Genetic evaluation of patients with Alström syndrome in the Polish population.

Alström syndrome (AS) is a rare syndromic form of obesity and type 2 diabetes (T2D) in children coexisting with retinal dystrophy and disorders of many organs caused by the mutations in ALMS1 gene. Aim of this study was to identify the causative mutations in ALMS1 in a group of 12 patients of Polish origin with clinical symptoms of AS, and their 21 first-degree relatives. Using DNA sequencing, nine different mutations including three novel were identified. These mutations were not present in 212 Polish individuals with no symptoms of AS, subjected to whole-exome sequencing and collected in a national registry. Looking for genotype-phenotype relationships, we confirmed a severe phenotype in a boy with homozygous mutation in exon 16, and a relationship between a presence of T2D and mutations in exon 19. Evaluation of the type of mutation and its clinical effects gives hope for earlier diagnosis of AS in future patients and more advanced therapeutic approaches for patients with already diagnosed AS.

HbA1c-based diabetes diagnosis among patients with glucokinase mutation (GCK-MODY) is affected by a genetic variant of glucose-6-phosphatase (G6PC2).

AIMS: Genetic variation at the rs560887 locus of the glucose-6-phosphatase, catalytic 2 gene (G6PC2) is known to affect regulation of fasting glycaemia. We determined the rs560887 genotype of patients with monogenic diabetes and glucokinase gene mutations (GCK-MODY) and correlated the genotypes with HbA(1c) levels. METHODS: Patients from families with GCK-MODY were recruited from two large cohorts from Poland (n = 128) and the Czech Republic (n = 154). Genotypes at the rs560887 polymorphic site in G6PC2 were examined using real-time quantitative polymerase chain reaction. The effect of rs560887 genotype on age at diagnosis of GCK-MODY and initial HbA(1c) levels were evaluated separately within both cohorts. Following that, a meta-analysis of rs560887 genotype-HbA(1c) associations of both Polish and Czech cohorts was performed to confirm homogeneity of findings and validate cohort-specific results. RESULTS: GG homozygosity at rs560887 was associated with marginally elevated HbA(1c) levels (P = 0.07 in both cohorts). The effects observed in both groups were very homogeneous (Q = 0.18; P = 0.68). Meta-analysis showed that GG homozygosity at rs560887 was associated with mean HbA(1c) levels higher by 2.4 mmol/mol (0.24%), 95% CI 0.5-4.4 mmol/mol (0.05-0.44%) than in individuals with other genotypes. Additionally, meta-analysis of both cohorts showed that GG homozygous individuals had higher odds of reaching the 48 mmol/mol (6.5%) diagnostic threshold of diabetes; (odds ratio 1.90; 95% CI 1.07-3.36; P = 0.03). No such effects were observed for age at diagnosis of diabetes. CONCLUSIONS: Variation at the rs560887 locus of G6PC2 is associated with worse glycated haemoglobin levels in individuals with GCK mutations; GG homozygotes are more likely to meet diagnostic criteria for diabetes based on HbA(1c) level.

Novel glucokinase mutations in patients with monogenic diabetes - clinical outline of GCK-MD and potential for founder effect in Slavic population.

Glucokinase (GCK) gene mutations are the causative factor of GCK-MD (monogenic diabetes) characterized by a mild clinical phenotype and potential for insulin withdrawal. This study presents the results of a nationwide genetic screening for GCK-MD performed in Poland. A group of 194 patients with clinical suspicion of GCK-MD and 17 patients with neonatal diabetes were subjected to GCK sequencing. Patients negative for GCK mutations were subjected to multiplex ligation-dependent probe amplification (MLPA) to detect deletions or insertions. A total of 44 GCK heterozygous mutations were found in 68 probands (35%). Among those, 20 mutations were novel ones: A282fs, D198V, E158X, G246V, G249R, I348N, L165V, L315Q, M115I, N254S, P284fs, Q338P, R377L, R43C, R46S, S212fs, S212P, T255N, V406A and Y214D. No abnormalities were detected in MLPA analysis. Homozygous D278E mutation was found in one patient with neonatal diabetes. The most frequently observed combinations of symptoms typical for GCK-MD were mild diabetes and/or fasting hyperglycaemia (98.3%), positive C-peptide at diagnosis (76%) and dominant mode of inheritance (59%). This study outlines numerous novel mutations of the GCK gene present in white Caucasians of Slavic origin. Thorough clinical assessment of known factors associated with GCK-MD may facilitate patient selection.

Doubling the referral rate of monogenic diabetes through a nationwide information campaign--update on glucokinase gene mutations in a Polish cohort.

In order to improve recruitment efficiency of patients with monogenic diabetes in Poland, in September 2010 a nationwide advertising campaign was launched to inform multiple target groups interested or participating in pediatric diabetologic care. Promotional actions aimed at informing physicians, patients, parents and educators were carried out through nationwide newspapers, medical and patient-developed websites and educational conference presentations. Recruitment efficiency was compared between September 2010 (publication of the first report on project's results) and the following 12 months. The number of families and patients referred to genetic screening was increased by 92% and 96% respectively nearly reaching the numbers recruited throughout the initial 4 years of the project. Participation of non-academic centers was also significantly increased from 2.3% to 7.5% (p = 0.0005). DNA sequencing and Multiplex Ligation-dependant Probe Amplification of the glucokinase gene resulted in finding 50 different mutations. Among those mutations, 19 were novel variants, which included: 17 missense mutations (predicted to be pathogenic according to bioinformatic analysis), 1 nonsense mutation and 1 mutation affecting a consensus intronic splice site. Advertising actions directed at increasing recruitment efficiency are a powerful and possibly neglected tool in screening for rare genetic disorders with a clinically defined phenotype.

The association between acyl-CoA synthetase (ACSL4) polymorphism and intramuscular fat content in (Landrace × Yorkshire) × Duroc pigs.

The aim of the study was to check whether different genotypes at acyl-CoA synthetase (ACSL4 locus, SNP G2645A) are associated with pork quality. 132 (Landrace × Yorkshire) × Duroc fatteners were genotyped by originally developed PCR-RFLP method. Upon the slaughter, the samples of longissimus lumborum muscle were taken from each carcass to determine the following parameters: content of water, protein and fat, pH (45 min, 24, 48, 96, and 144 h post mortem), electrical conductivity, drip loss, meat lightness, glycolytic potential, glycogen and lactate contents in meat. Among several associations observed, the highly significant (p<0.01) was found for intramuscular fat (IMF) content. Pigs with genotype GG revealed the highest content of IMF - 2.47%.

Wolfram syndrome in the Polish population: novel mutations and genotype-phenotype correlation.

OBJECTIVE: Wolfram syndrome is a rare form of diabetes mellitus associated with optic atrophy and disorders of different organs (e.g. diabetes insipidus, hearing loss, ataxia, anaemia and many others). This syndrome is caused by recessive mutations in the wolframin gene (WFS1) localized on chromosome 4p16·1. The aim of this study was to identify the causative mutations in WFS1 in a group of Polish patients with suspected Wolfram syndrome. PATIENTS AND MEASUREMENTS: Nine patients with clinical symptoms consistent with Wolfram syndrome (at least diabetes mellitus and optic atrophy) and 22 first-degree relatives were examined. The molecular analysis was carried out by direct sequencing of the exons, the exon-intron junctions, and the 5' and 3' untranslated regions of WFS1. RESULTS: Nine different mutations in WFS1 (five of them novel) were identified in the nine patients. Six patients were homozygous for the following mutations: V412fs, S443R, W539X, V659fs. They developed diabetes at a mean age of 5·2 years. Three patients were compound-heterozygous for the following mutations: S167fs, Q392X, Y513fs, W648X, V779G. They developed diabetes at a mean age of 6·5 years. CONCLUSIONS: Mean age of diagnosis of diabetes among the Polish patients was typical for Wolfram syndrome; however, compound-heterozygous patients were slightly older at diabetes onset.

Screening 52 single nucleotide polymorphisms for extreme value of glycolytic potential and drip loss in pigs.

Summary Low heritability of meat quality traits and the lack of their systematic registration in breeding programs have encouraged the search for single nucleotide polymorphisms (SNPs) located within genes coding the proteins involved in muscle and fat metabolism. In this report, a panel of 52 SNPs was used to find which alleles and genotypes are more/less frequent in groups of pigs differentiated by extreme value of glycolytic potential (GP) and drip loss (DL). The analysis was carried out in 52 fatteners (chosen from 246 pigs), of which 28 were Landrace and 27 Landrace x Yorkshire. Two designs were performed: I, fatteners were divided into two groups showing extreme value of GP (<125 versus >145), II, fatteners were divided into two groups showing extreme value of DL (<6.0 versus >6.0). Allele frequency differences between the phenotypic groups of extreme GL or DL were not influenced by the breed. The frequency of 52 SNPs alleles for each of group was calculated and a chi-squared test was used to estimate the significance of differences in allele frequencies between alternative groups in each experimental design. Three SNPs (DECR1, PPARGC1, MC4R) and another two (CYP21, SFRS1) showed significant differences between groups of extreme GP and DL, respectively. To exemplify and validate potential associations of candidate SNPs for GP and DL, 293 fatteners representing three commercial breeds/crosses (95 Landrace, 66 Landrace x Yorkshire and 132 Landrace x Yorkshire x Duroc were genotyped for DECR1 and CYP21 by PCR-RFLP assays. DECR1 showed significant associations with GP in Landrace and Landrace x Yorkshire x Duroc fatteners. CYP21 showed significant associations with DL in all breeds/crosses. Interestingly, the CYP21 polymorphism revealed adverse associations trend in Landrace x Yorkshire x Duroc pigs in comparison to Landrace and Landrace x Yorkshire fatteners.

The expression of genes PKM2 and CAST in the muscle tissue of pigs differentiated by glycolytic potential and drip loss, with reference to the genetic group.

The present studies aimed at an analysis of the expression level of genes PKM2 and CAST in Longissimus lumborum [LL] muscle tissue of pigs differing as regards the glycolytic potential [GP] and drip loss [DL] from the LL muscle, with reference to the genetic group. The studies covered a total of 65 pigs: 20 purebred Landrace [L], 22 crossbreeds of Landrace with the Yorkshire [L x Y] and 23 three-breed crosses (Landrace x Yorkshire) x Duroc [(L x Y) x D]. In the case of gene PKM2 one may observe in (L x Y) x D crossbreds, compared to L x Y crossbreds, an increased expression, closely related with the increase in dry matter content, including intramuscular fat, as well as a more favourable progress of glycolytic and energy metabolism during the early time post mortem (pH(45) and R(1)). Compared with Landrace animals, the lower expression of the CAST gene observed in (L x Y) x D pigs is manifested by a marked improvement of meat quality (R(1) pH(45) pH(24), pH(48)), arising from the rate of glycolytic and energy metabolism, typical for normal meat, that in effect results in its higher culinary and technological value.

The association between polymorphism of PKM2 gene and glycolytic potential and pork meat quality.

The objective of this study was to investigate the association of PKM2 gene with glycolytic potential and meat quality traits in three groups of fatteners - Landrace, Landrace x Yorkshire and (Landrace x Yorkshire) x Duroc. The present study was conducted on 243 fatteners, free of RYR1(T) gene, which 95 were of Landrace breed and the rest were the following crosses: 66 - Landrace x Yorkshire and 82 (Landrace x Yorkshire) x Duroc. It has been stated, that PKM2 gene (independently from the breed) was significantly associated with GP, lactate content, R(1) indicator, pH and drip loss. The presence of TT genotype may lead to increase of GP and lactate content and results in low pH(24) and pH(144) and bigger drip loss measured 96 and 144 h after the slaughter. Except for the landrace fatteners, the association of the PKM2 gene with the glycogen content has not been statistically confirmed. Statistically confirmed interaction shows, that the association of PKM2 gene with glycolytic potential and glycogen content concerns mainly the Landrace pigs. Moreover, a high (almost 89%) conformability of the genotype of PKM2 gene with the RN(-) phenotype, can serve as an additional argument in favour of the thesis.

The effect of interaction between genotype CAST/RsaI (calpastatin) and MYOG/MspI (myogenin) on carcass and meat quality in pigs free of RYR1(T) allele.

The purpose of the studies was to demonstrate to what degree genotypes of calpastatin (CAST/RsaI) and myogenin (MYOG) genes as well as the interaction between them may affect the carcass and meat quality of pigs. The investigations were conducted on 397 stress resistant pigs (free of RYR1(T) allele). It was demonstrated that the favourable effect of the variants of CAST and MYOG genes on carcass quality traits depends on the cut. The gene variant favourably affecting the weight of ham simultaneously had a negative effect on the weight of the loin. It was also shown that the interaction between CAST and MYOG genotypes has a significant effect on backfat thickness. The effect of a given combination of CAST and MYOG genotypes on carcass traits is related to the weight of a substantial cut (ham, loin). Genotypes at loci CAST/RsaI and MYOG have a significant effect on the value of certain traits and parameters of meat quality and its technological value (genotype CAST on pH at 35min and 2, 3, 24, 48, 96, 144h post-mortem (pH(35), pH(2), pH(3), pH(24), pH(48), pH(96), pH(144), respectively), R(1) (IMP/ATP), electrical conductivity at 3 and 4h post-mortem (EC(3), EC(4)), technological yield of meat in curing and thermal processing (TY) and protein content in the muscle tissue, while genotype MYOG on pH(48), EC(35), EC(3), EC(24) and dry matter content).