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

High Frequency of Haplotype HLA-DQ7 in Celiac Disease Patients from South Italy: Retrospective Evaluation of 5,535 Subjects at Risk of Celiac Disease

Celiac disease (CD) has a strong genetic component mainly due to HLA DQ2/DQ8 encoding genes. However, a minority of CD patients are DQ2/DQ8-negative. To address this issue, we retrospectively characterized HLA haplotypes in 5,535 subjects at risk of CD (either relatives of CD patients or subjects with CD-like symptoms) referred to our center during a 10-year period

Improving the Estimation of Celiac Disease Sibling Risk by Non-HLA Genes.

Celiac Disease (CD) is a polygenic trait, and HLA genes explain less than half of the genetic variation. Through large GWAs more than 40 associated non-HLA genes were identified, but they give a small contribution to the heritability of the disease. The aim of this study is to improve the estimate of the CD risk in siblings, by adding to HLA a small set of non-HLA genes. One-hundred fifty-seven Italian families with a confirmed CD case and at least one other sib and both parents were recruited. Among 249 sibs, 29 developed CD in a 6 year follow-up period. All individuals were typed for HLA and 10 SNPs in non-HLA genes: CCR1/CCR3 (rs6441961), IL12A/SCHIP1 and IL12A (rs17810546 and rs9811792), TAGAP (rs1738074), RGS1 (rs2816316), LPP (rs1464510), OLIG3 (rs2327832), REL (rs842647), IL2/IL21 (rs6822844), SH2B3 (rs3184504). Three associated SNPs (in LPP, REL, and RGS1 genes) were identified through the Transmission Disequilibrium Test and a Bayesian approach was used to assign a score (BS) to each detected HLA+SNPs genotype combination. We then classified CD sibs as at low or at high risk if their BS was respectively < or ≥ median BS value within each HLA risk group. A larger number (72%) of CD sibs showed a BS ≥ the median value and had a more than two fold higher OR than CD sibs with a BS value < the median (O.R = 2.53, p = 0.047). Our HLA+SNPs genotype classification, showed both a higher predictive negative value (95% vs 91%) and diagnostic sensitivity (79% vs 45%) than the HLA only. In conclusion, the estimate of the CD risk by HLA+SNPs approach, even if not applicable to prevention, could be a precious tool to improve the prediction of the disease in a cohort of first degree relatives, particularly in the low HLA risk groups

Improving the estimation of celiac disease sibling risk by non-HLA genes.

Celiac Disease (CD) is a polygenic trait, and HLA genes explain less than half of the genetic variation. Through large GWAs more than 40 associated non-HLA genes were identified, but they give a small contribution to the heritability of the disease. The aim of this study is to improve the estimate of the CD risk in siblings, by adding to HLA a small set of non-HLA genes. One-hundred fifty-seven Italian families with a confirmed CD case and at least one other sib and both parents were recruited. Among 249 sibs, 29 developed CD in a 6 year follow-up period. All individuals were typed for HLA and 10 SNPs in non-HLA genes: CCR1/CCR3 (rs6441961), IL12A/SCHIP1 and IL12A (rs17810546 and rs9811792), TAGAP (rs1738074), RGS1 (rs2816316), LPP (rs1464510), OLIG3 (rs2327832), REL (rs842647), IL2/IL21 (rs6822844), SH2B3 (rs3184504). Three associated SNPs (in LPP, REL, and RGS1 genes) were identified through the Transmission Disequilibrium Test and a Bayesian approach was used to assign a score (BS) to each detected HLA+SNPs genotype combination. We then classified CD sibs as at low or at high risk if their BS was respectively < or ≥ median BS value within each HLA risk group. A larger number (72%) of CD sibs showed a BS ≥ the median value and had a more than two fold higher OR than CD sibs with a BS value < the median (O.R = 2.53, p = 0.047). Our HLA+SNPs genotype classification, showed both a higher predictive negative value (95% vs 91%) and diagnostic sensitivity (79% vs 45%) than the HLA only. In conclusion, the estimate of the CD risk by HLA+SNPs approach, even if not applicable to prevention, could be a precious tool to improve the prediction of the disease in a cohort of first degree relatives, particularly in the low HLA risk groups

Flow-chart of the subjects at risk of CD from south Italy.

<p>Characteristics, age and presence/absence of HLA-DQ2/DQ8 in a population at risk of CD (relatives of CD- and with CD-like symptoms subjects) attending the Department of Laboratory Medicine of the University of Naples Federico II/CEINGE-Center of Advanced Biotechnology (Naples, Italy) between 2003 and 2013.</p

Frequencies of HLA-DQ genotypes (A) and haplotypes (B) detected in 666 CD patients from south Italy.

<p>CD, celiac disease</p><p>^aGenotypes were based on the presence of the following haplotypes: DQ2.5 = DQA1*05-DQB1*02 (DRB1*03) alleles; DQ2.2 = DQA1*02-DQB1*02 (DRB1*07) alleles; DQ2.3 = DQA1*03-DQB1*02 (DRB1*04/09/11) alleles; DQ8 = DQA1*03-DQB1*0302 (DRB1*04) alleles</p><p>^bStatistically significant differences, <i>p</i><0.001 at χ² test between males (M) and females (F)</p><p>^cDQX refers to: DQ7 = DQB1*0301 (DRB1*11/12/X) alleles; DQ4, DQ5, DQ6 and DQ9, were assigned if DQB1*04, DQB1*05, DQB1*06 and DQB1*0303 alleles were present, respectively</p><p>^dStatistically significant differences, <i>p</i><0.05 at χ² test, between DQ2/DQ8 (+) and DQ2/DQ8 (-) CD patients</p><p>Frequencies of HLA-DQ genotypes (A) and haplotypes (B) detected in 666 CD patients from south Italy.</p

Frequencies of HLA-DQ genotypes (A) and haplotypes (B) detected in unaffected subjects (n = 4869) from south Italy.

<p>CD, celiac disease</p><p>^aGenotypes were based on the presence of the following haplotypes: DQ2.5 = DQA1*05-DQB1*02 (DRB1*03) alleles; DQ2.2 = DQA1*02-DQB1*02 (DRB1*07) alleles; DQ2.3 = DQA1*03-DQB1*02 (DRB1*04/09/11) alleles; DQ8 = DQA1*03-DQB1*0302 (DRB1*04) alleles</p><p>^bStatistically significant differences between CD-relatives (n = 3662) and with CD-like symptoms (n = 1207) subjects; <i>p</i><0.001 at χ² test</p><p>^cDQX refers to: DQ7 = DQB1*0301 (DRB1*11/12/X) alleles; DQ4, DQ5, DQ6 and DQ9, if DQB1*04, DQB1*05, DQB1*06 and DQB1*0303 alleles were present, respectively</p><p>^dStatistically significant differences, <i>p</i><0.001 at χ² test, between DQ2/DQ8 (+) and DQ2/DQ8 (-) unaffected subjects.</p><p>Frequencies of HLA-DQ genotypes (A) and haplotypes (B) detected in unaffected subjects (n = 4869) from south Italy.</p

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