Glucokinase (GCK) Mutations and Their Characterization in MODY2 Children of Southern Italy

Type 2 Maturity Onset Diabetes of the Young (MODY2) is a monogenic autosomal disease characterized by a primary defect in insulin secretion and hyperglycemia. It results from GCK gene mutations that impair enzyme activity. Between 2006 and 2010, we investigated GCK mutations in 66 diabetic children from southern Italy with suspected MODY2. Denaturing High Performance Liquid Chromatography (DHPLC) and sequence analysis revealed 19 GCK mutations in 28 children, six of which were novel: p.Glu40Asp, p.Val154Leu, p.Arg447Glyfs, p.Lys458_Cys461del, p.Glu395_Arg397del and c.580-2A>T. We evaluated the effect of these 19 mutations using bioinformatic tools such as Polymorphism Phenotyping (Polyphen), Sorting Intolerant From Tolerant (SIFT) and in silico modelling. We also conducted a functional study to evaluate the pathogenic significance of seven mutations that are among the most severe mutations found in our population, and have never been characterized: p.Glu70Asp, p.His137Asp, p.Phe150Tyr, p.Val154Leu, p.Gly162Asp, p.Arg303Trp and p.Arg392Ser. These seven mutations, by altering one or more kinetic parameters, reduced enzyme catalytic activity by >40%. All mutations except p.Glu70Asp displayed thermal-instability, indeed >50% of enzyme activity was lost at 50°C/30 min. Thus, these seven mutations play a pathogenic role in MODY2 insurgence. In conclusion, this report revealed six novel GCK mutations and sheds some light on the structure-function relationship of human GCK mutations and MODY2

Prevalence of Y microdeletions in azoospermic and severe oligozoospermic men in Southern Italy: application of a rapid capillary electrophoresis method.

Male infertility is correlated with several genetic and non-genetic conditions. Microdeletions of Y chromosome are one of the most frequent genetic defects associated with male infertility. Evaluating this in infertile patients is important to assess an etiological diagnosis and possible prognosis of infertility, as well as to address clinical decision during treatment of infertility by intracytoplasmatic sperm injection, where the probability of success depends on the type and the number of deleted regions (azoospermia factor regions). To improve genetic counseling, it is useful to characterize Yq regions by a rapid and accurate method. In the current study, we evaluated the diagnostic efficiency and the time required of an in-house automated capillary electrophoresis method for Y microdeletions screening and applied it to estimate the prevalence of Y microdeletions in 100 infertile males affected by azoospermia or severe oligozoospermia (sperm count <5x10(6)/ml) and in 100 fertile male controls. In south Italian infertile men, the overall frequency of Y microdeletions was 9% (12.7% in azoospermic and 4.5% in severe oligozoospermic men). In conclusion, we think that the abovementioned procedure is suitable for the routine characterization of Y microdeletions

The Cause of Death of a Child in the 18th Century Solved by Bone Microbiome Typing Using Laser Microdissection and Next Generation Sequencing

The history of medicine abounds in cases of mysterious deaths, especially by infectious diseases, which were probably unresolved because of the lack of knowledge and of appropriate technology. The aim of this study was to exploit contemporary technologies to try to identify the cause of death of a young boy who died from a putative "infection" at the end of the 18th century, and for whom an extraordinarily well-preserved minute bone fragment was available. After confirming the nature of the sample, we used laser microdissection to select the most "informative" area to be examined. Tissue genotyping indicated male gender, thereby confirming the notary's report. 16S ribosomal RNA sequencing showed that Proteobacteria and Actinobacteria were more abundant than Firmicutes and Bacteroidetes, and that Pseudomonas was the most abundant bacterial genus in the Pseudomonadaceae family. These data suggest that the patient most likely died from Pseudomonas osteomyelitis. This case is an example of how new technological approaches, like laser microdissection and next-generation sequencing, can resolve ancient cases of uncertain etiopathology. Lastly, medical samples may contain a wealth of information that may not be accessible until more sophisticated technology becomes available. Therefore, one may envisage the possibility of systematically storing medical samples for evaluation by future generations

Internal deletions of amino acids 29-42 of human interleukin-6 (IL-6) differentially affect bioactivity and folding.

Internal deletions of the human interleukin-6 (IL-6) cDNA have been generated in the region encoding residues 29 to 42. Mutant proteins were produced by in vitro transcription-translation or in Escherichia coli and tested for their biological activity using the hybridoma growth factor (HGF) assay or a transcriptional activation assay on human hepatoma cells. The folding of the mutants was also checked by immunoprecipitation with conformation-specific monoclonal antibodies. The results show that only residues 29 to 34 are crucial for IL-6 activity and that the first two amino acids are probably involved in the definition of the IL-6 active site.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

GCK mutations detected in MODY2 children from South Italy.

a<p>GenBank: accession number (AH005826). ^bThe reference cDNA sequence was obtained from GenBank (NM_000162) and +1 corresponds to the A of the ATG translation initiation codon. ^cPolyphen prediction: probably damaging (1), benign (2), possibly damaging (3). SIFT score: <0.05 deleterious variant, ≥0.05 tolerated variant. ^dSwissprot accession number: P35557. ^eSibling pairs (MD19/20: two sisters; MD69/70: brother/sister).</p

Kinetic constants of human recombinant wild type-GCK and mutant β-cell GST-GCK fusion proteins.

<p>Data represent means ± SEM of 3 separate enzyme expressions each tested in duplicate. Note that the Hill coefficient (nH) and the relative activity index (I_a) are unit less. Kcat: GCK catalytic constant; S_0.5: affinity constant for glucose; nH: Hill coefficient; Km for ATP: affinity constant for ATP; I_a: GCK activity index. (*)<i>p</i><0.05, <i>t</i> test; (**)<i>p</i><0.005, <i>t</i> test.</p

Effect of temperature on the stability of GST-GCK mutants

<p>. Stock enzyme solutions were diluted to 250 µg/ml in storage buffer containing 30% glycerol, 50 mM glucose, 10 mM glutathione, 5 mM DTT, 200 mM KCl and 50 mMTris/HCl, pH 8.0. Panel A: The enzyme solutions were incubated for 30 min at temperatures ranging from 30 to 55°C and then assayed at 30°C as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038906#s4" target="_blank">Methods</a> section. Panel B: The enzyme solutions were incubated for periods of time from 5 to 60 min at 50°C. Results are means and SEM of three independent enzyme preparations for each mutant except for GST-GCK (Phe150Tyr) which corresponds to two independent enzyme preparations. (*) <i>p</i>≤0.03, (†) <i>p</i><0.008.</p