Familial young-onset forms of diabetes related to HNF4A and rare HNF1A molecular aetiologies.

OBJECTIVE: We have dissected the rare molecular anomalies that may affect hepatocyte nuclear factor-1a (HNF1A) and hepatocyte nuclear factor-4a (HNF4A) in patients with familial young-onset diabetes for whom HNF1A mutations have been excluded by sequence analysis. METHODS: Eighty-four unrelatedHNF1A-negative patients with diabetes diagnosed before the age of 40 years, a family history of diabetes and the absence of features suggestive of Type 2 diabetes were included. We analysed by sequencing the HNF4A promoter and coding regions, the HNF1A promoter region and specific regions of HNF1A(B) and HNF1A(C) isoforms and searched for large deletions of HNF1A and HNF4A by multiplex ligation-dependent probe amplification (MLPA). RESULTS: We identified five novel HNF4A mutations (5 / 84, 6%), including four missense and one in-frame deletion, and one mutation of the HNF1A promoter (1 / 84). Sequence analysis of isoform-specific coding regions of HNF1A did not reveal any mutation. We next identified two whole gene deletions of HNF1A and HNF4A, respectively (2 / 84, 2.4%). CONCLUSIONS: Altogether, the search for rare molecular events in HNF1A and HNF4A led us to elucidate 8 / 84 (9.5%) of our HNF1A-negative cases.This study shows that genetic aetiologies remain to be elucidated in familial young-onset diabetes. It also highlights the difficulty of the differential diagnosis with Type 2 diabetes because of the wide clinical expression of monogenic young-onset diabetes and the absence of specific biomarkers.

Calcium phosphate powder synthesis by out-of-phase pulsed sonoelectrochemistry.

High aspect ratio calcium phosphate (CaP) nanorods were achieved by out-of-phase pulsed sonoelectrodeposition from electrolytic aqueous bath composed of calcium nitrate, ammonium dihydrogenophosphate and surfactant at pH of 4.9. The nature of CaP phases was determined by powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR) and energy dispersive X-ray spectroscopy (EDX). The results reveal the predominantly presence of calcium deficient hydroxyapatite (CDHA). The transmission electron microscopy (TEM) analyzes highlighted that the nanorods are polycristalline and have an aspect ratio up to 30.

Search for DNA sequence variations using a MutS-based technology.

The search for DNA sequence variations (DSV) is emphasized with genetic studies of a large number of multifactorial diseases. Saturation of regions of interest with diallelic polymorphisms will be an essential step to pinpoint, through association studies, predisposing genes. We have developed a solid-phase method based on the ability of mismatch binding protein MutS to recognize single nucleotide mismatches. This approach was applied to the study of 83 sequence-tagged sites (STSs) extracted from an eight centimorgans (cM) chromosome 21 region. One-third of tested STSs were found to be polymorphic leading to a frequency of one DSV every 822 base pairs (bp). Sequencing of analyzed STSs showed the high reliability of the MutS-based technology for mismatches up to 2 bp in DNA fragments ranging in size from 200 bp to 1 kilobase (kb). The entire assay which is performed in a solid-phase format without the need of electrophoresis or sequencing, will provide an efficient tool for new polymorphism detection.

The clinical variability of maternally inherited diabetes and deafness is associated with the degree of heteroplasmy in blood leukocytes.

CONTEXT: Maternally inherited diabetes and deafness (MIDD) is a rare form of diabetes with a matrilineal transmission, sensorineural hearing loss, and macular pattern dystrophy due to an A to G transition at position 3243 of mitochondrial DNA (mtDNA) (m.3243A>G). The phenotypic heterogeneity of MIDD may be the consequence of different levels of mutated mtDNA among mitochondria in a given tissue. OBJECTIVE: The aim of the present study was thus to ascertain the correlation between the severity of the phenotype in patients with MIDD and the level of heteroplasmy in the blood leukocytes. PARTICIPANTS: The GEDIAM prospective multicenter register was initiated in 1995. Eighty-nine Europid patients from this register, with MIDD and the mtDNA 3243A>G mutation, were included. Patients with MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) or with mitochondrial diabetes related to other mutations or to deletions of mtDNA were excluded. RESULTS: A significant negative correlation was found between levels of heteroplasmy and age of the patients at the time of sampling for molecular analysis, age at the diagnosis of diabetes, and body mass index. After adjustment for age at sampling for molecular study and gender, the correlation between heteroplasmy levels and age at the diagnosis of diabetes was no more significant. The two other correlations remained significant. A significant positive correlation between levels of heteroplasmy and HbA(1c) was also found and remained significant after adjustment for age at molecular sampling and gender. CONCLUSIONS: These results support the hypothesis that heteroplasmy levels are at least one of the determinants of the severity of the phenotype in MIDD.

The conserved N-terminal region of the mitotic checkpoint protein BUBR1: a putative TPR motif of high surface activity.

BUBR1, a key component of the mitotic spindle checkpoint, is a multidomain protein kinase that is activated in response to kinetochore tension. Although BUB1 and BUBR1 play an important role in cell division, very little is known about their structural characteristics. We show that the conserved N-terminal region of BUBR1, comprising residues 1-204, is a globular domain of high alpha-helical content ( approximately 60%), stable in the pH range 4-9 and probably organized as a tetratricopeptide motif repeat (TPR), most closely resembling residues 16-181 of protein phosphatase 5. Because the latter presents a continuous amphipathic groove and is regulated by binding certain fatty acids, we compared the properties of BUBR1(1-204) and TPR-PP5(16-181) at air/water interfaces and found that both proteins exhibited a similar surface activity and formed stable, rigid monolayers. The deletion of a region that probably comprises several alpha-helices of BUBR1 indicates that long-range interactions are essential for the stability of the N-terminal domain. The presence of the putative TPR motif strongly suggests that the N-terminal domain of BUBR1 is involved in direct protein-protein interactions and/or protein-lipid interactions.

Aggregation of puroindoline in phospholipid monolayers spread at the air-liquid interface.

Puroindolines, cationic and cystine-rich low molecular weight lipid binding proteins from wheat seeds, display unique foaming properties and antimicrobial activity. To unravel the mechanism involved in these properties, the interaction of puroindoline-a (PIN-a) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers was studied by coupling Langmuir-Blodgett and imaging techniques. Compression isotherms of PIN-a/phospholipid monolayers and adsorption of PIN-a to lipid monolayers showed that the protein interacted strongly with phospholipids, especially with the anionic DPPG. The electrostatic contribution led to the formation of a highly stable lipoprotein monolayer. Confocal laser scanning microscopy and atomic force microscopy showed that PIN-a was mainly inserted in the liquid-expanded phase of the DPPC, where it formed an aggregated protein network and induced the fusion of liquid-condensed domains. For DPPG, the protein partitioned in both the liquid-expanded and liquid-condensed phases, where it was aggregated. The extent of protein aggregation was related both to the physical state of phospholipids, i.e., condensed or expanded, and to the electrostatic interactions between lipids and PIN-a. Aggregation of PIN-a at air-liquid and lipid interfaces could account for the biological and technological properties of this wheat lipid binding protein.

Mapping the whole human genome by fingerprinting yeast artificial chromosomes.

Physical mapping of the human genome has until now been envisioned through single chromosome strategies. We demonstrate that by using large insert yeast artificial chromosomes (YACs) a whole genome approach becomes feasible. YACs (22,000) of 810 kb mean size (5 genome equivalents) have been fingerprinted to obtain individual patterns of restriction fragments detected by a LINE-1 (L1) probe. More than 1000 contigs were assembled. Ten randomly chosen contigs were validated by metaphase chromosome fluorescence in situ hybridization, as well as by analyzing the inter-Alu PCR patterns of their constituent YACs. We estimate that 15% to 20% of the human genome, mainly the L1-rich regions, is already covered with contigs larger than 3 Mb.

Experimental studies of the liquid-glass transition in trimethylheptane

The molecular glass former trimethylheptane was studied by calorimetric, dielectric, ultrasonic, neutron scattering, Brillouin scattering, and depolarized light-scattering techniques. The molecular structure appears to be nearly spherical optically as indicated by the low depolarization ratio and dielectric susceptibility values. A preliminary mode-coupling theory (MCT) analysis of the light-scattering and neutron-scattering data indicates that T(C) greater, similar150 K, at least 25 K above T(G). The susceptibility minima were analyzed with the MCT interpolation equation, and disagreement between the light and neutron results was observed despite the apparent isotropy of the molecules.