O-0004 THE TGFBR1 GENE HAS A HIGHLY SIGNIFICANT MUTATION FREQUENCY AT SEVERAL SINGLE NUCLEOTIDE POLYMORPHISM (SNP) MARKER SITES IN CAUCASIAN PATIENTS WITH ADVANCED COLORECTAL CANCER AS COMPARED TO HEALTHY CONTROLS

Marc Pauly¹, Alain Menzel², Natacha Van der Taelem², Brigitte Metzger¹, Laetitia Chambeau¹, Jacques Kayser³, Carlo Faber³, Petr Nazarov⁴, Laurent Vallar⁴, Bernard Weber², Mario Antoine Dicato¹

¹Laboratoire de Recherche sur le Cancer et les Maladies du Sang, Fondation de Recherche Cancer et Sang, Luxembourg, Luxembourg, ²Laboratoires Réunis de Junglinster, Junglinster, Luxembourg, ³Clinique Ste-Thérèse, Luxembourg, Luxembourg, ⁴Microarray Center, Centre de Recherche Public de la Santé, Luxembourg, Luxembourg.

 

Abstract
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.

G. Assmann¹, J. Voswinkel¹, M. Mueller¹, J. Bittenbring¹, J. Koenig³, A. Menzel², M. Pfreundschuh¹, K. Roemer¹, I. Melchers⁴

¹Internal Medicine I and José-Carreras-Research Center, University of Saarland Medical School,Homburg/Saar, Germany; ²Laboratoires Réunis, Junglinster, Luxembourg; ³Institute for Medical Biostatistics, Epidemiology and Informatics, Johannes Gutenberg-University, Mainz, Germany;⁴Clinical Research Unit for Rheumatology, Department of Rheumatology and Clinical Immunology,University Medical Center Freiburg, Freiburg, Germany.

 

Abstract
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-inflammatory transcription factor nuclear factor kappa b (NFκB).

Methods

Genomic DNA was obtained from 221 patients with RA who fulfilled 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 significantly 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 significant 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.

 

[complete article]

Kremer JR, Langlet J, Skraber S, Weicherding P, Weber B, Cauchie HM, De Landtsheer S, Even J, Muller CP, Hoffmann L, Mossong J.

Institute of Immunology, National Health Laboratory and Centre de Recherche Public - Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Luxembourg Surveillance & Epidemiology of Infectious Diseases, National Health Laboratory, 42 rue du Laboratoire, L-191

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.

Bernard Weber¹, Alain Menzel¹, Marc Pauly², Natacha van der Taelem¹, Mario Dicato^2
1Laboratoires Réunis, Junglinster, Luxembourg; ²Laboratoire de Recherche sur le Cancer et les Maladies du Sang, Luxembourg

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.

Bernard Weber, Laboratoires Réunis, Junglinster, Luxembourg.
Email: Bernard.weber@labo.lu

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.

Bernard Weber, Laboratoires Réunis, Junglinster, Luxembourg.
Email: Bernard.weber@labo.lu

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".

Parisot F, Vander Taelem N, Menzel A, Weber B.

Clinical chemistry and laboratory medecine, 2008, vol.46, No 8, A136  

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.

Parisot F, Vander Taelem N, Menzel A, Weber B.  

MEDECINE SCIENCES 2008, HS1, vol 24, 36-37  

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.

(RA) Assmann G. 1, Müller M. 1, Bittenbring J. 1, Pfreundschuh M. 1, Roemer K. 1, Melcher I. 2, Kerschenmeyer L. 1, Voswinkel J. 1, Menzel A. 3 

(1) Medizinische Klinik I und Jose Carreras Forschungszentrum der Universitätsklinik des Saarlandes, (2) Medizinische Klinik, Abteilung Rheumatologie Uniklinik Freiburg, (3) Laboratories Reunis Langwies Luxembourg
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