rapidly when sufficient exogeneous creatine is available. The rise in CrP content precedes the rapid increase of CK and cessation of DNA synthesis found during cell fusion. The addition of creatine has no significant effect on the CK and protein content and on the fusion of the human muscle cells. ATP, AK, and protein increase gradually during proliferation and differentiation. When expressed per milligram of protein, ATP and AK are in the same range as in human skeletal muscle and are not correlated to the cell fusion. In contrast, CrP and CK per milligram protein are much higher in myotubes than in myoblasts but remain much lower than in muscle. These data indicate that only an early differentiation phase has been reached in the muscle cell cultures.

Uptake and stability of human and bovine acid alpha-glucosidase in cultured fibroblasts and skeletal muscle cells from glycogenosis type II patients.

Acid alpha-glucosidase (EC 3.2.1.20) was purified from human placenta and bovine testis by affinity chromatography using concanavalin A (conA) and Sephadex G 200. When added to the culture medium of human fibroblasts, the enzyme purified from bovine testis is taken up with a 200-fold higher efficiency than the enzyme from human placenta. Uptake of acid alpha-glucosidase from bovine testis is mediated by the mannose-6-phosphate receptor, whereas only a minor fraction of placental enzyme appears to be equipped with the mannose-6-phosphate recognition marker. Once internalized, both human and bovine acid alpha-glucosidase demonstrate a half-life of about 10 days in fibroblasts from control individuals and patients with different clinical forms of glycogenosis type II (Pompe's disease, acid alpha-glucosidase deficiency). Evidence is presented that the mannose-6-phosphate receptor is also present on the plasma membrane of the clonal myogenic skeletal muscle cell lines G8-1 and L6J1 (respectively from mouse and rat origin) and on cultured human skeletal muscle cells derived from a muscle biopsy. Addition of bovine testis acid alpha-glucosidase to skeletal muscle cell cultures from an adult patient with glycogenosis type II leads to complete correction of the enzyme deficiency.

Uptake of triiodothyronine into cultured human muscle cells.

The uptake of T3 was measured in cultured human muscle cells at 37 degrees C and pH 7.4 in a medium containing albumin and glucose. The initial up]take increased linearly when the total T3 concentration was varied from 10(-9) to 10(-4) M. At prolonged incubation time the uptake decreased to virtually zero in about 30 min. These data indicate a rapid passive transport mechanism of T3 and a fast equilibration of the cellular T3 with the surrounding medium. In agreement with these conclusions the efflux of T3 was rapid and the initial uptake was not altered by pre-incubation in a T3-containing medium.