Mutational analysis of candidate genes in psychiatric disorders

A genetic hypothesis for a disease presupposes the existence of variation in the DNA sequences of affected individuals. A series of techniques known together as “mutational analysis” can be applied towards identifying new sequence variations in selected genes. These techniques can screen a large series of individuals for mutations efficiently, so it is not necessary to determine the nucleotide sequence in every DNA sample. DNA samples suspected of harboring sequence variants are then sequenced. Denaturing gradient gel electrophoresis techniques, single stranded conformation polymorphism paradigms, and chemical cleavage of mismatches are 3 procedures widely used for the molecular screening of mutations today. We discuss each of these techniques for mutation screening. © 1993 Wiley-Liss, Inc.     

Single nucleotide polymorphisms of cytokine genes in the healthy Slovak population

Cytokines are molecules that control and modulate the activities of numerous target cells via binding to specific receptors. The observed differences in the cytokine production among individuals can be, at least partially, explained by gene polymorphisms. Several cytokine gene polymorphisms have been identified to play a role in susceptibility to various diseases, including autoimmune, infectious, allergic or cardiovascular diseases. The aim of the current study was to determine allele and genotype frequencies of 22 polymorphisms in 13 cytokine genes in the healthy Slovak population and to compare them with data available from six populations from Central and Southern Europe. A polymerase chain reaction with sequence-specific primers was used to genotype polymorphisms within genes encoding IL-1alpha, IL-1beta, IL-1R, IL-1RA, IL-4Ralpha, IL-12, IFN-gamma, TGF-beta, TNF-alpha, IL-2, IL-4, IL-6 and IL-10 in a sample of 140 unrelated Slovak subjects. The allelic distribution of all polymorphisms in the Slovak population was very close to that in the geographically and historically closest populations in Central Europe--the Czech and the Polish. However, several differences were found between the Slovak and four populations from Southern Europe. The obtained data represent a basis for further studies on association of cytokine gene polymorphisms with some diseases.     

Characterization of 458 single nucleotide polymorphisms of disease candidate genes in the Korean population

Single nucleotide polymorphisms (SNPs) are considered as very promising genetic markers for complex disease gene hunting. However, it has been demonstrated that there are significant ethnic differences in genetic variations. In order to investigate the genetic variations in the Korean population and their ethnic differences, a large number of SNPs of 161 disease candidate genes were collected from a publicly available SNP database and then tested for the distribution of allele frequency in the Korean population. Of all 458 SNPs tested, approximately 43.9% were polymorphic in the Korean population, whereas 44.5% were monomorphic. The remaining 11.6% were failed in the test. Significant differences have been observed when SNP allele frequency pattern of Koreans was compared with those of Caucasians and Africans, whereas this pattern was highly similar between Korean and Japanese populations. Our data indicate that although many of the SNPs available in publicly available database, especially coding-region SNPs (cSNPs), can be used as informative genetic markers for disease association studies, an extensive verification of public SNPs in a particular population studied should be undertaken prior to their association studies. Electronic Publication     

Genetic analysis of dyslexia candidate genes in the European cross-linguistic NeuroDys cohort

Dyslexia is one of the most common childhood disorders with a prevalence of around 5–10% in school-age children. Although an important genetic component is known to have a role in the aetiology of dyslexia, we are far from understanding the molecular mechanisms leading to the disorder. Several candidate genes have been implicated in dyslexia, including DYX1C1, DCDC2, KIAA0319, and the MRPL19/C2ORF3 locus, each with reports of both positive and no replications. We generated a European cross-linguistic sample of school-age children – the NeuroDys cohort – that includes more than 900 individuals with dyslexia, sampled with homogenous inclusion criteria across eight European countries, and a comparable number of controls. Here, we describe association analysis of the dyslexia candidate genes/locus in the NeuroDys cohort. We performed both case–control and quantitative association analyses of single markers and haplotypes previously reported to be dyslexia-associated. Although we observed association signals in samples from single countries, we did not find any marker or haplotype that was significantly associated with either case–control status or quantitative measurements of word-reading or spelling in the meta-analysis of all eight countries combined. Like in other neurocognitive disorders, our findings underline the need for larger sample sizes to validate possibly weak genetic effects.     

Serum total IgE levels in a representative sample of a Greek population

The distribution of IgE in a large randomly stratified Greek population sample was determined in 1187 subjects (793 men and 394 women) aged between 20 and 60 years. Skin prick testing was performed and serum total IgE expressed in iu/ml was measured by Phadebas PRIST: the data are presented as the geometric mean. Subjects were classified as atopic (257 men, 118 women) and nonatopic (536 men, 276 women) according to the results of skin testing with various aeroallergens. At any age, atopic males (120.5 vs 38 iu/ml) and females (99.8 vs 29.3 iu/ml) had higher mean IgE levels, as compared to nonatopic subjects ( P <0.0001). In our adult nonatopic sample, IgE levels did not differ with age ( P >0.05). At any age, nonatopic males had higher (38 iu/ml) mean IgE levels than nonatopic females (29.3 iu/ml) ( P <0.05). The comparison of normal IgE values (nonatopic subjects) from this study with those reported by other investigators revealed that Greek adult males and females had higher IgE levels than populations from other nations. Our results represent the first report on reference values regarding serum total IgE in Greek adults.     

ChIP-chip analysis of neurexins and other candidate genes for addiction and neuropsychiatric disorders

Abstract:

Several addiction susceptibility genes have been mapped by linkage and genomewide association. However, functional alleles associated with disease risk have not been identified, with a few possible exceptions. In addition, little is known about the cis- and trans-acting factors involved in regulating their expression. To address these issues, we used a ChIP-chip approach to identify regulatory elements in fetal-brain– targeting genes implicated in addiction and other neuropsychiatric conditions. Our data point to a number of putative regulatory elements, several of which, we show, are functionally significant. Many established or putative regulatory elements map near-disease–associated SNPs. These regions would be of interest to survey for patient-specific functional variants involved in disease susceptibility.     

高密度脂蛋白代谢相关基因单核苷酸多态性研究

目的探讨中国人群高密度脂蛋白代谢相关基因单核苷酸多态性(singlenucleotidepolymorphism,SNP)的分布特征。方法对象选自中国西部5省区健康汉族个体209人,男132名、女77名,平均年龄(59±10)岁。以蛋白酶K消化、苯酚及氯仿抽提以及异丙醇沉淀的方法提取全基因组DNA。应用聚合酶链反应、限制性内切酶片段长度多态性结合测序的方法检测ATP结合盒转运体(ATP-bindingcassettetransporter,ABCA1)、胆固醇酯转运蛋白(cholesteryleastertransferprotein,CETP)以及脂蛋白脂酶(lipoproteinlipase,LPL)等高密度脂蛋白代谢相关基因SNP。结果该研究群体等位基因ABCA1-A和G的基因频率分别为53.4%和46.6%;CETP-B1和B2分别为59.0%和41.0%;LPL-H(-)和LPL-H(+)分别为18.9%和81.1%;LPL-P(+)和LPL-P(-)分别为66.0%和34.0%,符合Hardy-Weinberg平衡定律。LPL基因多态位点Hind(+)与Pvu(+)之间存在显著的连锁不平衡。将性别因素作分层分析提示CETPTaq1B基因型频率存在明显的性别差异(χ2=9.94,P=0.0075)。测序表明上述限制性内切酶片段长度多态性均系内切酶识别区域内的单核苷酸碱基替换。中国人种与白人种基因多态性分布频率对照研究发现,CETP-Taq1B等位基因频率接近,而其余3种等位基因频率分布存在显著性差异。结论中国汉族人     

Climacteric symptoms in a representative Dutch population sample as measured with the Greene Climacteric Scale

OBJECTIVE: To measure climacteric symptoms in a population-based survey as assessed by the Greene Climacteric Scale and to obtain normative data for the total score and subscales (psychological, somatic, vasomotor, and sexual) of the Greene Climacteric Scale. METHODS: A sample representative of the Dutch female population is interviewed. The sample was drawn from the NIPO-Telepanel (with 269 women aged 45-65 years) and from the NIPO-CAPI@HOME database (a sample of 235 women aged 45-65 years). They all filled in the 21 items of the Greene Climacteric Scale. The women were divided in four groups according their menopausal status: premenopausal, perimenopausal, postmenopausal and posthysterectomy. RESULTS: The total score of the Greene Climacteric Scale (mean; SD) was in premenopausal women 10.53 +/- 7.36). The score in perimenopausal women (15.78 +/- 9.09) and postmenopausal women (15.33 +/- 9.01) were significant higher than in the premenopause. The same significant difference between pre and peri/postmenopausal women was observed in the psychological, somatic and vasomotor subscales. The depression subscale did not change significantly during the menopausal transition. Hysterectomized women had the same score as postmenopausal women, reflecting the rather high mean age of the hysterectomized women (55.8 years). CONCLUSIONS: Prevalence and intensity of climacteric symptoms as expressed in the Greene Climacteric Scale do increase during the menopausal transition and stay high during the postmenopause. Data presented can be considered normative for the Greene Climacteric Scale in a mainly Caucasian population.     

Single exon-resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes

Intellectual disability affects about 3% of individuals globally, with∼50% idiopathic. We designed an exonic-resolution array targeting all known submicroscopic chromosomal intellectual disability syndrome loci, causative genes for intellectual disability, and potential candidate genes, all genes encoding glutamate receptors and epigenetic regulators. Using this platform, we performed chromosomal microarray analysis on 165 intellectual disability trios (affected child and both normal parents). We identified and independently validated 36 de novo copy-number changes in 32 trios. In all, 67% of the validated events were intragenic, involving only exon 1 (which includes the promoter sequence according to our design), exon 1 and adjacent exons, or one or more exons excluding exon 1. Seventeen of the 36 copy-number variants involve genes known to cause intellectual disability. Eleven of these, including seven intragenic variants, are clearly pathogenic (involving STXBP1, SHANK3 (3 patients), IL1RAPL1, UBE2A, NRXN1, MEF2C, CHD7, 15q24 and 9p24 microdeletion), two are likely pathogenic (PI4KA, DCX), two are unlikely to be pathogenic (GRIK2, FREM2), and two are unclear (ARID1B, 15q22 microdeletion). Twelve individuals with genomic imbalances identified by our array were tested with a clinical microarray, and six had a normal result. We identified de novo copy-number variants within genes not previously implicated in intellectual disability and uncovered pathogenic variation of known intellectual disability genes below the detection limit of standard clinical diagnostic chromosomal microarray analysis.     

Single nucleotide polymorphisms in the regulatory regions of the HLA-DRB-expressed genes

DRB genes encode proteins that play an important role in the immune response, and their expressional regulation is crucial to the immune reaction. Sequence variation at the regulatory region can directly affect the gene expression level. The aim of the present study was to use Chinese samples to investigate the variation in the regulation region of the human leukocyte antigen (HLA)-DRB-expressed genes. Seventy- one single nucleotide polymorphisms (SNPs) were found in the four HLA-DRB-expressed genes. By comparing these data with SNPs in the U.S. National Center for Biotechnology Information dbSNP database, 69 SNPs (97.2%) were found to be novel. In addition, two genetic variations of insertion-deletion polymorphisms were discovered within the regulatory region of HLA-DRB1 gene. These polymorphisms can be used as resources of markers for association studies of complex diseases, for assessment of individual predisposition to diseases, and as research markers for population genetics and evolution.     

Allele frequencies for 15 short tandem repeat loci in representative sample of Croatian population

Aim

To study the distribution of allele frequencies of 15 short tandem repeat (STR) loci in a representative sample of the Croatian population.

Methods

A total of 195 unrelated Caucasian individuals born in Croatia, from 14 counties and the City of Zagreb, were sampled for the analysis. All the tested individuals were voluntary donors. Buccal swab was used as the DNA source. AmpFlSTR^® Identifiler^® was applied to simultaneously amplify 15 STR loci. Total reaction volume was 12.5 μL. The polymerase chain reaction (PCR) amplification was carried out in PE Gene Amp PCR System Thermal Cycler. Electrophoresis of the amplification products was preformed on an ABI PRISM 3130 Genetic Analyzer. After PCR amplification and separation by electrophoresis, raw data were compiled, analyzed, and numerical allele designations of the profiles were obtained. Deviation from Hardy-Weinberg equilibrium, observed and expected heterozygosity, power of discrimination, and power of exclusion were calculated. Bonferroni’s correction was used before each comparative analysis.

Results

We compared Croatian data with those obtained from geographically neighboring European populations. The significant difference (at P<0.01) in allele frequencies was recorded only between the Croatian and Slovenian populations for vWA locus. There was no significant deviation from Hardy-Weinberg equilibrium for all the observed loci.

Conclusion

Obtained population data concurred with the expected “STR data frame” for this part of Europe.In nearly thirty years since its first formal application (1), DNA analysis has been promoted into a core method for different types of examination, from routine paternity testing and massive identification of human remains to complicated forensic casework analysis (2). Introduction of a set of short tandem repeat (STR) loci as the markers induced a significant progress in this field of science (3-5). DNA typing for forensic, identification, and paternity testing purposes is based on the same techniques that are routinely employed in a wide range of medical and genetic situations, such as diagnosis, population genetic studies, and gene mapping (6). Therefore, importance of understanding the used marker heterogeneity within different populations is constantly emphasized (2,7).Different number and different sets of STR loci in a different number of the individuals of different geographical origin were used in previous studies of the Croatian population. Namely, 8 STR loci have already been employed in a study of the population of Republic of Croatia (8) and 13 CODIS STR loci in the analysis of the population of Southern Croatia (9). Here we have analyzed the distribution of allele frequencies at 15 STR loci in a representative sample of the Croatian population. Additionally, we compared these data with those obtained from geographically neighboring European populations.