A DRD2 and ANKK1 haplotype is associated with nicotine dependence.

To test the importance of the dopamine D2 receptor (DRD2) region in nicotine dependence, 150 smokers and 228 controls were genotyped for the DRD2 C957T, -141delC and ANKK1 TaqIA polymorphisms (rs6277, rs1799732 and rs1800497, respectively). The -141delC SNP did not show any association but both the C957T and TaqIA SNPs showed association at the allele, genotype, haplotype and combined genotype levels. The 957C/TaqI A1 haplotype was more than 3.5 times as likely to be associated with nicotine dependence compared with the 957T/TaqI A1 haplotype (P=0.003). Analysis of the combined genotypes of both SNPs revealed that individuals who were homozygous for the 957C-allele (CC) and had either one or two copies of the TaqI A1-allele were 3.3 times as likely to have nicotine dependence compared to all other genotype combinations (P=0.0003) and that these genotypes accounted for approximately 13% of the susceptibility to nicotine addiction in our population. Our findings suggest that the DRD2 C957T polymorphism and the ANKK1 TaqIA polymorphism are key contributors to the genetic susceptibility to nicotine dependence.

DRD2 C957T and TaqIA genotyping reveals gender effects and unique low-risk and high-risk genotypes in alcohol dependence.

AIMS: As recent conflicting reports describe a genetic association between both the C- and the T-alleles of the dopamine D2 receptor (DRD2) C957T polymorphism (rs6277) in alcohol-dependent subjects, our aim was to examine this polymorphism and TaqIA (rs1800497) in Australian alcohol-dependent subjects. METHODS: The C957T polymorphism was genotyped in 228 patients with alcohol dependence (72 females and 156 males) and 228 healthy controls. RESULTS: The C-allele and C/C genotype of C957T was associated with alcohol dependence, whereas the TaqIA polymorphism was not. When analysed separately for C957T, males showed an even stronger association with the C-allele and females showed no association. The C957T and TaqIA haplotyping revealed a strong association with alcohol dependence and a double-genotype analysis (combining C957T and TaqIA genotypes) revealed that the relative risk of different genotypes varied by up to 27-fold with the TT/A1A2 having an 8.5-fold lower risk of alcohol dependence than other genotypes. CONCLUSION: Decreased DRD2 binding associated with the C-allele of the DRD2 C957T polymorphism is likely to be important in the underlying pathophysiology of at least some forms of alcohol dependence, and this effect appears to be limited to males only.

Assessment of DNA degradation and the genotyping success of highly degraded samples.

DNA becomes progressively more fragmented as biological tissue degrades resulting in decreasing ability to gain a complete DNA profile. Successful identification of samples exhibiting very high levels of DNA degradation may be complicated by presenting in minute quantities. The industry standard method for human DNA identification utilising short tandem repeats (STR) may produce partial or no DNA profile with such samples. We report a comparative study of genotyping using STRs, mini-STRs and single nucleotide polymorphisms (SNPs) with template at different levels of degradation in varying amounts. Two methods of assessing quantity and quality of a DNA sample prior to genotyping were investigated. The QIAxcel capillary gel electrophoresis system provided a rapid, cost effective screening method for assessing sample quality. A real-time quantitative PCR (qPCR) assay was able to simultaneously quantify total human DNA, male DNA, DNA degradation and PCR inhibition. The extent of DNA degradation could be assessed with reasonable accuracy to 62.5 pg and genomic targets could be quantified to a lower limit of 15.6 pg. The qPCR assay was able to detect male DNA to a lower limit of 20 pg in a 1:1,000 background of female DNA. By considering the amount of DNA and the degradation ratio of a sample, a general prediction of genotyping success using AmpFlSTR® Profiler Plus®, MiniFiler™ kits and SNP analysis can be made. The results indicate mini-STRs and SNP markers are usually more successful in typing degraded samples and in cases of extreme DNA degradation (≤200 bp) and template amounts below 250 pg, mini-STR and SNP analysis yielded significantly more complete profiles and lower match probabilities than corresponding STR profiles.

The DRD2 gene 957C>T polymorphism is associated with posttraumatic stress disorder in war veterans.

BACKGROUND: Variations in genes related to the dopaminergic pathway have been implicated in neuropsychiatric disorders such as schizophrenia, substance misuse, Alzheimer's disease and Post Traumatic Stress Disorder (PTSD). A single nucleotide polymorphism (SNP) (957C>T) and a deletion polymorphism (-141delC) in the DRD2 gene and a SNP (Taq1A) in a gene directly downstream of DRD2 have all been implicated in dopamine functioning in the brain. METHODS: To test the importance of these three polymorphisms in PTSD susceptibility, a genetic screen was performed in 127 war veterans diagnosed with PTSD and 228 control individuals without a history of PTSD. RESULTS: No significant association was found between PTSD and the Taq1A or -141delC polymorphisms. However, a significant association was observed with PTSD and the 957C>T polymorphism. PTSD individuals were more likely to carry the C allele compared to the controls (P=0.021). CONCLUSIONS: Our findings suggest that the 957C>T polymorphism in the DRD2 gene is one of the genetic factors for susceptibility to PTSD.

Identification of genes differentially expressed by prematurely fused human sutures using a novel in vivo - in vitro approach.

Craniosynostosis is the premature fusion of calvarial sutures. It results from abnormal differentiation or proliferation of cells within the osteogenic fronts of growing calvarial bones. To date, research has focused on animal models and in vitro organ and tissue culture to determine the molecular mechanisms controlling calvarial suture morphogenesis. Here, we test a new, in vivo-in vitro approach based on the hypothesis that calvarial suture cells passaged in minimal medium exhibit a stable gene expression profile similar to undifferentiated osteoblastic cells that can provide a benchmark for comparison with in vivo expression of differentiated tissue. We show that tissue-specific expression is lost after the first passage and, using cDNA microarrays, compare expression between fused suture tissue from craniosynostosis patients and in vitro de-differentiated explant cells. A large number of differentially expressed genes were identified, including novel genes WIF1, LEF1, SATB2, RARRES1, DEFA1, DMP1, PTPRZ1, and PTPRC, as well as those commonly associated with human suture morphogenesis, e.g., FGF2, MSX2, and BMP2. Two differentially expressed genes, WIF1 and FGF2, were further examined in an in vivo-in vivo comparison between unfused and prematurely fused tissue. The same pattern of differential expression was observed in each case, further validating the ability of our in vivo-in vitro approach to identify genes involved in in vivo human calvarial tissue differentiation.

Validity of low copy number typing and applications to forensic science.

Low copy number (LCN) typing, particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg of template DNA. Generally, LCN typing simply can be defined as the analysis of any DNA sample where the results are below the stochastic threshold for reliable interpretation. There are a number of methodologies to increase sensitivity of detection to enable LCN typing. These approaches encompass modifications during the polymerase chain reaction (PCR) and/or post-PCR manipulations. Regardless of the manipulations, when processing a small number of starting templates during the PCR exaggerated stochastic sampling effects will occur. The result is that several phenomena can occur: a substantial imbalance of 2 alleles at a given heterozygous locus, allelic dropout, or increased stutter. With increased sensitivity of detection there is a concomitant increased risk of contamination. Recently, a commission reviewed LCN typing and found it to be "robust" and "fit for purpose." Because LCN analysis by its nature is not reproducible, it cannot be considered as robust as that associated with conventional DNA typing. The findings of the commission seem inconsistent with the nature of LCN typing. While LCN typing is appropriate for identification of missing persons and human remains and for developing investigative leads, caution should be taken with its use in other endeavors until developments are made that overcome the vagaries of LCN typing. A more in-depth evaluation by the greater scientific community is warranted. The issues to consider include: training and education, evidence handling and collection procedures, the application or purpose for which the LCN result will be used, the reliability of current LCN methods, replicate analyses, interpretation and uncertainty, report writing, validation requirements, and alternate methodologies for better performance.

Forensically relevant SNP classes.

Forensic samples that contain too little template DNA or are too degraded require alternate genetic marker analyses or approaches to what is currently used for routine casework. Single nucleotide polymorphisms (SNPs) offer promise to support forensic DNA analyses because of an abundance of potential markers, amenability to automation, and potential reduction in required fragment length to only 60-80 bp. The SNP markers will serve an important role in analyzing challenging forensic samples, such as those that are very degraded, for augmenting the power of kinship analyses and family reconstructions for missing persons and unidentified human remains, as well as for providing investigative lead value in some cases without a suspect (and no genetic profile match in CODIS). The SNPs for forensic analyses can be divided into four categories: identity-testing SNPs; lineage informative SNPs; ancestry informative SNPs; and phenotype informative SNPs. In addition to discussing the applications of these different types of SNPs, this article provides some discussion on privacy issues so that society and policymakers can be more informed.

Promoter polymorphisms in the MATP (SLC45A2) gene are associated with normal human skin color variation.

Human pigmentation is a complex physical trait in which the membrane-associated transporter protein (MATP) plays an important role as it is involved in intracellular processing and trafficking of melanosomal proteins. Recently, pathogenic mutations in MATP have been shown to cause oculocutaneous albinism type 4, while other polymorphisms are known to have a role in normal pigmentation variation. We previously reported significant associations of two coding region polymorphisms with hair, skin, and eye color in Caucasians. Here we characterize the promoter region of MATP identifying two new transcription start sites and a novel duplication (c.-1176_-1174dupAAT). A total of 700 individuals from five different population groups (529 Caucasians, 38 Asians, 46 African Americans, 47 Australian Aborigines, and 40 Spanish Basques) were genotyped for known promoter polymorphisms c.-1721C>G (rs13289) and c.-1169G>A (rs6867641), as well as c.-1176_-1174dupAAT. Allele frequencies of all three polymorphisms were significantly different between population groups. In Caucasians, the -1721G, +dup, and -1169A alleles were significantly associated with olive skin color. The three promoter polymorphisms were found to be in linkage disequilibrium with each other but not with the two previously reported coding region polymorphisms. Functional analyses in a melanoma cell line showed that the promoter haplotype -1721G, +dup, -1169A significantly decreased MATP transcription. This report provides further evidence for the involvement of MATP in normal pigmentation variation by identifying associations between MATP alleles and skin color variation in Caucasians and demonstrating a functional significance of these polymorphisms.

Unravelling the molecular control of calvarial suture fusion in children with craniosynostosis.

BACKGROUND: Craniosynostosis, the premature fusion of calvarial sutures, is a common craniofacial abnormality. Causative mutations in more than 10 genes have been identified, involving fibroblast growth factor, transforming growth factor beta, and Eph/ephrin signalling pathways. Mutations affect each human calvarial suture (coronal, sagittal, metopic, and lambdoid) differently, suggesting different gene expression patterns exist in each human suture. To better understand the molecular control of human suture morphogenesis we used microarray analysis to identify genes differentially expressed during suture fusion in children with craniosynostosis. Expression differences were also analysed between each unfused suture type, between sutures from syndromic and non-syndromic craniosynostosis patients, and between unfused sutures from individuals with and without craniosynostosis. RESULTS: We identified genes with increased expression in unfused sutures compared to fusing/fused sutures that may be pivotal to the maintenance of suture patency or in controlling early osteoblast differentiation (i.e. RBP4, GPC3, C1QTNF3, IL11RA, PTN, POSTN). In addition, we have identified genes with increased expression in fusing/fused suture tissue that we suggest could have a role in premature suture fusion (i.e. WIF1, ANXA3, CYFIP2). Proteins of two of these genes, glypican 3 and retinol binding protein 4, were investigated by immunohistochemistry and localised to the suture mesenchyme and osteogenic fronts of developing human calvaria, respectively, suggesting novel roles for these proteins in the maintenance of suture patency or in controlling early osteoblast differentiation. We show that there is limited difference in whole genome expression between sutures isolated from patients with syndromic and non-syndromic craniosynostosis and confirmed this by quantitative RT-PCR. Furthermore, distinct expression profiles for each unfused suture type were noted, with the metopic suture being most disparate. Finally, although calvarial bones are generally thought to grow without a cartilage precursor, we show histologically and by identification of cartilage-specific gene expression that cartilage may be involved in the morphogenesis of lambdoid and posterior sagittal sutures. CONCLUSION: This study has provided further insight into the complex signalling network which controls human calvarial suture morphogenesis and craniosynostosis. Identified genes are candidates for targeted therapeutic development and to screen for craniosynostosis-causing mutations.

Association of functionally different RUNX2 P2 promoter alleles with BMD.

UNLABELLED: RUNX2 gene SNPs were genotyped in subjects from the upper and lower deciles of age- and weight-adjusted femoral neck BMD. Of 16 SNPs in RUNX2 and its two promoters (P1 and P2), only SNPs in the P2 promoter were significantly associated with BMD. These P2 promoter SNPs were functionally different in gel-shift and promoter activity assays. INTRODUCTION: Specific osteoblast genes are induced by Runx2, a cell-specific transcription factor that is a candidate gene for controlling BMD. We tested the hypothesis that RUNX2 genetic variation is associated with BMD. MATERIALS AND METHODS: From a population repository of normal subjects, the age- and weight-adjusted femoral neck BMD was ranked, and the upper and lower deciles (n = 132 each) were taken to represent the adjusted extremes of the population distribution. In these 264 subjects, we identified 16 allelic variations within the RUNX2 gene and promoters (P1 and P2) through DNA sequencing and denaturing high-performance liquid chromatography. Characterization of these alleles was performed through allele-specific cloning, transfection into ROS 17/2.8 cells, luciferase reporter analysis, and electrophoretic mobility shift assays. RESULTS: Within the P2 promoter were three polymorphic nucleotides for which the minor alleles were over-represented in the upper decile of BMD (0.117 and 0.064 in the upper and lower deciles, respectively). These alleles are in near complete linkage disequilibrium with each other and represent a haplotype block that is significantly associated with increased BMD. The common and rare P2 promoter alleles were cloned upstream of luciferase, and when transfected into osteoblast-like cells, the construct representing the rare haplotype showed significantly greater P2 promoter activity than the common haplotype. CONCLUSIONS: Because the high BMD allele had higher P2 promoter activity, the data suggest that greater RUNX2 P2 promoter activity is associated with higher BMD.

An alternate method for extracting DNA from environmentally challenged teeth for improved DNA analysis.

A grinding-free method to extract DNA from teeth via a direct minimal-invasive retrograde approach to the pulp cavity and dentine was compared to a standard grinding/pulverisation method. This alternate method uses endodontic dental files to access the root canals and pulp cavity for tissue and dentine harvest via the apical end of the roots and avoids mechanical damage to the crown and root morphology. In contrast, other methods require pulverisation of the whole root or tooth, transection or destruction of the occlusal surface to gain access to the DNA in the root canals and pulp chamber. This study compared two methods for preparing dentine powder from the roots of environmentally challenged teeth for forensic DNA analysis. We found that although the filing method was more laborious, and produced less dentine powder, the amount of amplifiable DNA per milligram of powder was substantially higher with the filing method compared to grinding the entire root. In addition, the number of short tandem repeat (STR) alleles detected and the peak height ratios of the STR profiles were notably higher. Although several other methods of extracting DNA-rich tissue from the pulp chamber of teeth have previously been reported, the method presented in this study is minimally invasive, thereby allowing the preservation of tooth and crown morphology.

Single nucleotide polymorphisms in the MATP gene are associated with normal human pigmentation variation.

Human physical pigmentation is determined by the type and amount of melanin and the process of pigmentation production probably involves more than 100 genes. A failure to synthesize melanin results in oculocutaneous albinism (OCA). A recently identified form of OCA results from mutations in the Membrane Associated Transporter Protein (MATP) gene. The role of MATP in human pigmentation is not clear. We investigated the role of two nonpathogenic nonsynonymous single nucleotide polymorphisms (SNPs) in the MATP gene to determine if they are associated with normal human skin, hair, and eye color variation. A total of 608 individuals from four different population groups (456 Caucasians, 31 Asians, 70 African-Americans, and 51 Australian Aborigines) were genotyped for c.814G>A (p.Glu272Lys) and c.1122C>G (p.Phe374Leu). Results indicate that the allele frequencies of both polymorphisms are significantly different between population groups. The two alleles, 374Leu and 272Lys, are significantly associated with dark hair, skin, and eye color in Caucasians. The odds ratios (ORs) of the LeuLeu genotype for black hair and olive skin are 25.63 and 28.65, respectively, and for the LysLys genotype are 43.23 and 8.27, respectively. The OR for eye color is lower at 3.48 for the LeuLeu and 6.57 for LysLys genotypes. This is the first report of this highly significant association of MATP polymorphisms with normal human pigmentation variation.

The C/C genotype of the C957T polymorphism of the dopamine D2 receptor is associated with schizophrenia.

The T allele of the human dopamine D2 receptor (DRD2) gene C957T polymorphism is associated with reduced mRNA translation and stability. This results in decreased dopamine induced DRD2 upregulation and decreased in vivo D2 dopamine binding. Conversely, the C allele of the C957T polymorphism is not associated with such changes in mRNA leading to increased DRD2 expression. PET and postmortem binding studies show that schizophrenia is often associated with increased DRD2 availability. We report that on the basis of comparing the frequencies of the C/C and T/T genotypes of 153 patients with schizophrenia and 148 controls that schizophrenia is associated with the C/C genotype. The C957T shows a population attributable risk for schizophrenia of 24% and an attributable risk in those with schizophrenia of 42%. Increased expression of D2 receptors associated with the C allele is likely to be important in the underlying pathophysiology of at least some forms of schizophrenia. Enhanced understanding of schizophrenia afforded by this finding may lead to advances in treatment and prevention.

Gene polymorphisms and their effects in the melanocortin system.

In addition to its role in human pigmentation, components of the melanocortin system regulate appetite, energy homeostasis and hormone production. Recent studies have suggested possible roles of this system in immunity, transmission of pain signals, and reproductive potential. A number of polymorphisms have been identified in genes of the melanocortin system and are associated with pigmentation in humans, as well as being causative of disorders of adrenal hormone production and obesity. This review gives an outline of these polymorphisms, their functional significance and possible application to or impact on diagnosis and pharmacotherapy based on melanocortin pathways.

Mouse models of obesity.

Insights into the etiology of human obesity have arisen from the study of animal models. Animal models of obesity are also important for the development of future treatments of obesity. An agouti mouse mutation resulting in obese, yellow mice was described over a century ago and in 1992 agouti was cloned, making it the first obesity gene characterized at the molecular level. The lethal yellow mouse mutation is one of five dominant agouti mutations and is an excellent model for human obesity. The molecular categorization of agouti was responsible for the elucidation of the melanocortin system's involvement in hypothalamic weight regulation. As genetic knowledge increases many transgenic mice have been created with genes either over-expressed or deleted, models which further enhance the understanding of obesity.

Linear amplification of target prior to PCR for improved low template DNA results.

Forensic analysis of genetic material is often limited by the quantity and quality of DNA available for examination. Stochastic effects associated with low amounts of starting template can lead to a reduction in the quality of the result, making interpretation difficult. This paper presents an amplification method to copy target DNA in a linear fashion prior to short tandem repeat (STR) analysis to increase the available starting template without introducing the amplification bias seen in other methods used to increase the sensitivity of PCR. Results show that implementing the pre- PCR procedure allows for greater allele recovery in multiplex STR analysis compared with samples that were not subjected to prior processing.

Comment on Kokshoorn, B, and Blankers, BJ 'Response to Grisedale, KS and van Daal, A: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method'.

Kokshoorn and Blankers responded to our recent article by saying that replicate analysis and consensus profiling of low template samples was best in terms of reliability and objectivity. We agree that the consensus approach has benefits, particularly in eliminating non-repeating spurious alleles from the final profile. However, with the development of statistical models that can accommodate stochastic effects and allele drop in, it may be beneficial to perform a single amplification with three times the amount of template, since much information is lost from the profile using the consensus approach.

Comparison of STR profiling from low template DNA extracts with and without the consensus profiling method.

BACKGROUND: The consensus profiling method was introduced to overcome the exaggerated stochastic effects associated with low copy number DNA typing. However, little empirical evidence has been provided which shows that a consensus profile, derived from dividing a sample into separate aliquots and including only alleles seen at least twice, gives the most informative profile, compared to a profile obtained by amplifying the entire low template DNA extract in one reaction. Therefore, this study aimed to investigate the quality of consensus profiles compared to profiles obtained using the whole low template extract for amplification. METHODS: A total of 100 pg and 25 pg DNA samples were amplified with the PowerPlex® ESI 16 Kits using 30 or 34 PCR cycles. A total of 100 pg and 25 pg DNA samples were then divided into three aliquots for a 34-cycle PCR and a consensus profile derived that included alleles that appeared in at least two of the replicates. Profiles from the non-split samples were compared to the consensus profiles focusing on peak heights, allele drop out, locus drop out and allele drop in. RESULTS: Performing DNA profiling on non-split extracts produced profiles with a higher percentage of correct loci compared to the consensus profiling technique. Consensus profiling did eliminate any spurious alleles from the final profile. However, there was a notable increase in allele and locus drop out when a LTDNA sample was divided prior to amplification. CONCLUSIONS: The loss of information that occurs when a sample is split for amplification indicates that consensus profiling may not be producing the most informative DNA profile for samples where the template amount is limited.

Extracting evidence from forensic DNA analyses: future molecular biology directions.

Molecular biology tools have enhanced the capability of the forensic scientist to characterize biological evidence to the point where it is feasible to analyze minute samples and achieve high levels of individualization. Even with the forensic DNA field's maturity, there still are a number of areas where improvements can be made. These include: enabling the typing of samples of limited quantity and quality; using genetic information and novel markers to provide investigative leads; enhancing automation with robotics, different chemistries, and better software tools; employing alternate platforms for typing DNA samples; developing integrated microfluidic/microfabrication devices to process DNA samples with higher throughput, faster turnaround times, lower risk of contamination, reduced labor, and less consumption of evidentiary samples; and exploiting high-throughput sequencing, particularly for attribution in microbial forensics cases. Knowledge gaps and new directions have been identified where molecular biology will likely guide the field of forensics. This review aims to provide a roadmap to guide those interested in contributing to the further development of forensic genetics.