INTRODUCTION

Standard recommendations for screening for colorectal cancer (CRC) specify a colonoscopy at age 50 and if normal a repeat after 10 years. However, the yield of the follow-on colonoscopy in this setting is poor. The yield of screening could be improved with a preliminary test to identify subjects with a higher risk of CRC. We are interested in the role of blood-based genetic markers in CRC pre-screening because of their low cost and relative non-intrusiveness.

METHODS

Blood samples were taken from 187 CRC patients and 793 healthy controls from a European Caucasian population. After gDNA extraction, selected sequences were amplified by PCR followed by melting curve analysis. The SNP status for TGFBR1 (rs334348) was determined for each subject. The association between allele frequency for the SNPs and CRC status was evaluated by logistic regression. Both additive and dominance effects were considered. The high-risk group would become the focus of further screening and early detection of cancer. Results: For TGFBR1, there was a highly significant additive association between the G allele and colorectal cancer. The AA "normal" genotype is the commonest amongst the controls i.e. 54.4%. In contrast the GG variant was rare among the controls, i.e. 6.9%. In CRC cases the AA genotype was rarest (17.6%), while the GG genotype was commonest (42.7%). In both cases the AG genotype was intermediate (controls 38.7%, cases 39.6%). Thus for CRC the odds ratio for GG as a risk factor relative to the normal AA is 19.00 (confidence interval of 95%: 11.6 to 31.1) with a highly significant P value of < 0.000001. The AG genotype was once again intermediate with an odds ratio (OR) for AA versus AG of 3.15 (CI of 95%: 2.04 - 4.87) and OR of AG versus GG of 6.03 (CI of 95%: 3.94 - 9.25) both with a highly significant P value of < 0.000001.

CONCLUSION

Presently the yield of mass screening for colorectal cancer has by its nature a large number of persons of whom the majority will not have cancer. Thus repeat screening after a normal first colonoscopy has a low yield and nevertheless misses a certain number of intermediary cancers. By being able to predict a clearly higher risk group, the repeat colonoscopy might have to be done earlier than stated by guidelines for these individuals. Furthermore, this inexpensive germline test, needing to be done only once, can be carried out at any age, including much earlier than is recommended by current guidelines

INTRODUCTION

An adaptive feature to the 24h lasting earth rotation is the resulting circadian life cycle of all known organisms living on this planet. These adaptive rhythms are not only a simple response to the 24h changes in the physical environment but they are internally created in a specialized part of the hypothalamus, the suprachiasmatic nucleus (SCN), which is called the central clock. This clock has to be reset every 24h to zero by a mechanism that utilizes external triggers as light and temperature. A disruption of the circadian clock is linked to different disease such as sleep disorder, diabetes, obesity, elevated cancer risk etc. Interestingly these biological clocks do not only exist in the brain which gets light signals by photoreceptors but they occur in every organ, even in every cell which is not exposed to light. For example, pancreas, liver, lung etc. How do these clocks know which time it is? These peripheral clocks, also called metabolic clocks, have an impact on the SCN and vice versa. They consist of oscillating transcription machineries composed of repressors and activators.

METHODS

The molecular basis of the circadian clock was discovered in 1971 by Konopka and Benzer, who isolated mutants in Drosophila melanogaster which had either an advanced or a delayed activation cycle. The same gene was responsible for both behaviors and was cloned in 1984 by Michael Rosbash, it was the period2 gene (PER2). Only in 1997 the mouse homolog was cloned by positional cloning by the group of J.Takashi, the gene was named CLOCK. Experiments proving that clock genes were also active in peripheral tissues were done in cell cultures using genes cloned and expressed in vectors containing firefly luciferase as a reporter. Due to chromatin immune precipitation in combination with next generation sequencing the epigenetic effects and influences like histone methylation and histone acetylation during a 24h period can be analyzed nowadays.

RESULTS

The circadian clock being an adaptation to the 24h earth rotation is a self-regulating and recurrent mechanism which has to be reset every day. Very well-known examples of inner clocks are behavioral traits as the migration of fish. Anadromous fish, e.g. salmon migrate from the sea into fresh water to spawn, and catadromous fish as eels migrate from fresh water down into the sea to spawn. The normal 24 h life cycle of a human being is characterized by a period of physical activity which correlates with exposure to light and feeding and an 8h resting period, during which individuals sleep and are not physically active but fast. These clocks are universal, they can be observed in paramecium, in caenorhabditis elegans, in arabidopsis, in drosophila melanogaster in mouse as well as in homo sapiens. Regarding the sleep behavior different phenotypes are known. The conventional sleeper with a sleeping period of approximately 8 h, going to sleep at 10pm to 12 pm and waking up in the morning at 6am or 8 am. But there are also the so called "night owls" with a sleeping period of 8 h, but their period is shifted to later hours and we also know ",morning larks" where the period is shifted to early hours. But also the phenotype of the "short sleeper" is known, where the period of sleep is reduced due to a point mutation in a single gene, to 6 or even 4 hours. A famous example of a natural short sleeper is the former British Prime Minister Margaret Thatcher. All these phenotype are normal and are not linked to disease. But a disruption of the clock is linked to many diseases as sleep disturbance, disturbance of learning and memory formation, disease of the GI-tract, cardiovascular- and pulmonary disease, disturbance of the immune function and problems in the cell cycle.

The question in which researches are interested for many years is the underlying molecular mechanism of the functioning of such a clock. It was especially mysterious how organs, yes even single cells of the pancreas, the lung, the liver etc. know when it is bright and when it is dark. In contrary to the superchiasmatic nucleus (SCN) in the brain these peripheral cells are not directly in contact with a light/dark trigger. Nowadays we believe that the underlying mechanism of the clock is a transcriptional oscillator which acts in every cell of the body.

In simplifying we can distinguish four clocks (which of course in nature are linked):

1. The central clock of SCN (CLOCK, BMAL, PER, CRY)
2. A metabolic clock consisting of nuclear transcription factors which control the expression of BMLA (ROR, REVERB, BMLA)
3. A peripheral clock involved mainly in protein degradation and phosphorylation in the cytoplasm (PER,CRY, CK1 epsilon & delta, FBXL21)
4. A peripheral clock working on protein degradation in the cell nucleus (PER,CRY, BMLA,CLOCK,CK1 epsilon & delta, FBXL3 & FBXL21) .

In summary, the central molecular clock works like this: An activator which is composed of the proteins CLOCK and BMLA work as a heterodimer. They bind as a transcription factor to an E-box recognition site controlling the expression of PER1, 2, 3 as well as of cryptochrome, CRY1 and 2. The mRNAs of these genes are translocated into the cytoplasm where they are translated into proteins. This process begins in the morning, continues during the day so that in the beginning of the evening the concentration of PER and CRY is very high, they translocate into the nucleus and bind as homo- or heterodimers to BMLA-CLOCK. As a consequence the transcription factors are removed from the E-BOX, the repressors PER and CRY are not transcribed anymore and their concentration is diminished in the cytoplasm. In the beginning of the morning PER and CRY are degraded, the concentration of BMLA and CLOCK raises and the oscillator begins a new round. The same principle is realized in peripheral clocks. For example the activator of the central clock BMLA is regulated by the oscillatory binding of REVERB as a repressor and of ROR as an activator binding to an RRE-response element located 5'- upstream of BMLA. ROR is expressed constitutively and REVERB is regulating the system. The repressors PER and CRY are phosphorylated by the kinases CK1epsilon and - delta, which are than activating FBLXL21 in the cytoplasm and consequently are degraded in the proteasome. In the nucleus the repressors are degraded in a process involving FBXL3 in competition with FBXL21.

Although the underlying molecular mechanisms of the central and the peripheral clocks are mainly the same, both are regulated by transcriptional oscillators, the effects of the mutated genes are different depending on the tissue. If, e.g. CRY1 is mutated in cells and the CRY2 gene is intact in the peripheral clock, the CRY1 cells show a disruption of the clock, consequently the patient has a predisposition for different disease I the periphery but the clock of the SCN acts as an integrator so that the CRY1 mutation has no impact on the sleep behavior.

CONCLUSION

The circadian clocks consist of different types of clocks. The central clock in the brain as well as the metabolic clocks in the periphery are influencing each other. The understandings of the underlying molecular mechanisms will not only help in the future to better understand metabolic disease but it will reveal a new class of druggable targets.

LITERATURE

1. Cho H, Zhao X, Hatori M, Yu RT,. Barish GD, Lam MT, Chong LW, DiTacchio L, Atkins AR, Glass CK, Liddle C, Auwerx J, Downes M, Panda S,. Evans RM (2012) "Regulation of Circadian Behavior and Metabolism by Rev-erb? and Rev-erb?": Nature 485, 123-127
2. Kaasik K, Allen JJ, Kivimae S, Chalkley R, Huang Y, Kissel H, Burlingame AL, Shokat KM, Ptá?ek LJ, Fu YH (2013) "Reciprocal regulation of Circadian Clock through GSK3? and O-linked N-acetylglucosaminylation": Cell Metabolism 17, 291-302
3. Koike N, Yoo SH, Huang HC, Vivek Kumar V, Lee C, Kim TK, Takahashi JS (2012) "Transcriptional Architecture and Chromatin Landscape of the Core Circadian Clock in Mammals": Science 338, 123-127
4. Lakin-Thomas P (2000) "Circadian rhythms: New functions for old clock genes?: TIG 16, 135-142
5. Merrow M, Spoelstra K, Roenneberg T (2005) "The circadian cycle: daily rhythms from behavior to genes": EMBO reports 6, 930-935
6. Pellegrino R; Kavakli IH; N; Christopher J. Cardinale CJ; Dinges DF; Kuna ST; Maislin G;. Van Dongen HP; Tufik S; Hogenesch JB; Hakonarson H;. Pack AI (2014) "A Novel BHLHE41 Variant is Associated with Short Sleep and Resistance to Sleep Deprivation in Humans": Sleep 37, 1327-1336
7. Shimomura K, Kumar V, Koike N, Kim TK, Chong J, Buhr ED, Whiteley AR, Low SS, Omura C, Fenner D, Owens JR, Richards M, Yoo SH, Hong HK, Vitaterna MH, Bass J, Pletcher MT, Wiltshire T, Hogenesch J, Lowrey PL, Takahashi JS (2013) "Usf1, a suppressor of the circadian Clock mutant, reveals the nature of the DNA-binding of the CLOCK:BMAL1 complex in mice": eLife 00426, 1-25
8. Zhang L, Abraham D, Nishino S, Oster H, Fujiki N, Gregor Eichele, Fu YH, Ptá?ek LJ. (2012) "PKC? Participates in the Entrainment of the Cerebral Circadian Clocks by Feeding": Proc Natl Acad Sci 109, 20679-84.

1 Research of Cancer and Blood Research, Luxembourg, Luxembourg

2 Laboratoires Réunis, Junglinster, Luxemnourg

Introduction

Screening for colorectal cancer (CRC) is currently based on colonoscopy and other methods such as the faecal occult blood test. We are interested in the role of blood-based genetic markers in CRC screening because of their low cost and relative non-intrusiveness. However, the performance of test strategies involving different numbers of markers of different properties needs further study. The trade-off between specificity and sensitivity may be explored in receiver operating characteristic (ROC) analysis.

Methods

We have shown an association between colorectal cancer (185 cases vs. 93 controls) ans SNP status for TGFBR1 rs334348, INSR rs12459488, telRNA rs12696304, LRCC1 rs16847897, CHR8 rs7014346 and FOXO1 rs2721068, and deletion status for GSTT1 and GSTM1. The information from all these tests was combined using a discriminant function. All information for each subject was condensed into a single index which gives the bast discrimination between cases and controls. The index could be used to distinguish high-risk subjects from low-risk ones, so that the high-risk group could become the focus of further screening and early detection of cancer. Furthermore, ROC analysis could be used to optimise the economic performance of the test for various relative costs of false positives or false negatives.

Results

We have used data on the SNPs and deletions to enable us to evaluate the performance of the test based on different numbers of variants. The addition of variants often led to increased sensitivity and specificity of the test. For example, with all eight markers the sensitivity was found to be 74.6% and the specificity 77.4% for equal costs of false positives and false negatives. With only three variants (TGFBR1, GSTT1 and INSR), sensitivity was 70.3% and specificity 67.7%. However, the two ROC curves were not widely separated so that the optimal panel might depend on the costs of the tests themselves.

Conclusions

Presently the yield of mass screening for colorecral cancer has by its nature a vast number of persons of which the majority will not have cancer. Based on this fact, repeat screening after a normal first colonoscopy has a low yield and still misses a certain number of intermediary cancers. By being able to predict a higher risk group, the repeat colonoscopy might have to be done earlier than stated by guidelines for these individuals. Although the cost of the test with a panel of biomarkers depends on the number of markers and the particular ones chosen, a first estimate would be in the range 50 Euro to 100 Euro per individual, much less than the cost of a colonoscopy. Furthermore, the test could be carried out at any age, including much earlier than is recommended for colonoscopy.

For additional information refer to the interview in medscape witf Dr Dicato:

http://www.medscape.com/viewarticle/847467

Life a deadly game (genetic and epigenetic elements of longevity)

Background

Colonoscopy (CS) used for colorectal cancer (CRC) screening is relatively expensive and intrusive. We have shown that CRC in Caucasian patients have a high frequency of certain TGFBR1 SNP's relative to normal controls. Here we extend the results to these SNP frequencies in normal tissue of the patients and the concordance of three different SNP's in the TGFBR1 gene across patients and subjects.

Methods

Tumor samples were obtained from 188 CRC patients and from 98 healthy control. After gDNA extraction, selected amplicons were amplified by PCR, followed by melting curve analysis. The statistical evaluation of the genotype frequency for the different SNPs comparing CRC patients with controls was carried out using logistic regression.

Results

Results previously reported were confirmed:- a highly significant association between TGFBR1 SNP genotype and colorectal cancer (P<0.000001). For example, with the TGFBR1 SNP rs334348, the frequency of the GG genotype was 43% for patients but 11% for controls, while the AA frequency was only 18% for patients and 43% for controls. All CRC patients and controls were typed for three SNP's in the TGFBR1 gene and the concordance was found to be perfect. That is, only 3 of the 27 possible genotypes were observed:- (i) rs334348, AA; rs334349, AA; rs1591, CC; (ii) rs334348, AG; rs334349, AC; rs1591, CT; and (iii) rs334348, GG; rs334349, CC; rs1591, TT. Blood and tumor samples from patients had the same identical SNP pattern.

Conclusions

The perfect concordance of the three SNP types in tumors and leukocytes shows that it reflects the underlying genotype of the patient rather than a somatic mutation in the tumour. Thus SNP type is a characteristic of subjects and a potential genetic marker for CRC susceptibility. We suggest that subjects aged 50 years or older be offered a test for a TGFBR1 variant. In case of a positive result, subjects should be informed that they are at risk of CRC, and strongly encouraged to have a colonoscopy. For this suggestion to become a definitive recommendation a cost-effectiveness analysis of the use of TGFBR1 SNP typing would be useful ( although such typing is inexpensive) and a prospective study of colonoscopy with TGFBR1 typing is mandatory.

Background

In order to evaluate various SNPs in different genes and chromosomal locations in caucasian patients as markers linked to the predisposition of the colorectal cancer (CRC) disease, we analyzed the frequency of different tumour-associated single-nucleotide polymorphisms in the following two genes: PAI (5G vs. 4G, rs1799899) and TGFbR1 (rs334348, A>G; rs334349, G>A; rs1591, A>C), chromosomal locations of caucasian CRC patients as compared to a healthy caucasian population.

Method

Tumour samples were obtained from 188 caucasian CRC patients at the adenocarcinoma stage and from 92 healthy caucasian individuals. After gDNA extraction, selected amplicons were amplified by PCR, followed by melting curve analysis. The statistical evaluation of the mutation frequency at the different SNPs while comparing CRC patients versus healthy individuals was calculated following the Chi-squared test.

Results

When analyzing 188 tumour samples and comparing with a healthy population to estimate the genotype distribution and mutation frequency in CRC cases, a significant frequency at the PAI (5G vs. 4G, rs1799899, P=0.025) locus. We had previously found a highly significant mutation frequency at three TGFbR1 SNPs (rs334348, rs334349, rs1591, P<0.005). Analysing the distribution of genotypes for TGFbR1 and PAI simultaneously did not lead to a significant reduction in chi-squared, suggesting that the effects of these variants were independent.

Conclusion

PAI (plasminogen activator inhibitor) was revealed as a significant marker and thus risk factor linked to the predisposition and/or occurrence of CRC in caucasian patients. The association seemed to be independent of that reported for SNPs rs334348, rs334349, rs1591 in the tumour growth factor beta receptor 1 (TGFbR1) gene.

Background

In order to evaluate various SNP's in different genes and chromosomal locations in Caucasian patients as markers of the predisposition to the colorectal cancer (CRC) disease, we analyzed the frequency of different tumour-associated single-nucleotide polymorphisms in several genes including p53 and SMAD7 of Caucasian CRC patients as compared to a healthy Caucasian control population.

Method

Tumour samples were obtained from 188 CRC patients at the adenocarcinoma stage and from 98 healthy control individuals. After gDNA extraction, selected amplicons were amplified by PCR, followed by melting curve analysis. The statistical evaluation of the difference in genotype frequency at the different SNPs comparing CRC patients with healthy controls was carried out using the Chi-squared test.

Results

When analyzing 188 tumour samples and comparing with 98 healthy controls, a significant difference in the genotype distribution of the G429C SNP in the p53 gene was observed (P=0.046). Similarly, a significant difference was found for rs4939827 C>T in the SMAD7 gene (P=0.037), although no significant differences were found for rs4464148 T>C (P=0.585) or rs12953717 C>T (P=0.197) also in SMAD7. Differences in genotype distribution for CHR9 C>A rs719725 and CHR8 G>A rs7014346 were almost significant (P=0.050 and P=0.054 respectively). Significant results previously reported for two other genes were confirmed:- PAI, 5G vs. 4G, rs1799899 (P=0.047) and TGFBR1 A>G rs334348, G>A rs334349 and A>C rs1591 (P<0.0001). No significant differences were found for SNP's in 9 other genes.

Conclusion

The results for p53, SMAD7, PAI, CHR8 and CHR9 are suggestive rather than conclusive and invite confirmation through further study. Concerning the association between the TGFBR1 SNP's and colorectal cancer, on the other hand, the results are already clear. For example, with the TGFBR1 SNP rs334348, the frequency of the GG genotype was as much as 43% for the CRC patients but 11% for the controls, while the frequency of the AA genotype was only 18% for the patients and 43% for the controls.

Background

In order to evaluate various SNPs in different genes and chromosomal locations in caucasian patients as markers linked to the predisposition of the colorectal cancer (CRC) disease, we analyzed the frequency of different tumour-associated single-nucleotide polymorphisms in the following genes: p53 [G429C], mdm2 [G309T], TGFßR1 (rs334348, A>G; rs334349, G>A; rs1591, A>C), SMAD7 (rs4464148, T>C; rs12953717, C>T; rs4939827, C>T), FLJ (rs3802842, A>C) as well as CHR8 (rs7014346, G>A; 8q24 rs6983267, T>G) and CHR9 (rs719725, C>A) chromosomal locations of 100 caucasian CRC patients as compared to a caucasian healthy population.

Methods

Tumour samples were obtained from caucasian CRC patients at the adenocarcinoma stage and from caucasian healthy individuals. After gDNA extraction, selected amplicons were amplified by PCR, followed by melting curve analysis. The statistical evaluation of the mutation frequency at the different SNPs while comparing CRC patients versus healthy individuals was calculated following the Chi-squared test.

Results

When analyzing 100 tumour samples and comparing with a healthy population to estimate the genotype distribution and mutation frequency in CRC cases, the most highly significant mutation frequency occurred at three TGFßR1 SNPs (rs334348, rs334349, rs1591, p=0,00000) and a still significant frequency at the chromosomal CHR8 (rs7014346, p=0,03452) and CHR9 (rs719725, p=0,02867) locations. However, the mutation frequencies at all the other analyzed SNP sites were not significant.

Conclusion

As shown by this study, SNPs rs334348, rs334349, rs1591 in the tumor growth factor beta receptor 1 (TGFßR1) revealed as the most significant markers and thus strong risk factors linked to the predisposition and/or occurrence of CRC in caucasian patients, followed by SNPs rs719725 and rs7014346 at chromosome 9 and 8, respectively.

Objective

This study examines two common, functional, single nucleotide polymorphisms (SNP) in the genes coding the human homolog of murine-double-minute-2 (MDM2) and p53 in patients with rheumatoid arthritis (RA) based on the hypothesis that p53 may be an important negative regulator of the pro-in?ammatory transcription factor nuclear factor kappa b (NF?B).

Methods

Genomic DNA was obtained from 221 patients with RA who ful?lled at least 4 ACR criteria and from 521 healthy controls. Mdm2 SNP309 and p53 P72R were genotyped by polymerase chain reaction and restriction enzyme analysis.

Results

In RA patients the frequencies of the mdm2 SNP309 G allele and both G-containing genotypes were signi?cantly reduced (G allele: OR: 0.75, 95% CI: 0.59–0.95, p=0.016; genotype TG: OR: 0.71, 95% CI: 0.50–1.00; genotype GG: OR. 0.58, 95% CI: 0.34–0.99; both: p=0.049). Concerning p53 P72R, no differences in allele or genotype frequencies were detected. A combined analysis of both polymorphisms revealed a signi?cant interaction between them (p=0.046). In individuals carrying ?1 p53 72R allele, MDM2 had a protective effect, whereas in individuals homozygous for p53 72P, MDM2 had the opposite effect.

Conclusion

The function of MDM2 depends on the p53 P72R genotype, resulting in either an increased or reduced risk for RA. We suggest that in most cases MDM2 stabilizes the conformation of p53, whereas in p53 PP-positive subjects MDM2 supports the degradation of p53.

Key words

Rheumatoid arthritis, p53, MDM2, polymorphism, transcription factor.

Link

[complete article]

The genetic diversity of norovirus strains obtained from gastroenteritis outbreaks, sporadic case surveillance, and wastewater plants was compared in Luxembourg from October 2008 until June 2009. Except for GI.6 and GIV.1 strains detected exclusively in wastewater, all other genotypes were also found in human samples. Of the 9 NoV genotypes detected, only three (GII.4, GIIb/II.3, GIIc/II.12) were associated with institutional outbreaks. The majority of sequences from all sources belonged to genotype GII.4 including two potentially new sub-variants. Strains collected in the context of outbreaks may significantly underrepresent the overall genetic diversity of NoVs circulating in a country.

Colorectal cancer (CRC) occurs either in the colon or rectum. It is the third most common cancer and the second leading cause of cancer-related death in Western Europe. The average lifetime risk of developing CRC is about 5%. Any factor leading to increased cell division in the large intestine can potentially increase the probability of developing CRC. Environmental factors such as a diet that is poor in vegetables and rich in fat may promote cell growth. Additionally cigarette smoke, excessive alcohol consumption and inflammatory bowel disorders such as Crohn's disease and ulcerative colitis are known to cause overgrowth of colon cells. The majority of CRCs are sporadic cases whereas about 25% of the cases have an inherited compound. Mutations in the genes MSH2 and MLH1 account for only one fifth of the inherited cases of CRC. The rest of the individual colorectal cancer risk is probably due to common genetic variants that, individually, do not contribute much to an increased risk of CRC. However the presence of many variants of these SNPs can contribute to the development of the disease. In this case, the risk can be largely reduced by changing the lifestyle.

In the present study, SNPs related to cell gowth and divison were analysed in 100 patients withan advanced CRC. The carcinomas had a histological staining equal or greater than pT1 and were thus belonging to the adenoma-carcinoma or to the carcinoma stage. The control group consisted of 100 patient not carrying a CRC. Genotyping was performed by melting curve analysis on DNA isolated from whole blood-samples.

The analysed SNPs were: p53 (rs1042522), MDM2 (rs2279744), TGFBR1 (rs334348, rs334349, rs1590), TGFBR4 (rs7871490), SMAD7 (rs4464148, rs12953717, rs493927), PROC3 (rs6983267), FLJ (rs3802842), CHR9 (rs719725) and CHR8 (rs7014346).

Our results show that especially polymorphisms related to TGFBR and SMAD7 seem to be promising CRC predispostion markers. Further studies, which focus on sequencing of the complete genes, will elucidate which role spontaneous mutations as well as known and newly discovered polymorphisms play in the development of CRC.

Background

Hormone replacement therapy (HRT) relieves menopausal symptoms and protects against osteoporosis. However, HRT also has risks. It can increase the risk of breast cancer, heart disease and stroke. Certain types of HRT have a higher risk, and each woman's own risks can vary depending upon her health history and lifestyle.

Aim of the Study

Prevention and treatment of osteoporosis can be individualized by considering the fact that the individual response to HRT, calcium suplementation, inflammation modulation and nutrition is influenced by genetic polymorphisms. Depending on the genetic background the dosage of therapeutic or chemopreventive agents needs to be adapted. Predictive genetics permits also the assessment of the relative risk of HRT by excluding high risk alleles for venous thrombosis (Factors II, V, MTHFR, PAI-1) and sporadic breast cancer.

Materials and Methods

Genotyping of clinically relevant single nucleotide polymorphisms (SNPs) and interpretation of results is performed with an expert system which integrates lifestyle and risk factors and which provides personalized recommendations for reducing the circulating levels of potentially carcinogenic and oxidative stress inducing metabolites.

Results

Result interpretation is based on metaanalaysis studies and on data from replicative and confirmatory studies. The impact of polymorphisms of genes on the risk of adverse events and treatment response is assessed by calculation of a score based on the cumulative and quantitative analysis of the impact each single low penetrance allele in combination with a standardized questionnaire on lifestyle and nutrition factors

Conclusions

The results from genotype profiles permit a personalized management of the RR including prevention, HRT and nutrigenetic recommendations. Together with familial and individual risk factors, genotyping of multiple alleles can permit a stratification of the relative risk for adverse events and identify those patients who should profit from HRT or be careful with HRT.

Background

Androgenetic alopecia (AGA), also known as male pattern baldness, is the most common form of hair loss in humans. Its prevalence is highly age-dependent and its pathogenesis is dependent on androgenic hormones. Not only men but also women are affected by this disease. An association of AGA with a variety of clinical phenotypes has been suggested, including coronary heart disease, benign prostatic hyperplasia, prostate cancer and disorders associated with insulin resistance. From twin studies it is known that AGA has a heritability of approximately 80%.

Aim of the Study

Two polymorphic gene clusters may explain the main part of AGAs genetic components.

1. Since 2005 a polymorphism of the androgen receptor (AR) gene located at band q12 on the X chromosome is known to play a crucial role in the development of AGA.
2. In 2008 three polymorphisms located on chromosome 20 could also be correlated to AGA although the function of the gene products is not known.

The AR E211(rs6152) G>A polymorphism does not result in an amino acid change and is therefore unlikely to convey a direct effect on protein expression, structure, or function. This silent marker is located 401 bases downstream of a functional trinucleotide repeat. It is therefore possible that the association between lowered risk of AGA and the presence of the A allele may reflect the effect of the repeat status. The fact that the gene, which encodes the AR, is located on the X chromosome, means that men inherit only one copy of the gene, and that this copy always comes from their mothers' side. This may explain the observation that male pattern baldness often skips a generation. But the AR gene doesn't completely explain the inheritance of male pattern baldness and indeed in 2008, evidence was provided in two independent studies that also polymorphisms of a gene located chromosome 20 were associated to AGA.

Materials and Methods

The ALOPECIA genetic test for Male Pattern Hair Loss provides information on the presence of four specific variations: AR (rs6152) located on chromosome X and three other variations located on chromosome 20 (rs2180439, rs1160312, rs91063).

Results

Depending on the combination of the different variations there are 9 haplotypes resulting, which can be assigned to different risks of developing male pattern hair loss. This test can identify patients susceptible to develop AGA prior to the onset of symptoms.

Conclusions

A diagnosis in an early, respectively in a preclinical stage, allows a treatment to be initiated at a time when intervention has a greater likelihood of success. The fact that high risk patients for AGA can be identified offers the opportunity for early medical intervention prior to the appearance of visible signs of hair loss, when stabilization is most cosmetically beneficial. "The earlier you can predict pattern hair loss, the more likely you are to save your hair".

ABSTRACT

The aim of the present study was to investigate the impact of polymorphisms of the vitamin K epoxyde reductase 1 (VKORC1) and cytochrome CYP2C9 and CYP2A6 on the average therapeutic dose of acenocoumarol (Sintrom®) in a collective consisting of 164 Caucasian patients with therapeutic INR (International Normalized Ratio) values ranging between 2 and 4. Each DNA sample was genotyped for the following SNPs: VKORC1 [G-1639A] [C1173T], CYP2C9*2/*3 and CYP2A6*2/*3*4 by using melting curve analysis with hybridization probes after real-time PCR amplification. Among the variants described for the VKORC1 gene, two are known to be involved in the response to anticoagulant treatment: [G-1639A] located inside the gene promoter and [C1173T] situated in the coding region. Our study collective showed a complete linkage (100%) between these two SNPs. Homozygous wild-type patients for VKORC1 [G-1639A] were treated with 2.97mg of a daily mean dose of Sintrom®(3.0-4.0, CI95%) in order to achieve INR values ranging between 2 and 4. The mean doses were only 2.09mg (1.93-2.27, CI95%) and 0.91mg(0.68-1.15, CI95%) for heterozygous [G/A] (n=71; 43.3%) and homozygous carriers [A/A] (n=21; 12.8%) respectively. Overall for 56.1% of our patients the mean dose of Sintrom® was reduced due to the presence of the VKORC1 G1639A variant allele. The two main CYP2C9 variants (*2/*3) predispose to a decreased enzymatic activity of approximately 12%and 5%, respectively. Genetic variation in one of these two alleles was associated with reduced daily doses of Sintrom® (2.08 mg (1.89-2.28, CI 95%)) in comparison to wild type (2.56 mg (2.28-2.84, CI 95%)). For homozygous carriers (n=5; 3.1%) the mean dose was 1.38 mg (0.25-2.51, CI 95%). The 2 CYP2C9 polymorphisms were associated with a reduced Sintrom® dose regimen in 44% (n=72) of the patients. The three main CYP2A6 SNPs (*2/*3 and *4) are associated with reduced enzymatic activity and increased half-life time of the drug. No correlation could be determined in this study between anticoagulation therapy dosage and these polymorphisms. All wild-type patients for all the analyzed alleles had an average dose of 3.50 mg (3.0-3.99, CI 95%) of Sintrom® per day (19.5% of the study cohort). For homozygous variant carriers of one SNP either for VKORC1 or CYP2C9 the mean dose was only 1.08 mg (0.82-1.34, CI 95%) per day of Sintrom® (15.85% of the study collective). In conclusion, our data demonstrate clearly the impact of genetic polymorphisms of VKORC1 and CYP2C9 on the mean dosage of Sintrom® and these genetic variants showed a high prevalence in a collective of Luxembourg patients.

ABSTRACT

Deux catégories de gènes sont connues pour jouer un rôle dans le dosage des anti-vitamines K. D’une part, ceux impliqués dans l’inhibition des anti-vitamines K, VKORC1 : (Vitamin K Epoxide Reductase Multiprotein Complex 1) et, d’autre part, ceux impliqués dans le métabolisme des antagonistes de la vitamine K (CYP2) Cette étude a porté sur un panel de 164 patients d’origine caucasienne, traités par Sintrom dans le but de déterminer le ou les polymorphismes impliqués dans la détermination de la dose thérapeutique adaptée au patient. Suite au traitement, les patients du panel présentent tous une valeur d’INR (International Normalized Ratio) comprise dans la norme thérapeutique entre 2 et 4. Le génotypage des SNPs : VKORC1 [G-1639A] [C1173T], CYP2C9*2/*3 et CYP2A6*2/*3*4 a été réalisé pour chaque patient. Ce génotypage a été réalisé grâce à l’analyse de la courbe de fusion effectuée après la PCR en temps réel. Parmi les différentes mutations décrites pour le gène du VKORC1, les deux plus importantes pour le dosage des anticoagulants sont : [G-1639A] dans le promoteur du gène et [C1173T] dans sa séquence codante. Dans ce panel d’étude, les résultats obtenus pour ces deux polymorphismes sont reliés à 100 %. Ainsi la détermination d’un polymorphisme est suffisante.Les résultats ont montré que les patients de génotype homozygote sauvage sur le VKORC1 [G-1639A] utilisent une dose moyenne hebdomadaire de 2,97 mg (3,0-4,0, CI 95 %) de Sintrom. Cette dose moyenne est diminuée à 2,09 mg (1,93-2,27, CI 95 %) pour les hétérozygotes [G/A] (n = 71 ; 43,3 %) respectivement à 0,91 mg (0,68-1,15, CI 95 %) chez les porteurs homozygotes [A/A] (n = 21 ; 12,8 %). L’analyse de ce polymorphisme permet d’expliquer la nécessité de diminuer la dose thérapeutique chez 56,1 % des cas de notre panel d’étude. Le cytochrome P450 CYP2C9 est une enzyme synthétisée par le foie. Elle intervient dans le métabolisme des anticoagulants. Les deux variantes (SNPs) principales de ce cytochrome sont CYP2C9*2 et CYP2C9*3. Elles prédisposent à une réduction de l’activité enzymatique et ont été mises en évidence dans 12 % et 5 % des cas respectivement. La détection d’une mutation (hétérozygote) dans l’un de ces deux allèles permet d’expliquer la nécessité de diminuer la dose hebdomadaire de prise de Sintrom de 2,56 mg (2,28-2,84, CI 95 %) à 2,08 mg (1,89-2,28, CI 95 %). Lorsque les patients sont porteurs homozygotes (n = 5 ; 3,1 %) la dose moyenne est abaissée à 1,38 mg (0,25-2,51, CI 95 %). L’analyse de ce polymorphisme permet d’expliquer la nécessité de diminuer la dose thérapeutique chez 44 % (n = 72) des cas du panel d’étude. Les trois SNPs principaux du cytochrome CYP2A6 sont CYP2A6*2/*3 et *4. Ils prédisposent à une réduction de l’activité enzymatique et donc une augmentation de la demi-vie du médicament. Cette étude n’a pas permis d’établir de manière significative un lien entre la dose hebdomadaire de Sintrom et le génotype des patients pour ces allèles. Il a également été montré que les patients sauvages sur l’ensemble des allèles étudiés prennent en moyenne 3,50 mg (3,0-3,99, CI 95 %) de Sintrom hebdomadaire (représente 19,5 % du panel d’étude), alors que la présence d’une mutation homozygote explique la nécessité de prendre seulement 1,08 mg (0,82-1,34, CI 95 %) en moyenne de Sintrom par semaine chez ces patients (représente 15,85 % du panel d’étude). Le polymorphisme du VKORC1 s’est révélé être aussi important que celui de CYP2C9 pour expliquer les sensibilités interindividuelles face à la prise d’acénocoumarol. Selon cette étude, il est donc recommandé d’établir un schéma génétique sur les allèles : VKORC1 [G-1639A] et CYP2C9*2/*3 chez les patients avant le début d’un traitement anticoagulant basé sur l’acénocoumarol. Cette analyse génétique éviterait le surdosage de ces patients les premiers jours de leur traitement.

Einleitung

Verschiedene Formen von Zellstress erzeugen intrazellulär eine Vermehrung und Aktivierung vom p53-Protein. Das p53- Protein zeigt eine antagonisierende Wirkung auf den Transcriptionsfaktor Nucler Factor kappa B (NFkB), der wiederum eine Schlüsselrolle in der Entzündungsmediation der Synovialis bei Rheumatoider Arthritis (RA) hat. Das Murin-doubleminute2- Gen (MDM2)kodiert für ein Protein, das als der wichtigste Negativregulator für das p53-Protein über Ubiquitin- Ligasen fungiert. Der p53-Gen-Polymorphismus (p53-GPM) mit Basenaustausch an Stelle 72 mit G statt T und der Aminosäure-Kodierung von Prolin statt Argenin führt zur verminderten Aktivität des p53-Proteins. Der MDM2-Gen- Polymorphismus (MDM2-GPM) am Basenpaar 309 hat eine stärkere Inaktivierung des p53-Proteins im Vergleich zum Wildtyp zur Folge. In der mitteleuropäischen Normalbevölkerung hat der p53-GPM eine Häufigkeit von 33% heterozygot und 10% homozygot, der MDM2-GPM von 47% versus 14%. Material und Methoden: 200 RA-Patienten gemäß ACR-Kriterien wurden hinsichtlich des Vorliegens des MDM2-GPM und 129 RA-Patienten hinsichtlich des p53-GPM untersucht. Das Verteilungsmuster wurde mit dem der Normalbevölkerung verglichen. Hierzu erfolgte aus einer peripheren Blutprobe im EDTA-Röhrchen die DNA-Isolierung, der anschließende Nachweis des jeweiligen Genpolymorphismus mit jeweils spezifischer PCR-Amplifizierung, DNA-Restriktion und Gelelektrophorese. Es wurde eine Subgruppenanalyse nach Seropositivität (positiver Rheumafaktor (RF)) und erosiv-destruierendem Verlauf durchgeführt.
Ergebnisse: 200 RA-Patienten zeigen zu 49% heterozygot und 10% homozygot den MDM2-GPM, 129 RA-Patienten zu 37% heterozygot und zu 8% homozygot den p53-GPM. 119 von 200 RA-Patienten mit positivem RF waren zu 55% heterozygot und zu 7% homozygot mit MDM2-GPM, 59 von 129 RA-Patienten zu 24% heterozygot und zu 9% homozygot mit p53-GPM. 108 von 200 RA-Patienten zeigten zu 53% heterozygot und zu 9% homozygot den MDM2- GPM, 43 von 129 RA-Patienten heterozygot zu 33% und homozygot zu 8% den p53-GPM. Zusammenfassung: Es zeigt sich kein vermehrtes Auftreten des MDM2-GPM bei 200 RA-Patienten und des p53-GPM bei 129 RA-Patienten im Vergleich mit der Normalbevölkerung inklusive der Subgruppenanalyse hinsichtlich Seropositivität und erosivdestruierenden Verlaufs bei RA-Patienten (Wilcoxon-Test-Verfahren).  

35. Kongress der DGRh und 21. Jahrestagung der ARO Hamburg, 19. - 22. September 2007