MODY type 2 P59S GCK mutant: founder effect in South of Italy

Clin Genet 2013: 83: 83 87 Printed in Singapore. All rights reserved Short Report 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd CLINICAL GENETICS doi: 10.1111/j.1399-0004.2012.01856.x MODY type 2 P59S GCK mutant: founder effect in South of Italy Delvecchio M, Ludovico O, Bellacchio E, Stallone R, Palladino T, Mastroianno S, Zelante L, Sacco M, Trischitta V, Carella M. MODY type 2 P59S GCK mutant: founder effect in South of Italy. Clin Genet 2013: 83: 83 87. John Wiley & Sons A/S. Published by Blackwell Publishing Ltd, 2012 Mutations in the glucokinase (GCK ) gene are the most frequent cause of maturity onset diabetes of the young (MODY) in Italy. We evaluated GCK mutations in 32 unrelated patients younger than 18 years who had been diagnosed with MODY. Eleven different GCK heterozygous mutations were identified in 22 (68.7%) of the 32 probands. Nine mutations were missense and two were nonsense. Three of these mutations (E17X, P59S and E372X) have not been described previously and were shown to be associated with hyperglycaemia. Several prediction methods suggested that the E17X and E372X mutations result in a premature truncated protein and that the P59S mutation is pathogenic. This idea was further supported by evidence suggesting that Proline 59 is a highly conserved amino acid residue and that the P59S mutation does not appear to be present in non-diabetic controls and in sequence variant databases. Furthermore, this mutation was found in six (27.3%) of the patients from the same geographical area, Gargano, pointing to the existence of a founder effect, which was confirmed by microsatellite analysis. Conflictofinterest Nothing to declare. M Delvecchio a, O Ludovico b, E Bellacchio c, R Stallone d, T Palladino d, S Mastroianno b, L Zelante d, M Sacco a, V Trischitta e,f and M Carella d a Paediatrics Unit, and b Endocrine Unit, Hospital, San Giovanni Rotondo, Italy, c Research Laboratories, Bambino Gesù Children s Hospital, IRCCS, Rome, Italy, d Medical Genetics Unit, and e Research Unit of Diabetes and Endocrine Diseases, Hospital, San Giovanni Rotondo, Italy, and f Department of Experimental Medicine, Sapienza University, Rome, Italy Key words: diabetes founder effect GCK-MODY genetics paediatrics These authors equally contributed to this article. Corresponding author: Maurizio Delvecchio, MD, PhD, Paediatrics Unit, Hospital, Viale Cappuccini 1, San Giovanni Rotondo, 71013 San Giovanni Rotondo (FG), Italy. Tel.: +39 3393 379630; fax: +39 0882 416200; e-mail: mdelvecchio75@gmail.com Received 27 October 2011, revised and accepted for publication 13 February 2012 Maturity onset diabetes of the young (MODY; MIM# 606391) is a genetically and clinically heterogeneous form of diabetes mellitus, usually defined as non-insulin-dependent diabetes. MODY is characterized by hyperglycaemia or overt diabetes in at least two or three consecutive generations, onset before the age of 25 years and the absence of antipancreatic antibodies. There are at least seven different subtypes of MODY (1), each of which is associated with a mutated gene. In Italy, the glucokinase (GCK ) gene is the most frequent mutation, with the heterozygous-inactivating mutations causing a subtype of MODY [subtype glucokinase (GCK-MODY)] formally known as MODY2 (2), which accounts for up to 60% of all cases, at least in childhood (3). GCK plays a critical role in blood glucose homeostasis through the regulation of insulin secretion by pancreatic β-cells and glucose uptake and storage in the liver (4). Hundreds of GCK -inactivating mutations have been shown to segregate with GCK-MODY (5, 6). To evaluate GCK mutations in a cohort of children and adolescents diagnosed with MODY, we analysed 83

Delvecchio et al. Table 1. GCK gene mutations reported in the study Region Nucleotide change Protein effect Number of probands (this report) References Exon 2 c.49g>t p.glu17x 1 Novel c.175c>t p.pro59ser 6 Novel Exon 3 c.218a>g p.asp73gly 1 Massa et al. (7) Exon 4 c.401t>c p.leu134pro 1 Massa et al. (7) Exon 5 c.571c>t p.arg191trp 1 Ellard et al. (20) Exon 6 c.667g>a p.gly223ser 7 Massa et al. (7) c.676g>a p.val226met 1 Velho et al (21) Exon 7 c.683c>t p.thr228met 1 Stoffel et al (22) c.704t>c p.met235thr 1 Gloyn et al (23) Exon 8 c.866a>g p.tyr289cys 1 Osbak et al. (5) Exon 9 c.1114g>t p.glu372x 1 Novel Fig. 1. MView output of protein sequence alignment performed with WU-Blast program (http://www.ebi.ac.uk/tools/sss/wublast/) using the glucokinase protein sequence (Homo sapiens; NP_000153.1) as input. The organism and the sequence accession number are indicated on the left; the numbers on the top indicate the amino acid position on the input sequence. The grey shading indicates Glu17, Pro59 and Glu372. the coding and the promoter region of the gene. In this study, we report on three novel mutations (E17X, P59S and E372X), focusing our attention on one, P59S, which was particularly frequent in our samples and which showed a founder effect in our population. Materials and methods Thirty-two unrelated children and adolescents (2.5 17.9 years old) who satisfied the diagnostic criteria for MODY (1) were recruited. The criteria were (i) incidental hyperglycaemia (blood glucose >100 mg/dl) confirmed on a second blood sampling; (ii) the absence of islet cell antibodies, of insulin autoantibodies, of IA-2 antigens, and of GAD antibodies; (iii) three consecutive generations with hyperglycaemia diagnosed before age 25 years. Informed consent for the GCK gene mutational analysis was obtained from all of the probands parents. Genomic DNA was extracted from peripheral blood lymphocytes using Biorobot EZ1 (QIAGEN GmbH, Hilden, Germany). The coding and promoter regions of the GCK gene (exons 1a and 2 10; RefSeq NM_000162.3) were amplified by polymerase chain reaction (PCR) using gene-specific oligonucleotide primers (sequences available on request), and all of the amplicons were sequenced using a BigDye Seq kit 1.1 (Applied Biosystems) according to the manufacturer s recommendations. The reactions were analysed on an ABI 3130xl DNA sequencer (Applied Biosystems, Foster City, CA), and the results were analysed with Sequencher software v5.0 (GeneCodes, Ann Arbor, MI). All of the mutations were validated at least once on a second PCR product. To assess whether the P59S mutation was derived from a common ancestor, we analysed three polymorphic markers spanning approximately 7.5 cm on the GCK locus (D7S678, D7S2488 and D7S2427). The sense set of the PCR primers was labelled fluorescently, 84

MODY type 2 P59S GCK mutant Fig. 2. Haplotype analysis of the three markers surrounding the glucokinase (GCK ) gene in the six families carrying the P59S mutation; the marker order is telomere, D7S678, P59S-GCK, D7S2488, D7S2427, centromere. In relation to the GCK-P59S allele, N denotes no mutation and M denotes mutation. The arrow denotes the proband. Hyperglycaemic individuals are indicated with filled symbols; normoglycaemic individuals are indicated by open symbols. The number under each member represents the sample identifier. and the PCR products of these reactions were analysed using the GeneMapper system of ABI PRISM 3130xl (Applied Biosystems). Protein sequence alignment was performed with WU-Blast program (http://www.ebi.ac.uk/tools/sss/ wublast/) using the GCK protein sequence (Homo sapiens; NP_000153.1) as input. Results Eleven different GCK heterozygous mutations were identified in 22 (68.7%) of the 32 probands. Nine of the mutations (P59S, D73G, L134P, R191W, G223S, V226M, T228M, M235T and Y289C) were missense, and two (E17X and E372X) were nonsense. The most frequent gene mutation was G223S (in 7 of 22 patients, 31.8%) and P59S (in 6 of 22 patients, 27.3%). All of the other mutations were detected in single pedigrees (Table 1). To the best of our knowledge, three of these mutations (E17X, P59S and E372X) have not been described previously. These mutations were shown to segregate with hyperglycaemia in the family studies. They were not detected in 100 nondiabetic controls and were not reported in singlenucleotide polymorphism databases, the 1000 genomes database (www.1000genomes.org) or the lovd database (www.lovd.nl). Protein sequence alignments with multiple species indicated a very strong degree of conservation of the altered amino acid in the three mutations (Fig. 1). Glutamic acid at position 17 was conserved in all of the species tested, except for Actinopterygii and Amphibia, where this amino acid was an aspartic acid. Proline at position 59 was highly conserved and was never replaced with a serine residue. Glutamic acid at position 372 was also highly conserved. The novel E17X and E372X mutations were predicted to result in a premature truncated protein, whereas several prediction methods indicated that P59S is pathogenic. PolyPhen classified this mutation as probably damaging, whereas SIFT predicted this substitution as damaging. P59S was found in 6 (27.3%) of the 22 patients, all of whom originated from a small area in Italy known as Gargano, suggesting the 85

Delvecchio et al. Table 2. Clinical characteristics of families carrying the P59S mutation a Subject Age at DM diagnosis (years) BMI (kg/m 2 ) c-peptide (ng/ml) BP (mmhg) FPG level (mg/dl) HbA 1c (%) Medication Family A Proband, III:2 13 20.4 95/70 113 5.8 Diet II:1 26 25.1 120/60 137 6.5 Diet II:2 55 33.1 121/79 178 10.0 Sulfonylurea II:3 115/70 89 II:4 110/73 97 II:6 27 22.8 100/70 113 5.7 Diet II:8 20 21.1 110/82 134 6.8 Diet III:1 113/62 84 4.7 Family B Proband, III:5 6.9 15.8 0.8 108 5.4 None II:3 21 22.2 111 5.9 None II:5 25 0.7 115 5.8 None III:2 7 14.6 0.9 105 5.3 None III:3 10.3 0.7 116 5.8 None III:4 84 Family C Proband, III:1 5.5 14.2 0.8 110 5.7 None II:1 24 23.0 113 6.0 None II:2 27 21.5 118 6.1 Diet II:3 81 5.1 III:2 78 5.1 Family D Proband, II:1 5.2 18.2 107 5.8 None I:1 24 21.3 110 5.7 None I:2 83 II:2 82 Family E Proband, III:2 2.5 13.2 0.8 106 5.7 None I:2 45 135 Diet II:1 24 22.0 129 None II:2 78 III:1 1.9 17.0 1.2 110/65 104 5.6 None Family F Proband, III:1 11 21.7 1.6 90/65 112 5.9 None II:2 32 20.4 1.4 110/75 134 6.3 None II:3 1.4 83 4.7 II:4 26 28.4 2.1 120/80 137 6.5 None II:6 28 1.5 130/80 132 6.1 None BMI, body mass index; BP, blood pressure; DM, diabetes mellitus; FPG, fasting plasma; HbA 1c, glycated haemoglobin. a Mean ± SD was 118.1 ± 11.7 mg/dl in P59S carriers and 83.9 ± 5.6 in normoglycaemic non-mutated subjects (p between groups was <0.001, ANOVA test). existence of a founder effect. Three microsatellite markers flanking the GCK gene were tested within the six families. All of the 13 mutation carriers, but none of the nine healthy relatives of the families, shared a common haplotype, confirming a founder effect for the P59S mutation. The six pedigrees carrying the P59S mutation are shown in Fig. 2, and the clinical features of each subjects are presented in Table 2. At recruitment, none of the patients carrying the P59S mutation showed any complication due to hyperglycaemia. The subject II.2 in family A was hyperglycaemic, but her GCK sequence was normal. She was classified as having type 2 diabetes mellitus based on her clinical data (age at recruitment 55 years, undergoing treatment with sulfonylurea, body mass index 33.1 kg/m 2 ) and her biochemistry (HbA 1c 10%, cholesterol 225 mg/dl). Normoglycaemic subjects (fasting plasma glucose <100 mg/dl in two different samples), if available, were tested for GCK mutations and found to be negative. The probands with the wild-type GCK sequence are currently undergoing further screening to identify mutations in other MODY genes (results not yet available). Discussion GCK-MODY is the most frequent subtype of MODY in children and adolescents with asymptomatic 86

MODY type 2 P59S GCK mutant hyperglycaemia in South and Central Europe (8 11). In our study, more than 60% of the subjects were diagno6ed with GCK-MODY, confirming data already described in much larger studies, including Italian populations (3, 7). Overall, in 22 unrelated probands affected by GCK- MODY, we detected 11 mutations in the GCK gene. Among these, three, i.e. E17X, P59S and E372X, have not been described previously. The novel E17X and E372X mutations were detected in only one pedigree and are predicted to lead to a premature truncated protein. The P59S mutation, which results in the replacement of Proline 59 with Serine, was quite prevalent in our sample, being observed in about 30% of the GCK-MODY patients. The highly conserved Proline 59 residue in the species, the absence of Serine 59 in the non-diabetic controls and in the sequence variant databases as well as the SIFT and PolyPhen predictions support the hypothesis of the P59S pathogenetic effect. Furthermore, we showed that this mutation cosegregates with hyperglycaemia in all of the affected families. From a metabolic point of view, the patients carrying the P59S mutation showed the typical GCK-MODY phenotype, which is characterized by mild fasting hyperglycaemia and a small 2-h OGTT blood glucose increase (12). In common with previous studies (12 15), the most frequent mutation in our sample was G223S. Interestingly, although all of the mutations were detected in subjects from various parts of the study region, the P59S mutation was only found in six families living in a small area called Gargano. A more detailed familial history revealed that all of these have ancestors from the same small town, suggesting a founder effect. To support this, we performed microsatellite analysis which confirmed the hypothesis. To the best of our knowledge, most of the GCK mutations are private, with just a few previous reports suggesting the possibility of a founder mutation for the six gene variants (13, 16 19). The microsatellite analysis supported a founder effect for only one of these mutations, E339G (18). In summary, our paper describes the genetic analysis of GCK in children and adolescents with MODY in Italy and confirms that GCK is the most frequently mutated gene in Italian MODY patients. We describe three novel GCK mutations, one of which, P59S, showed a founder effect. This is the second-ever report of a genetically proven founder effect for GCK-MODY. Acknowledgements The authors thank Francesco Gallo, Maria Susanna Coccioli, Giuliana Cardinale, Elvira Piccino, Cataldo Torelli and Raffaele Gurrado for clinical evaluation; Mattia Miroballo for microsatellite analysis and Pietro Palumbo for pathogenetic prediction analysis. References 1. Hattersley A, Bruining J, Shield J, Njolstad P, Donaghue KC. 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