A novel mitochondrial DNA deletion in a patient with Kearns-Sayre syndrome: a late-onset of the fatal cardiac conduction deficit and cardiomyopathy accompanying long-term rGH treatment.

BACKGROUND: Kearns-Sayre Syndrome (KSS) is a multisystem disorder caused by a dysfunction of the oxidative phosphorylation system within mitochondria. Mitochondrial DNA (mtDNA) rearrangements are a key molecular feature of this disease, which manifest a broad phenotypic spectrum. CASE PRESENTATION: Here, we present a boy with KSS whose symptoms included cardiac conduction deficit, cardiomyopathy and growth hormone (GH) deficiency. The patient showed typical symptoms for KSS from early childhood (chronic progressive external ophthalmoplegia, retinopathy, short stature). Long-range PCR analysis disclosed a 7663-base pair heteroplasmic deletion in the mtDNA encompassing nucleotides 6340-14003. At 12 years of age, GH deficiency was recognized and recombinant growth hormone (rGH) therapy was started. At 15 years of age, a complete atrioventicular block was diagnosed and the patient received a pacemaker. During the following 6 months, progressive deterioration of the left ventricle was observed and an echocardiogram showed features of dilated cardiomyopathy. The rGH treatment was then discontinued at a final height of 163 cm. Unfortunately, due to multi-organ insufficiency and inflammation, the patient died at the age of 18 years. CONCLUSIONS: The response to rGH therapy in the patient was very satisfactory. The large mtDNA deletion had no apparent impact on the response to rGH. Cardiac disturbances occurred as part of the syndrome and were not related to rGH therapy; however, the progression of the disease led to death.

Mismatch repair-independent increase in spontaneous mutagenesis in yeast lacking non-essential subunits of DNA polymerase ε.

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.

[Molecular analysis of the corticotropin-releasing hormone receptor type 2 gene fragment in anorexia nervosa]. / Analiza molekularna fragmentu genu dla drugiego typu receptora kortykoliberyny w jadlowstrecie psychicznym.

INTRODUCTION: There is strong evidence for the importance of genetic factors in the aetiology of anorexia nervosa (AN). Stress factors can be also associated with the clinical manifestation of AN. The hypothalamic-pituitary-adrenal axis plays an important role in stress control. The increased activity of the hypothalamic-pituitary-adrenal axis in AN is caused by corticotropin-releasing hormone hypersecretion. CRH concentration in cerebrospinal fluid is increased in patients with AN. Intracerebral administration of CRH suppresses appetite. CRH receptor type 2 (CRHR 2) is involved in the appetite suppression effects of CRH. Thus CRHR 2 gene can be a candidate gene for AN. Three CRHR 2 splicing isoforms are known: alpha, b and g. In the hypothalamus, the main appetite control centre, only the isoform CRHR 2alpha is expressed, whose mRNA is characterised by one unique exon 1alpha. AIM: The aim of the study was the screening for mutations in exon 1alpha of the CRHR 2 gene in patients with AN, which could play a role in the pathogenesis of the disease. METHODS: The molecular analysis has been performed in 20 patients with AN and 10 healthy controls. DNA was isolated from peripheral blood leukocytes. DNA fragments were amplified by polymerase chain reaction (PCR) and sequencing was performed. RESULTS: After screening, no genetic variants have been found in the analysed region, including exon 1alpha and the untranslated region, both in anorectic patients as in controls. CONCLUSION: The results do not confirm the hypothesis that the analysed region of the CRHR 2 gene is involved in the pathogenesis of AN.

Galectin-3 is not an universal marker of malignancy in thyroid nodular disease in children and adolescents.

Current methods of research into thyroid nodular disease (TND) based on the polymerase chain reaction (PCR) and reverse-transcription (RT) permit the detection of some markers, even in poorly cellular biological material. The findings from fine-needle aspiration biopsy (FNAB), the most commonly used procedure in TND, do not always correlate with the postoperative histopathological diagnosis, sometimes giving a false negative result. The aim of this present study was to improve the classical cytological evaluation of the material obtained with ultrasound-guided biopsy with a RT-PCR based technique in order to detect carcinoma even in a minimally invasive form. Aspirate from the 30 patients included in the study was smeared for conventional cytology (H+E and MGG staining) and the leftover material in the needle was frozen for subsequent PCR analysis. Fine-needle aspiration specimens were evaluated for the presence of galectin-3 (GAL-3), the most promising molecular marker of malignancy. As a positive control for cells of follicular origin, thyroglobulin (Tg) gene expression was performed. RT-PCR was performed on extracted RNA and with specific primers for the screened genes, based on a one-step reaction with a Biometra PCR machine. Tg expression was observed in 23 aspirates, among which 10 were positive for the expression of GAL-3 [3 cases of PTC, 1 an oxyphilic variant of FTC, 1 an oxyphilic variant of follicular adenoma (FA), 1 a foetal variant of FA, 2 of Hashimoto thyroiditis (HT) and 2 of HT with coexisting FA]. Our results are the first evidence that GAL-3 expression, previously documented in thyroid carcinoma of follicular origin, is also present in Hashimoto thyroiditis. This study reveals some limitations in nodule or multiple nodules of benign character. If the diagnosis of HT is excluded, then the usefulness of the method in the diagnosis of malignancy may still be very high.