Ex vivo proton NMR analysis and characterization of thymus lipid metabolites and their variation with age in C57BL/6 mice.

Application (1)H NMR spectroscopy techniques to the ex vivo study of thymus of 0.5-24 month-old C57BL/6 mice allowed the identification, the quantification and the estimation of some structural parameters (length and unsaturation) of various lipids in the thymus and their changes with age. An initial decrease of lipid metabolites in the thymus from 0.5 to 1 month of age was followed by large raises on further ageing, with 14, 8, and 4 fold increases for the total lipid content, fatty acids and glycerides, respectively, which correlated positively with age and negatively with thymus involution. The estimated average number of methylene groups per lipid chain essentially doubled its value from approximately 4-5 for the youngest mice to around 8 for the elderly, while the values obtained for the average number of double bonds per chain decreased with age from about 1-0.9 at 0.5/1 months of age to 0.6-0.7 for 18/24 months-old mice. The combination of NMR and histological data allowed studying the age-associated changes of the contribution of adipose- derived lipids to the total lipid content of the thymus and the amount of adipose tissue infiltrating the thymus. Both parameters initially showed a decrease from 0.5 months of age to (the adipose-free thymus at) 1 month of age. Afterwards, a continuous increase was observed on ageing: at 2 months about 55% of the lipids in the thymus were adipose-derived, while at 24 months they amounted to as much as about 95% of the total lipid content; for the same age-period, the estimated minimum adipose lipid content in the thymus changed from about 0.26% to 4.5%.

Prevalence and functional analysis of the S107P polymorphism (rs6647476) of the monocarboxylate transporter 8 (SLC16A2) gene in the male population of north-west Spain (Galicia).

OBJECTIVE: Mutations in SLC16A2, the gene encoding the thyroid hormone (TH)-specific transporter monocarboxylate transporter 8 (MCT8), result in a thyroid phenotype and severe mental retardation caused by neuronal TH deficiency. These mutational effects raise the question of whether polymorphic variation in SLC16A2 may also be associated with differences in serum levels of TH and/or TSH. DESIGN: This is the first major study of the frequency of the SLC16A2 rs6647476 single nucleotide polymorphism (SNP) (amino acid change Ser107Pro). We also studied the relationships of SLC16A2 genetic variants with serum levels of TSH, T4 and T3, with their mRNA expression and with expression of the TH-responsive genes ZAKI-4 and BTEB in white blood cells. Experiments in cultured fibroblasts were carried out to ascertain the dynamics of the T3 response. METHODS: A total of 276 men were studied. Genotyping of the S107P SNP was carried out using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP); serum hormone levels were determined by chemiluminescence; expression of mRNA was quantified by real-time PCR. RESULTS: The SLC16A2 S107P SNP was found in 36% of Galician males. With the present sample size we did not find any association of this polymorphism with variability in serum levels of TSH, free T4 (fT4) or fT3, or with basal expression of mRNA for SLC16A2 or the two TH-responsive genes ZAKI-4 and BTEB, either in white blood cells or in cultured human fibroblasts from either Ser107 or Pro107 genotypes under T3 stimulation. CONCLUSIONS: The S107P change in MCT8 is frequent in the male population in Galicia. In the population studied in this report an association with a thyroid phenotype was not demonstrated, even though the S107P SNP causes an important amino acid change.

Bases genéticas de los procesos tiroideos benignos / Genetic bases of benign thyroid processes

Los avances experimentados en la última década en la técnicas de estudio de los genes han supuesto una simplificación sin precedentes en el estudio de las bases genéticas de las enfermedades, acelerando el descubrimiento de genes causales o implicados en su desarrollo, y se vislumbra un horizonte en el que la secuenciación del genoma de cada ser humano será posible con rapidez y a un bajo coste. Conocemos bien las bases genéticas de los defectos en la formación de las hormonas tiroideas y conocemos parcialmente las bases genéticas de los defectos en la ontogenia tiroidea; en los últimos 4 años se han descubierto los genes causales de 2 nuevos síndromes que cursan con una disminución en la sensibilidad de la acción de las hormonas tiroideas y que afectan a su transporte (mutaciones en MCT8) y su metabolismo intracelular (mutaciones en SECISBP2); conocemos las bases genéticas de los adenomas tóxicos y bocios multinodulares tóxicos adenomatosos, y se han identificado varios genes implicados en el desarrollo de adenomas foliculares del tiroides. Sin embargo, aún no conocemos todos los genes implicados en la ontogenia tiroidea, desconocemos las bases genéticas del bocio multinodular hiperplásico, tan prevalente en algunas áreas geográficas del estado, así como de la mayoría de los trastornos tiroideos autoinmunitarios. Tenemos por delante retos importantes en cuanto a conocer las bases genéticas de los procesos tiroideos benignos que, unido a su elevada prevalencia y a la potencia de la tecnología de que disponemos o dispondremos en poco tiempo, nos ofrece un futuro prometedor y excitante en este campo de la investigación (AU)

The advances made in the last decade in gene analysis techniques have greatly simplified the study of the genetic bases of disease, hastening identification of the genes causing or involved in disease development. Rapid and low-cost genome sequencing in all individuals may become a reality. The genetic bases of defects in thyroid hormone formation have been well defined, and those of defects in thyroid ontogeny have been partially defined; in the last 4 years, the genes responsible for 2 new syndromes causing reduced sensitivity of the action of thyroid hormone and affecting thyroid hormone transport (MCT8 mutations) and intracellular metabolism (SECISBP2 mutations) have been discovered. The genetic bases of toxic adenomas and toxic multinodular goiters have been determined and several genes involved in the development of follicular thyroid adenomas have been identified. However, not all the genes involved in thyroid ontogeny have been identified and the genetic bases of multinodular hyperplastic goiter, highly prevalent in some regions of Spain, as well as those of most autoimmune thyroid disorders, are unknown. Major challenges remain in the characterization of the genetic bases of benign thyroid processes, which, together with their high prevalence and the current and future potential of technology, suggest a promising and exciting future in this field of research (AU)

Prematurely aged children: molecular alterations leading to Hutchinson-Gilford progeria and Werner syndromes.

Ageing is thought to be a polygenic and stochastic process in which multiple mechanisms operate at the same time. At the level of the individual organism ageing is associated with a progressive deterioration of health and quality of life, sharing common features such as: alopecia and grey hair, loss of audition, macular degeneration, neurodegeneration, cardiovascular diseases, osteoporosis, cataract formation, type-2 diabetes, lipodystrophies; a generally increased susceptibility to infection, autoimmune disorders and diseases such as cancer; and an impaired ability to cope with stress. Recent studies of mechanisms involved in the ageing process are contributing to the identification of genes involved in longevity. Monogenic heritable disorders causing premature ageing, and animal models have contributed to the understanding of some of the characteristic organism-level features associated with human ageing. Werner syndrome and Hutchinson-Gilford progeria syndrome are the best characterized human disorders. Werner syndrome patients have a median life expectancy of 47 years with clinical conditions from the second decade of life. Hutchinson-Gilford progeria syndrome patients die at a median age of 11-13 years with clinical conditions appearing soon after birth. In both syndromes, alterations in specific genes have been identified, with mutations in the WRN and LMNA genes respectively being the most closely associated with each syndrome. Results from molecular studies strongly suggest an increase in DNA damage and cell senescence as the underlying mechanism of pathological premature ageing in these two human syndromes. The same general mechanism has also been observed in human cells undergoing the normal ageing process. In the present article the molecular mechanisms currently proposed for explaining these two syndromes, which may also partly explain the normal ageing process, are reviewed.

Genetic bases of benign thyroid processes.

The advances made in the last decade in gene analysis techniques have greatly simplified the study of the genetic bases of disease, hastening identification of the genes causing or involved in disease development. Rapid and low-cost genome sequencing in all individuals may become a reality. The genetic bases of defects in thyroid hormone formation have been well defined, and those of defects in thyroid ontogeny have been partially defined; in the last 4 years, the genes responsible for 2 new syndromes causing reduced sensitivity of the action of thyroid hormone and affecting thyroid hormone transport (MCT8 mutations) and intracellular metabolism (SECISBP2 mutations) have been discovered. The genetic bases of toxic adenomas and toxic multinodular goiters have been determined and several genes involved in the development of follicular thyroid adenomas have been identified. However, not all the genes involved in thyroid ontogeny have been identified and the genetic bases of multinodular hyperplastic goiter, highly prevalent in some regions of Spain, as well as those of most autoimmune thyroid disorders, are unknown. Major challenges remain in the characterization of the genetic bases of benign thyroid processes, which, together with their high prevalence and the current and future potential of technology, suggest a promising and exciting future in this field of research.

Pendred syndrome in two Galician families: insights into clinical phenotypes through cellular, genetic, and molecular studies.

CONTEXT: We studied two families from Galicia (northwest Spain) with Pendred syndrome (PS) and unusual thyroid phenotypes. In family A, the proposita had a large goiter and hypothyroxinemia but normal TSH and free T3 (FT3). In family B, some affected members showed deafness but not goiter. OBJECTIVE: Our objective was to identify the mutations causing PS and molecular mechanisms underlying the thyroid phenotypes. INTERVENTIONS: Interventions included extraction of DNA and of thyroid tissue. PATIENTS: Propositi and 10 members of the two families participated in the study. MAIN OUTCOME MEASURES: Main outcome measures included SLC26A4 gene analysis, deiodinase activities in thyroid tissue, and c.416-1G-->A effects on SLC26A4 splicing. In addition, a primary PS thyrocyte culture, T-PS2, was obtained from propositus B and compared with another culture of normal human thyrocytes, NT, by Western blotting, confocal microscopy, and iodine uptake kinetics. RESULTS: Proposita A was heterozygous for c.578C-->T and c.279delT, presented with goiter, and had normal TSH and FT3 but low FT4 attributable to high type 1 and type 2 iodothyronine deiodinase activities in the goiter. Propositus B bore c.279delT and a novel mutation c.416-1G-->A; some deaf relatives were homozygous for c.416-1G-->A but did not present goiter. The c.279delT mutation was associated with identical haplotype in the two families. T-PS2 showed truncated pendrin retained intracellularly and high iodine uptake with low efflux leading to iodine retention. CONCLUSIONS: c.279delT is a founder mutation in Galicia. Proposita A adapted to poor organification by increasing deiodinase activities in the goiter, avoiding hypothyroidism. Lack of goiter in subjects homozygous for c.416-1G-->A was due to incomplete penetrance allowing synthesis of some wild-type pendrin. Intracellular iodine retention, as seen in T-PS2, could play a role in thyroid alterations in PS.

A novel phenotypic expression associated with a new mutation in LMNA gene, characterized by partial lipodystrophy, insulin resistance, aortic stenosis and hypertrophic cardiomyopathy.

BACKGROUND: Lipodystrophies are a heterogeneous group of diseases characterized by abnormal fat distribution. Familial partial lipodystrophy 2 (FPLD2) is due to mutations in the LMNA gene. Previous studies have suggested that LMNA mutations 5' to the nuclear localization signal (NLS) are more likely to underlie laminopathies with cardiac or skeletal muscle involvement, while mutations 3' to the NLS are more likely to underlie lipodystrophy and progeroid syndromes. OBJECTIVE: To study the clinical and molecular features of a subject with FPLD. SUBJECTS AND METHODS: We carried out mutational analysis of LMNA gene in a woman with FPLD phenotype and in her relatives. Insulin resistance was evaluated by minimal model. Body composition was evaluated by dual-energy X-ray absorptiometry (DEXA). Echocardiography was done in affected subjects. 3T3-L1 preadipocytes were transfected with wild-type or mutant prelamin A constructs. In transfected cells, lamin A was detected using a Cy3-conjugated monoclonal anti-FLAG antibody. RESULTS: The patient showed atypical fat distribution, insulin resistance, severe aortic stenosis and hypertrophic cardiomyopathy. She has an affected 11-year-old son, not yet lipodystrophic but with an incipient aortic disease. LMNA sequencing showed that mother and son were both heterozygous for a novel c.1772G > T missense mutation in exon 11, which causes the substitution of the cysteine at residue 591 by a phenylalanine (C591F). In mouse preadipocytes transfected with the mutant human LMNA gene, the mutant lamin A isoform was mislocated in the nucleus. CONCLUSIONS: This patient shows a novel clinical form of FPLD2, due to a mutation affecting lamin A only, with cardiac involvement.

Stress-induced alterations in the programmed natural cycles of post-natal lymphoid organ development in C57BL/6 mice: Evidence for a regulatory feedback relationship between bone marrow and thymus.

This study investigated some effects of weaning and immobilization stress in C57BL/6 mice aged 22-68 days, i.e., over a period including activation of the hypothalamus-pituitary-adrenal (HPA) axis and puberty. Specifically, the study evaluated the evolution, over the referred age interval, of a set of variables (body, thymus, spleen and axillary lymph nodes weights, the proportion of lymphoid cells in the bone marrow, the relative chemoattraction capacity of thymic supernatants for lymphoid cells and the migratory capacity of bone marrow lymphoid cells) in either weaned mice or weaned mice subjected to immobilization stress, compared to "non-stressed" unweaned mice. Cyclic patterns, observed for most variables in unweaned mice, were especially pronounced in two cases: the relative migratory capacity of bone marrow lymphoid cells collected at different ages towards neonatal thymic supernatant, and the relative chemoattraction capacity of thymic supernatants of different ages as tested against a sample of bone marrow lymphoid cells from mice aged 35 days. Weaning stress tended to intensify the involution stages of the cycles in thymus, spleen and lymph node weight, but increased the relative proportion of lymphoid cells in the bone marrow cell population. Both types of exogenous stress tended to affect cycle phase, i.e., cycle peaks and troughs were shifted in time. Correlations were observed between patterns seen in the thymus and bone marrow, suggesting the existence of an autoregulatory feedback loop governing pre-T cell migration and bone marrow/thymus homeostasis. These results also suggest that exogenous stress acts as a non-programmed regulator, modulating the naturally programmed cyclic patterns.

Evolution of the thymus size in response to physiological and random events throughout life.

During embryogenesis and in the early stages of life, the thymus is a crucial organ for the generation of the T cell repertoire. T cells are generated from hematopoietic stem cells already differentiated to precursor T cells in the bone marrow. These cells enter the thymus guided by chemotactic factors secreted by this organ. The complex maturation process takes place that ensures self-tolerance and homeostasis. Thymocytes that show autoreactivity do not leave the thymus, but rather die by apoptosis. The final percentage of mature T cells that survive to migrate from the thymus to the periphery is very low: at most 5%, under optimal conditions. The highest migration occurs in childhood and adulthood, at least in mice and humans; however, it declines throughout life and is minimal in the elderly. Under normal circumstances, the thymus commences involution soon after birth, and this involution correlates with the capacity to export mature T cells to the periphery. Hormones, cytokines, and neurotransmitters all play a role in this age-associated process, but the reasons for and mechanisms of this involution remain unknown. Apart from physiological conditions that change throughout life and govern age-related thymus evolution, random states and events provoked by intrinsic or extrinsic factors can induce either thymus involution, as in reversible transient thymic hypoplasias, or thymic hyperplasias. The age-associated involution, unlike transient involutions, follows a regular pattern for all individuals, though there are clear differences between the sexes. Nevertheless, even the age-associated involution seems to be reversible, raising the possibility of therapeutic strategies aimed at enhancing thymus function in the elderly.