No abstract

Rett syndrome is a severe neurodevelopmental disorder, almost exclusively affecting females and characterized by a wide spectrum of clinical manifestations. Both the classic and atypical forms of Rett syndrome are primarily due to mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with atypical Rett syndrome, X-linked infantile spasms sharing common features of generally early-onset seizures and mental retardation. CDKL5 is known as serine/threonine protein kinase 9 (STK9) and is mapped to the Xp22 region. It has a conserved serine/threonine kinase domain within its amino terminus and a large C-terminal region. Disease-causing mutations are distributed in both the amino terminal domain and in the large C-terminal domain. We have screened the CDKL5 gene in 44 patients with atypical Rett syndrome who had tested negative for MECP2 gene mutations and have identified 6 sequence variants, out of which three were novel and three known mutations. Two of these novel mutations p.V966I and p.A1011V were missense and p.H589H a silent mutation. Other known mutations identified were p.V999M, p.Q791P and p.T734A. Sequence homology for all the mutations revealed that the two mutations (p.Q791P and p.T734A) were conserved across species. This indicated the importance of these residues in structure and function of the protein. The damaging effects of these mutations were analysed in silico using PolyPhen-2 online software. The PolyPhen-2 scores of p.Q791P and p.T734A were 0.998 and 0.48, revealing that these mutations could be deleterious and might have potential functional effect. All other mutations had a low score suggesting that they might not alter the activity of CDKL5. We have also analysed the position of the mutations in the CDKL5 protein and found that all the mutations were present in the C-terminal domain of the protein. The C-terminal domain is required for cellular localization through protein-protein interaction; any mutations in this domain might alter this function of the protein. This is the first report from India showing the mutation in CDKL5 gene in Indian cases of Rett syndrome. Our study emphasizes the role of CDKL5 mutation screening in cases of atypical Rett syndrome with congenital seizure variant.

Hemoglobin-A2 (HbA2) measurement in human hemolysates has great significance, since its level can indicate beta-thalassemia carrier status in otherwise healthy individuals. An ELISA for HbA2 using antiserum monospecific to the delta chain of HbA2 and affinity purified antirabbit gamma globulins (ARGG) conjugated to horseradish peroxidase (HRP) have been developed. The monospecific antiserum used does not cross react with other hemoglobins. Hemolysates from volunteers are used for measurement of HbA2. In a limited trial for beta-thalassemia carrier screening (n = 350), the results obtained with the developed ELISA are comparable with those obtained with a micro-column chromatography method (r > or = 0.89). The developed ELISA is simple, accurate, precise, inexpensive, and several samples can be processed simultaneously with ease, making this system a suitable candidate for transforming into a user friendly kit.

Metformin, an antidiabetic drug, has recently been repositioned in the treatment of several nondiabetic disorders including reproductive disorders such as polycystic ovarian syndrome (PCOS), where it improves endometrial functions. In vitro studies employing supratherapeutic concentrations (5-20 mM) of metformin, have reported anti-proliferative effects on endometrial epithelial and stromal cells. However, animal and human studies have revealed that therapeutic serum concentrations of metformin range between 20-70 µM. In the present study, the effect of therapeutic concentrations of metformin was studied on endometrial epithelial cells (EECs). Therapeutic concentrations of metformin induced proliferation in Ishikawa and HEC-1A cells. The proliferation of EECs was found mTOR dependent. Interestingly, therapeutic metformin concentrations were not able to activate the classical AMPK signaling. On the contrary, supratherapeutic metformin concentration (10 mM) inhibited mTOR and activated AMPK signaling. Microarray analysis of metformin-treated HEC-1A cells revealed dose-dependent differential effects on biological pathways associated with translation, ribosomal RNA processing, mitochondrial translation and cell proliferation. Therapeutic concentrations of metformin upregulated mitochondrial number as demonstrated by increased MitotrackerTM Red staining and enhanced succinate dehydrogenase (SDHD) expression; however higher concentration (10 mM) abrogated the same. Our results suggest that therapeutic concentrations of metformin augment mitochondrial strength and induce mTOR dependent endometrial cell proliferation.