Dionisios Chrysis

Clinical Phenotype and Mutant TRα1

A father and daughter with a mutation in the nuclear receptor gene for thyroid hormone (THRA) have abnormal levels of thyroid hormone, normal thyrotropin levels, growth retardation, and mildly delayed motor and cognitive development.

Interleukin-1β and TNF-α Act in Synergy to Inhibit Longitudinal Growth in Fetal Rat Metatarsal Bones†

We hypothesized that pro‐inflammatory cytokines can act locally in the growth plate to impair longitudinal growth. In a model of cultured fetal rat metatarsal bones, we found that IL‐1β and TNF‐α act in synergy to inhibit longitudinal growth, an effect linked to decreased proliferation and increased apoptosis of growth plate chondrocytes. IGF‐I could partially reverse all these effects.

Doxorubicin Induces Apoptosis in Germ Line Stem Cells in the Immature Rat Testis and Amifostine Cannot Protect against This Cytotoxicity

The underlying primary damage to the seminiferous epithelium caused by chemotherapeutic regimens at childhood is largely unknown. The present investigation was designed to identify acute cytotoxic events in the testis caused by a single dose of doxorubicin. Male rats at 6, 16, and 24 days of age were injected with doxorubicin (3 mg/kg, i.p.) or vehicle (saline) alone and 24 and 48 hours later, the germ cell types and apoptotic cells in the seminiferous epithelium were examined. As indicated by microscopy and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, an 8-fold increase in the number of apoptotic germ cells in the testes of 6-day-old rats was observed 48 hours after doxorubicin treatment. Spermatogonia migrating to the basement membrane were the primary cell type undergoing this induced apoptosis. A single dose of amifostine (200 mg/kg) administered i.p. 15 minutes before injection of doxorubicin provided no protection against this enhanced apoptosis. Under the same conditions, testicular levels of p53 and activated caspase 8 were elevated, whereas the level of murine double minute-2 was lowered. In contrast, doxorubicin treatment did not result in any significant change in the physiologic, stage-specific germ cell apoptosis occurring in the testes of 16- and 24-day-old rats. These observations suggest that the initiation phase of spermatogenesis is highly sensitive to doxorubicin-induced apoptosis. Gonocytes and early spermatogonia are the cell types that are vulnerable to this p53-trigged apoptosis, which results in a decrease in the size of the pool of germ-line stem cells. Amifostine fails to protect the germ cells against this cytotoxic insult.

Increased Apoptosis Occurring During the First Wave of Spermatogenesis Is Stage-Specific and Primarily Affects Midpachytene Spermatocytes in the Rat Testis

Abstract The physiological apoptosis that occurs in immature testis appears to be necessary for the maturation of this tissue. Thus, inhibition of the early apoptotic wave associated with the first round of spermatogenesis is followed by accumulation of spermatogonia and infertility later in life. To identify the cell types undergoing apoptosis in immature rat testis and to characterize the relationship between this apoptosis and progression of the first wave of spermatogenesis, sequential viable segments of seminiferous tubules from 8-, 18-, and 26-day-old rats were examined under a phase-contrast microscope. One novel observation was the existence of pronounced stage-specificity during the peak of apoptosis at the very early postnatal ages of 18 and 26 days. Increased apoptosis of pachytene spermatocytes in stages VII–VIII was the major feature that distinguished immature spermatogenesis from the corresponding adult process. The frequency of apoptosis among type A spermatogonia in immature stages IX–I was also elevated in comparison to the corresponding mature stages. The age-related peak of apoptosis was mediated by caspase 3; furthermore, stage-dependent expression of Bax in midpachytene spermatocytes was observed in the 18- and 26-day-old testis. These observations suggest that this Bax-regulated, caspase 3-mediated, increased apoptosis of midpachytene spermatocytes during the first wave of immature spermatogenesis represents a major difference in comparison to apoptosis occurring in the mature testis, and it may play an important regulatory role in establishing spermatogenesis in the rat testis.

Resistance to thyroid hormone caused by a mutation in thyroid hormone receptor (TR)α1 and TRα2: clinical, biochemical, and genetic analyses of three related patients

BACKGROUND The thyroid hormone receptor α gene (THRA) transcript is alternatively spliced to generate either thyroid hormone receptor (TR)α1 or a non-hormone-binding variant protein, TRα2, the function of which is unknown. Here, we describe the first patients identified with a mutation in THRA that affects both TRα1 and TRα2, and compare them with patients who have resistance to thyroid hormone owing to a mutation affecting only TRα1, to delineate the relative roles of TRα1 and TRα2. METHODS We did clinical, biochemical, and genetic analyses of an index case and her two sons. We assessed physical and radiological features, thyroid function, physiological and biochemical markers of thyroid hormone action, and THRA sequence. FINDINGS The patients presented in childhood with growth failure, developmental delay, and constipation, which improved after treatment with thyroxine, despite normal concentrations of circulating thyroid hormones. They had similar clinical (macrocephaly, broad faces, skin tags, motor dyspraxia, slow speech), biochemical (subnormal ratio of free thyroxine:free tri-iodothyronine [T3], low concentration of total reverse T3, high concentration of creatine kinase, mild anaemia), and radiological (thickened calvarium) features to patients with TRα1-mediated resistance to thyroid hormone, although our patients had a heterozygous mis-sense mutation (Ala263Val) in both TRα1 and TRα2 proteins. The Ala263Val mutant TRα1 inhibited the transcriptional function of normal receptor in a dominant-negative fashion. By contrast, function of Ala263Val mutant TRα2 matched its normal counterpart. In vitro, high concentrations of T3 restored transcriptional activity of Ala263Val mutant TRα1, and reversed the dominant-negative inhibition of its normal counterpart. High concentrations of T3 restored expression of thyroid hormone-responsive target genes in patient-derived blood cells. INTERPRETATION TRα1 seems to be the principal functional product of the THRA gene. Thyroxine treatment alleviates hormone resistance in patients with mutations affecting this gene, possibly ameliorating the phenotype. These findings will help the diagnosis and treatment of other patients with resistance to thyroid hormone resulting from mutations in THRA. FUNDING Wellcome Trust, NIHR Cambridge Biomedical Research Centre, Marie Curie Actions, Foundation for Development of Internal Medicine in Europe.

Clinical Phenotype of a New Type of Thyroid Hormone Resistance Caused by a Mutation of the TRα1 Receptor: Consequences of LT4 Treatment

CONTEXT Recently the first patients with inactivating mutations in T₃ receptor (TR)-α1 have been identified. These patients have low free T₄, low T₄, high T₃, low rT₃, and normal TSH serum levels, in combination with growth retardation, delayed bone development, and constipation. OBJECTIVE The aim of the current study was to report the effects of levothyroxine (LT4) treatment on the clinical phenotype of 2 patients (father and daughter) with a heterozygous inactivating mutation in TRα1. SETTING AND PARTICIPANTS Both patients were treated with LT4 for the last 5 years. To evaluate the effect of LT4 treatment, LT4 was withdrawn for 35 days and subsequently reinitiated. Data were collected from medical records, by reanalysis of serum collected over the last 6 years, and by a detailed clinical evaluation. RESULTS Treatment with LT4 resulted in a suppression of serum TSH and normalization of serum free T₄ and rT₃, whereas T₃ levels remained elevated in both patients. In addition, there was a normalization of the dyslipidemia as well as a response in serum IGF-I, SHBG, and creatine kinase in the index patient. All these parameters returned to pretreatment values when LT4 was briefly stopped. LT4 also resulted in an improvement of certain clinical features, such as constipation and nerve conductance. However, cognitive and fine motor skill defects remained. CONCLUSION This study reports the consequences of LT4 treatment over a prolonged period of time in 2 of the first patients with a heterozygous mutation in TRα1. LT4 therapy leads to an improvement of certain but not all features of the clinical phenotype.

Apoptosis is developmentally regulated in rat growth plate.

Apoptosis occurs in the growth plate during normal and abnormal longitudinal growth. To investigate the role of apoptosis during growth plate maturation, apoptosis and apoptosis-related proteins were studied in rat tibial growth plates at 2, 4, 8, and 11 wk of age. Apoptosis was studied by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end-labeling (TUNEL) method, and immunohistochemistry was used to detect p53, caspase-3 and -6, the antiapoptotic proteins Bcl-2 and Bcl-x, and the proapoptotic proteins Bax and Bad. In all age groups studied, most apoptotic chondrocytes were terminal hypertrophic chondrocytes (THPCs) with a significant increase during development. At 2 wk, 0.108±0.026 THPCs were found to be apoptotic per millimeter of growth plate width; at 4 wk, 0.355±0.048; at 8 wk, 0.394±0.043; and at 11 wk, 1.084±0.069 (p<0.001; 11 wk vs 2, 4, and 8 wk). THPCs were negative for p53 immunoreactivity at 2 and 4 wk, whereas some THPCs were positive at 8 and 11 wk. Caspase-3 and -6 were found in proliferative and early hypertrophic cells at 2 wk, whereas mature hypertrophic cells and THPCs were negative. At later stages of development, mature hypertrophic cells and THPCs were stained for both caspase-3 and -6. Bcl-2 and Bcl-x were present in proliferative and early hypertrophic cells at 2 wk, whereas at older ages a decrease in staining was observed. At 2 wk of age, Bax and Bad immunoreactivities were localized in proliferative and early hypertrophic cells, whereas at 8 and 11 wk many mature hypertrophic cells and THPCs were immunoreactive for Bax and Bad. Our results show that apoptosis is developmentally regulated in the rat growth plate. In older animals, with decreased growth rate and growth plate height, apoptosis is significantly increased, especially in THPCs.

Mutations in SLC34A3/NPT2c Are Associated with Kidney Stones and Nephrocalcinosis

Compound heterozygous and homozygous (comp/hom) mutations in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium (Na(+))-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a disorder characterized by renal phosphate wasting resulting in hypophosphatemia, correspondingly elevated 1,25(OH)2 vitamin D levels, hypercalciuria, and rickets/osteomalacia. Similar, albeit less severe, biochemical changes are observed in heterozygous (het) carriers and indistinguishable from those changes encountered in idiopathic hypercalciuria (IH). Here, we report a review of clinical and laboratory records of 133 individuals from 27 kindreds, including 5 previously unreported HHRH kindreds and two cases with IH, in which known and novel SLC34A3 mutations (c.1357delTTC [p.F453del]; c.G1369A [p.G457S]; c.367delC) were identified. Individuals with mutations affecting both SLC34A3 alleles had a significantly increased risk of kidney stone formation or medullary nephrocalcinosis, namely 46% compared with 6% observed in healthy family members carrying only the wild-type SLC34A3 allele (P=0.005) or 5.64% in the general population (P<0.001). Renal calcifications were also more frequent in het carriers (16%; P=0.003 compared with the general population) and were more likely to occur in comp/hom and het individuals with decreased serum phosphate (odds ratio [OR], 0.75, 95% confidence interval [95% CI], 0.59 to 0.96; P=0.02), decreased tubular reabsorption of phosphate (OR, 0.41; 95% CI, 0.23 to 0.72; P=0.002), and increased serum 1,25(OH)2 vitamin D (OR, 1.22; 95% CI, 1.05 to 1.41; P=0.008). Additional studies are needed to determine whether these biochemical parameters are independent of genotype and can guide therapy to prevent nephrocalcinosis, nephrolithiasis, and potentially, CKD.

Proteasome Inhibition Up-regulates p53 and Apoptosis-Inducing Factor in Chondrocytes Causing Severe Growth Retardation in Mice

Proteasome inhibitors (PI), a novel class of anticancer drugs, are relatively well tolerated and have recently been introduced into the clinic for the treatment of multiple myeloma. The tumor selectivity and low toxicity of PIs are surprising, given the crucial role of the ubiquitin/proteasome system in a multitude of cellular processes. Here, we show that systemic administration of PIs specifically impairs the ubiquitin/proteasome system in growth plate chondrocytes. Importantly, young mice displayed severe growth retardation during treatment as well as 45 days after the cessation of treatment with clinically relevant amounts of MG262 (0.2 micromol/kg body weight/injection) or bortezomib (1.0 mg/kg body weight/injection). Dysfunction of the ubiquitin/proteasome system was accompanied by the induction of apoptosis of stem-like and proliferative chondrocytes in the growth plate. These results were recapitulated in cultured fetal rat metatarsal bones and chondrocytic cell lines (rat, human). Apoptosis was associated with up-regulation of the proapoptotic molecules, p53 and apoptosis-inducing factor (AIF), both in vitro and in vivo. In addition to the observation that AIF is expressed in the growth plate, we also provide evidence that AIF serves as a direct target protein for ubiquitin, thus explaining its prominent up-regulation upon proteasome inhibition. Suppression of p53 or AIF expression with small interfering RNAs partly rescued chondrocytes from proteasome inhibition-induced apoptosis (35% and 41%, respectively). Our observations show that proteasome inhibition may selectively target essential cell populations in the growth plate causing significant growth failure. These findings could have important implications for the use of proteasome inhibitors in the treatment of childhood cancer.

Novel application of IGF‐I and IGFBP‐3 generation tests in the diagnosis of growth hormone axis disturbances in children with β‐thalassaemia

Children with β‐thalassaemia major (β‐thal) frequently have growth retardation in the presence of low serum IGF‐I and a normal GH response to pharmacological stimulation suggesting that they have GH insensitivity (GHIS). This study was carried out to study the cause of their growth retardation.