Inheritance of HNF1a and GCK Mutations in a Family With MODY
Inheritance of HNF1a and GCK Mutations in a Family With MODY
A total of 10 members of two unrelated families (family-1 and family-2) were recruited in the Departments of Paediatrics and Endocrinology of the 'Hospital Universitario de Guadalajara' (Spain), for genetic diagnosis of MODY 2 or MODY 3. A control group of 50 nondiabetic individuals was also tested for the presence of the novel mutation identified in the families. All subjects included in the study were native Spanish. The study followed the tenets of the Declaration of Helsinki, and informed consents were obtained from all the participants. The probands were clinically evaluated by experienced endocrinologists and/or paediatricians and met all the following criteria established by the Spanish Diabetes Society for MODY genetic testing: (i) autosomal dominant inheritance; (ii) hyperglycaemia diagnosis before the age of 25 years in at least one family member, and/or detectable C-peptide levels in patients with or without insulin treatment at least 3 years after the clinical diagnosis and (iii) negative pancreatic autoimmunity. Additional clinical parameters such as fasting blood glucose, blood glucose 2 h after OGTT (75 g of anhydrous glucose after an overnight fast), glycosylated haemoglobin (HbA1c), body mass index, follow-up therapy and evidence of diabetic retinopathy or nephropathy were also obtained.
Genomic DNA was extracted from the peripheral leucocytes of all the subjects with the E.Z.N.A. Blood DNA kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer's instructions. The promoter (nucleotides -487 to -1356 in GCK and -226 to -414 in HNF1A), 5' UTR and the 10 exons of both GCK and HNF1A genes were screened for mutations by automated PCR-terminator cycle sequencing using the BigDye® (version 3·1) kit (Applied Biosystems, Foster City, CA, USA). The sequencing reaction products were analysed in an automated DNA sequencer (ABI Prism 3130 genetic analyzer; Applied Biosystems). Intronic primers were designed to allow for the analysis of splicing consensus sequences (Table S1). Mutations were named based on the cDNA reference sequence ENST00000403799 for GCK and ENST00000257555 for HNF1A genes (http://www.ensembl.org/index/html). We denoted the first nucleotide of the translation initiation site as nucleotide + 1.
Multiple alignments of amino acid sequences were performed using clustal x software (http://www.clustal.org/). Prediction of mRNA secondary structure was carried out using centroidfold software (http://www.ncrna.org/centroidfold/).
Patients and Methods
Subjects
A total of 10 members of two unrelated families (family-1 and family-2) were recruited in the Departments of Paediatrics and Endocrinology of the 'Hospital Universitario de Guadalajara' (Spain), for genetic diagnosis of MODY 2 or MODY 3. A control group of 50 nondiabetic individuals was also tested for the presence of the novel mutation identified in the families. All subjects included in the study were native Spanish. The study followed the tenets of the Declaration of Helsinki, and informed consents were obtained from all the participants. The probands were clinically evaluated by experienced endocrinologists and/or paediatricians and met all the following criteria established by the Spanish Diabetes Society for MODY genetic testing: (i) autosomal dominant inheritance; (ii) hyperglycaemia diagnosis before the age of 25 years in at least one family member, and/or detectable C-peptide levels in patients with or without insulin treatment at least 3 years after the clinical diagnosis and (iii) negative pancreatic autoimmunity. Additional clinical parameters such as fasting blood glucose, blood glucose 2 h after OGTT (75 g of anhydrous glucose after an overnight fast), glycosylated haemoglobin (HbA1c), body mass index, follow-up therapy and evidence of diabetic retinopathy or nephropathy were also obtained.
Molecular Genetic Analysis
Genomic DNA was extracted from the peripheral leucocytes of all the subjects with the E.Z.N.A. Blood DNA kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer's instructions. The promoter (nucleotides -487 to -1356 in GCK and -226 to -414 in HNF1A), 5' UTR and the 10 exons of both GCK and HNF1A genes were screened for mutations by automated PCR-terminator cycle sequencing using the BigDye® (version 3·1) kit (Applied Biosystems, Foster City, CA, USA). The sequencing reaction products were analysed in an automated DNA sequencer (ABI Prism 3130 genetic analyzer; Applied Biosystems). Intronic primers were designed to allow for the analysis of splicing consensus sequences (Table S1). Mutations were named based on the cDNA reference sequence ENST00000403799 for GCK and ENST00000257555 for HNF1A genes (http://www.ensembl.org/index/html). We denoted the first nucleotide of the translation initiation site as nucleotide + 1.
In Silico Analyses
Multiple alignments of amino acid sequences were performed using clustal x software (http://www.clustal.org/). Prediction of mRNA secondary structure was carried out using centroidfold software (http://www.ncrna.org/centroidfold/).