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1.
Forensic Sci Int Genet ; 17: 87-90, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25864157

ABSTRACT

The rise of DNA evidence to the forefront of forensic science has led to high sample numbers being submitted for profiling by investigators to casework laboratories: bottleneck effects are often seen resulting in slow turnaround times and sample backlog. The ParaDNA(®) Screening and Intelligence Tests have been designed to guide investigators on the viability of potential sources of DNA allowing them to determine which samples should be sent for full DNA analysis. Both tests are designed to augment the arsenal of available forensic tests for end users and be used concurrently to those commonly available. Therefore, assessing the impact that common forensic tests have on such novel technology is important to measure. The systems were tested against various potential inhibitors to which samples may be exposed as part of the investigative process. Presumptive test agents for biological materials (blood, semen and saliva) and those used as fingerprint enhancement agents were both used. The Screening Test showed a drop in performance following application of aluminium powder and cyanoacrylate (CNA) on fingerprints samples; however this drop in performance was not replicated with high template DNA. No significant effect was observed for any agent using the Intelligence Test. Therefore, both tests stand up well to the chemical agents applied and can be used by investigators with confidence that system performance will be maintained.


Subject(s)
DNA/analysis , Forensic Genetics/methods , Specimen Handling/methods , Body Fluids/chemistry , DNA/blood , DNA Fingerprinting/methods , Dermatoglyphics , Humans , Male , Polymerase Chain Reaction , Semen/chemistry
2.
J Forensic Sci ; 60(3): 690-2, 2015 May.
Article in English | MEDLINE | ID: mdl-25739746

ABSTRACT

Seminal fluid represents a common biological material recovered from sexual assault crime scenes. Such samples can be prescreened using different techniques to determine cell type and relative amount before submitting for full STR profiling. The ParaDNA(®) Screening System is a novel forensic test which identifies the presence of DNA through amplification and detection of two common STR loci (D16S539 and TH01) and the Amelogenin marker. The detection of the Y allele in samples could provide a useful tool in the triage and submission of sexual assault samples by enforcement authorities. Male template material was detected on a range of common sexual assault evidence items including cotton pillow cases, condoms, swab heads and glass surfaces and shows a detection limit of 1 in 1000 dilution of neat semen. These data indicate this technology has the potential to be a useful tool for the detection of male donor DNA in sexual assault casework.


Subject(s)
DNA Fingerprinting/instrumentation , DNA/isolation & purification , Forensic Medicine/instrumentation , Semen/chemistry , Alleles , Amelogenin/genetics , Chromosomes, Human, X , Chromosomes, Human, Y , DNA Fingerprinting/methods , DNA Probes , Fluorescence , Genetic Markers , Humans , Male , Microsatellite Repeats , Reproducibility of Results , Sex Offenses , Software
3.
Diabetes Care ; 35(7): 1482-4, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22611063

ABSTRACT

OBJECTIVE: To demonstrate the importance of using a combined genetic and functional approach to correctly interpret a genetic test for monogenic diabetes. RESEARCH DESIGN AND METHODS: We identified three probands with a phenotype consistent with maturity-onset diabetes of the young (MODY) subtype GCK-MODY, in whom two potential pathogenic mutations were identified: [R43H/G68D], [E248 K/I225M], or [G261R/D217N]. Allele-specific PCR and cosegregation were used to determine phase. Single and double mutations were kinetically characterized. RESULTS: The mutations occurred in cis (double mutants) in two probands and in trans in one proband. Functional studies of all double mutants revealed inactivating kinetics. The previously reported GCK-MODY mutations R43H and G68D were inherited from an affected father and unaffected mother, respectively. Both our functional and genetic studies support R43H as the cause of GCK-MODY and G68D as a neutral rare variant. CONCLUSIONS: These data highlight the need for family/functional studies, even for previously reported pathogenic mutations.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Glucokinase/genetics , Female , Genetic Testing , Heterozygote , Humans , Male , Mutation, Missense , Pedigree
4.
PLoS One ; 7(4): e34541, 2012.
Article in English | MEDLINE | ID: mdl-22493702

ABSTRACT

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ∼65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113-1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) -mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC(50) 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Glucokinase/genetics , Mutation, Missense , Adolescent , Adult , Child , Child, Preschool , DNA Mutational Analysis , Diabetes Mellitus, Type 2/diagnosis , Enzyme Stability , Female , Genetic Testing , Genotype , Heterozygote , Humans , Male , Middle Aged , Pedigree , Phenotype , Slovakia
5.
Mol Endocrinol ; 24(1): 171-7, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19934346

ABSTRACT

Posttranslational activation of glucokinase (GCK) through S-nitrosylation has been recently observed in the insulin-secreting pancreatic beta-cell; however, the function of this molecular mechanism in regulating the physiology of insulin secretion is not well understood. To more fully understand the function of posttranslational regulation of GCK, we examined two naturally occurring GCK mutations that map to residues proximal to the S-nitrosylated cysteine and cause mild fasting hyperglycemia (maturity-onset diabetes of the young; subtype glucokinase). The kinetics of recombinantly generated GCK-R369P and GCK-V367M were assessed in vitro. The GCK-R369P protein has greatly reduced catalytic activity (relative activity index 0.05 vs. 1.00 for wild type), whereas the GCK-V367M has near normal kinetics (relative activity index 1.26 vs. 1.00 for wild type). Quantitative imaging and biochemical assays were used to assess the effect of these mutants on the metabolic response to glucose, GCK activation, and S-nitrosylation of GCK in betaTC3 insulinoma cells. Expression of either mutant in betaTC3 cells did not affect the metabolic response to 5 mM glucose. However, expression of either mutant blocked the effects of insulin on glucose-stimulated nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate reduction, suggesting defects in posttranslational regulation of GCK. Each of these mutations blocked GCK activation, and prevented posttranslational cysteine S-nitrosylation. Our findings link defects in hormone-regulated GCK S-nitrosylation to hyperglycemia and support a role for posttranslational regulation of GCK S-nitrosylation as a vital regulatory mechanism for glucose-stimulated insulin secretion.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Enzyme Activation , Glucokinase/genetics , Glucokinase/metabolism , Point Mutation , Protein Processing, Post-Translational , Age of Onset , Animals , Cell Line, Tumor , Enzyme Activation/drug effects , Fluorescence Resonance Energy Transfer , Glucokinase/chemistry , Glucose/metabolism , Humans , Insulin/pharmacology , Insulin/physiology , Insulin-Secreting Cells/enzymology , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/physiology , Kinetics , Luminescent Proteins/chemistry , Luminescent Proteins/isolation & purification , Luminescent Proteins/metabolism , Mice , Molecular Imaging/methods , Mutant Proteins/chemistry , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Protein Processing, Post-Translational/drug effects , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Transfection
6.
Hum Mol Genet ; 18(21): 4081-8, 2009 Nov 01.
Article in English | MEDLINE | ID: mdl-19643913

ABSTRACT

Genome-wide association studies have identified a number of signals for both Type 2 Diabetes and related quantitative traits. For the majority of loci, the transition from association signal to mutational mechanism has been difficult to establish. Glucokinase (GCK) regulates glucose storage and disposal in the liver where its activity is regulated by glucokinase regulatory protein (GKRP; gene name GCKR). Fructose-6 and fructose-1 phosphate (F6P and F1P) enhance or reduce GKRP-mediated inhibition, respectively. A common GCKR variant (P446L) is reproducibly associated with triglyceride and fasting plasma glucose levels in the general population. The aim of this study was to determine the mutational mechanism responsible for this genetic association. Recombinant human GCK and both human wild-type (WT) and P446L-GKRP proteins were generated. GCK kinetic activity was observed spectrophotometrically using an NADP(+)-coupled assay. WT and P446L-GKRP-mediated inhibition of GCK activity and subsequent regulation by phosphate esters were determined. Assays matched for GKRP activity demonstrated no difference in dose-dependent inhibition of GCK activity or F1P-mediated regulation. However, the response to physiologically relevant F6P levels was significantly attenuated with P446L-GKRP (n = 18; P

Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Blood Glucose/metabolism , Glucokinase/metabolism , Liver/metabolism , Triglycerides/blood , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Substitution , Catalysis/drug effects , Fasting/blood , Fructosephosphates/pharmacology , Gene Expression Profiling , Glucokinase/genetics , Glucose/metabolism , Glucose/pharmacology , Humans , Islets of Langerhans/metabolism , Kinetics , Mutation , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction
7.
J Pediatr ; 153(1): 122-6, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18571549

ABSTRACT

OBJECTIVE: To evaluate the clinical response to sulphonylurea treatment in a child with a homozygous T168A GCK (glucokinase) mutation, causing permanent neonatal diabetes mellitus (PNDM). STUDY DESIGN: Oral glibenclamide was given for 3 months. Pancreatic beta cell function was assessed by a glucagon stimulation test. Mutant and wild-type (WT) GCK were characterized. RESULTS: Sulphonylurea treatment resulted in a 12-fold increase in basal and stimulated C-peptide levels. HbA1c levels were reduced from 9.4% to 8.1% on a reduced insulin dose (0.85 to 0.60 U/kg/day). Mutant T168A-GST-GCK showed reduced kinetic activity (0.02 fold) compared to WT. CONCLUSIONS: Sulphonylureas can close the adenosine triphosphate (ATP)-sensitive potassium channel and elicit insulin secretion, but the ATP generated from metabolism is insufficient to fully restore insulin secretory capacity. Nonetheless, sulphonylurea treatment should be tried in patients with GCK-PNDM, particularly those with mutations resulting in less severe kinetic defects, in whom improved glycemic control may be obtained with lower doses of insulin.


Subject(s)
Diabetes Mellitus/genetics , Glucokinase/genetics , Mutation , Sulfonylurea Compounds/therapeutic use , Administration, Oral , Family Health , Glucokinase/chemistry , Glyburide/therapeutic use , Homozygote , Humans , Infant, Newborn , Insulin/therapeutic use , Male , Mutation, Missense , Protein Conformation , Sulfonylurea Compounds/administration & dosage , Treatment Outcome
8.
Biochem Soc Trans ; 36(Pt 3): 306-11, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18481947

ABSTRACT

There are well-documented examples in the literature of where determining the genetic aetiology of a disorder has provided insights into important regulatory pathways and protein interactions, and, more recently, has led to improved treatment options for patients. The studies of monogenic forms of beta-cell dysfunction are no exception. Naturally occurring mutations in the gene for the beta-cell enzyme glucokinase (GCK) result in both hyper- and hypo-glycaemia. Over 200 mutations have been described, and careful study of the mutational mechanisms for a number of these has provided important insights into glucokinase regulation. Increased understanding of post-translational regulatory mechanisms holds the promise of novel pharmacotherapeutic options for the treatment of T2DM (Type 2 diabetes mellitus). It is well established that common genetic variation in genes involved in monogenic forms of beta-cell dysfunction contributes to susceptibility to T2DM. Recent genome-wide scans for association have identified a number of novel T2DM susceptibility genes which probably influence beta-cell mass and/or function. Their identification allows the investigation of the role of rare mutations in monogenic beta-cell dysfunction. Current results indicate the importance of these genes in pancreatic development and suggest that mutations which result in a severe functional defect could be lethal.


Subject(s)
Genetic Predisposition to Disease , Glucokinase/genetics , Insulin-Secreting Cells/enzymology , Insulin-Secreting Cells/pathology , Animals , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/genetics , Glucokinase/chemistry , Humans , Mutation/genetics
9.
Eur J Endocrinol ; 159(1): 27-34, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18450771

ABSTRACT

OBJECTIVE: Activating glucokinase (GCK) mutations are a rarely reported cause of congenital hyperinsulinism (CHI), but the prevalence of GCK mutations is not known. METHODS: From a pooled cohort of 201 non-syndromic children with CHI from three European referral centres (Denmark, n=141; Norway, n=26; UK, n=34), 108 children had no K(ATP)-channel (ABCC8/KCNJ11) gene abnormalities and were screened for GCK mutations. Novel GCK mutations were kinetically characterised. RESULTS: In five patients, four heterozygous GCK mutations (S64Y, T65I, W99R and A456V) were identified, out of which S64Y was novel. Two of the mutations arose de novo, three were dominantly inherited. All the five patients were medically responsive. In the combined Danish and Norwegian cohort, the prevalence of GCK-CHI was estimated to be 1.2% (2/167, 95% confidence interval (CI) 0-2.8%) of all the CHI patients. In the three centre combined cohort of 72 medically responsive children without K(ATP)-channel mutations, the prevalence estimate was 6.9% (5/72, 95% CI 1.1-12.8%). All activating GCK mutations mapped to the allosteric activator site. The novel S64Y mutation resulted in an increased affinity for the substrate glucose (S(0.5) 1.49+/-0.08 and 7.39+/-0.05 mmol/l in mutant and wild-type proteins respectively), extrapolating to a relative activity index of approximately 22 compared with the wild type. CONCLUSION: In the largest study performed to date on GCK in children with CHI, GCK mutations were found only in medically responsive children who were negative for ABCC8 and KCNJ11 mutations. The estimated prevalence (approximately 7%) suggests that screening for activating GCK mutations is warranted in those patients.


Subject(s)
Congenital Hyperinsulinism/genetics , Glucokinase/genetics , Mutation , Cohort Studies , Congenital Hyperinsulinism/drug therapy , Congenital Hyperinsulinism/epidemiology , Denmark/epidemiology , Enzyme Activation/drug effects , Gene Frequency , Genotype , Glucokinase/metabolism , Glucose/metabolism , Heterozygote , Humans , Norway/epidemiology , Prevalence , Substrate Specificity , United Kingdom/epidemiology
10.
Mol Biochem Parasitol ; 153(1): 59-65, 2007 May.
Article in English | MEDLINE | ID: mdl-17350696

ABSTRACT

Overexpression of P-glycoproteins (Pgps) is assumed to be a principal mechanism of resistance of nematodes and arthropods to macrocyclic lactones. Quantitative RT-PCR (Q-RT-PCR) was used to demonstrate changes in transcription levels of two putative P-glycoprotein genes, designated here as SL0525 and SL-Pgp1, in sea lice (Lepeophtheirus salmonis) following exposure to emamectin benzoate (EMB). Pre-adult L. salmonis were challenged in an EMB bioassay for 24h and gene expression was studied from lice surviving EMB concentrations of 0, 10, and 30ppb. Gene expression was measured using Q-RT-PCR with elongation factor 1 (eEF1alpha) as an internal reference gene. The results show that both target genes, SL0525 and SL-Pgp1, had significantly increased levels of expression with exposure to 10ppb EMB (p=0.11 and p=0.17, respectively) whereas the group exposed to 30ppb was on the verge of being significant (p=0.053) only in the expression of SL-Pgp1. Gene expression for SL0525 and SL-Pgp1 were increased over five-fold at 10ppb EMB. Therefore, the upregulation of these target genes may offer protection by increasing Pgp expression when lice are exposed to EMB. Our optimized Q-RT-PCR can be used to determine if over-expression of these genes could be the basis for development of resistance in sea lice and thus allow suitable alternative chemotherapeutic options to be assessed.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antiparasitic Agents/pharmacology , Copepoda/drug effects , Copepoda/genetics , Ivermectin/analogs & derivatives , Animals , Base Sequence , Copepoda/metabolism , Copepoda/pathogenicity , DNA Primers/genetics , Drug Resistance/genetics , Ivermectin/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic/drug effects
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