Pharmacogenetic Variation at CYP2D6, CYP2C9, and CYP2C19 : Population Genetic and Forensic Aspects

Pharmacogenetics deals with genetically determined variation in drug response. In this context, three phase I drug-metabolizing enzymes, CYP2D6, CYP2C9, and CYP2C19, have a central role, affecting the metabolism of about 20-30% of clinically used drugs. Since genes coding for these enzymes in human populations exhibit high genetic polymorphism, they are of major pharmacogenetic importance. The aims of this study were to develop new genotyping methods for CYP2D6, CYP2C9, and CYP2C19 that would cover the most important genetic variants altering the enzyme activity, and, for the first time, to describe the distribution of genetic variation at these loci on global and microgeographic scales. In addition, pharmacogenetics was applied to a postmortem forensic setting to elucidate the role of genetic variation in drug intoxications, focusing mainly on cases related to tricyclic antidepressants, which are commonly involved in fatal drug poisonings in Finland. Genetic variability data were obtained by genotyping new population samples by the methods developed based on PCR and multiplex single-nucleotide primer extension reaction, as well as by collecting data from the literature. Data consisted of 138, 129, and 146 population samples for CYP2D6, CYP2C9, and CYP2C19, respectively. In addition, over 200 postmortem forensic cases were examined with respect to drug and metabolite concentrations and genotypic variation at CYP2D6 and CYP2C19. The distribution of genetic variation within and among human populations was analyzed by descriptive statistics and variance analysis and by correlating the genetic and geographic distances using Mantel tests and spatial autocorrelation. The correlation between phenotypic and genotypic variation in drug metabolism observed in postmortem cases was also analyzed statistically. The genotyping methods developed proved to be informative, technically feasible, and cost-effective. Detailed molecular analysis of CYP2D6 genetic variation in a global survey of human populations revealed that the pattern of variation was similar to those of neutral genomic markers. Most of the CYP2D6 diversity was observed within populations, and the spatial pattern of variation was best described as clinal. On the other hand, genetic variants of CYP2D6, CYP2C9, and CYP2C19 associated with altered enzymatic activity could reach extremely high frequencies in certain geographic regions. Pharmacogenetic variation may also be significantly affected by population-specific demographic histories, as seen within the Finnish population. When pharmacogenetics was applied to a postmortem forensic setting, a correlation between amitriptyline metabolic ratios and genetic variation at CYP2D6 and CYP2C19 was observed in the sample material, even in the presence of confounding factors typical for these cases. In addition, a case of doxepin-related fatal poisoning was shown to be associated with a genetic defect at CYP2D6. Each of the genes studied showed a distinct variation pattern in human populations and high frequencies of altered activity variants, which may reflect the neutral evolution and/or selective pressures caused by dietary or environmental exposure. The results are relevant also from the clinical point of view since the genetic variation at CYP2D6, CYP2C9, and CYP2C19 already has a range of clinical applications, e.g. in cancer treatment and oral anticoagulation therapy. This study revealed that pharmacogenetics may also contribute valuable information to the medicolegal investigation of sudden, unexpected deaths.Farmakogenetiikka tutkii yksilöiden lääkeainevasteeseen vaikuttavia perinnöllisiä tekijöitä. Lääkeaineita metaboloivat entsyymit CYP2D6, CYP2C9 ja CYP2C19 ovat keskeisiä farmakogeneettisessä tutkimuksessa, sillä niiden kautta metaboloituu noin 20-30% kliinisesti käytetyistä lääkeaineista ja ne ovat geneettisesti polymorfisia. Entsyymien aktiivisuus vaihtelee hitaasta ultranopeaan, minkä seurauksena yksilöt voivat altistua esimerkiksi lääkeaineiden yllättäville sivu- ja yhteisvaikutuksille. Tämän tutkimuksen tavoitteena oli kehittää CYP2D6-, CYP2C9- ja CYP2C19-geeneille genotyypitysmenetelmä, joka kattaa tärkeimmät entsyymien aktiivisuuteen vaikuttavat mutaatiot, ja kuvata näiden geenien variaatio erilaisissa ihmisväestöissä ensimmäistä kertaa maailmanlaajuisessa mittakaavassa. Lisäksi farmakogenetiikkaa sovellettiin oikeuslääketieteeseen tutkimalla geneettisen variaation merkitystä kuolemaan johtaneissa lääkeainemyrkytyksissä. Kehitetyt genotyypitysmenetelmät perustuivat polymeraasiketjureaktioon ja minisekvensointiin ja ne osoittautuivat luotettaviksi ja edullisiksi. Tutkimuksen aineisto, joka koostui 138 (CYP2D6), 129 (CYP2C9) ja 146 (CYP2C19) populaationäytteestä, kerättiin sekä genotyypittämällä uusia näytteitä että keräämällä aineistoa kirjallisuudesta. CYP2D6-geenin variaation molekulaarinen analysointi osoitti, että sen maailmanlaajuinen vaihtelu ihmisväestöissä on verrattavissa neutraalien genomisten markkereiden variaatioon: suurin osa diversiteetistä oli yksilöiden välillä ja vaihtelu eri maantieteellisillä alueilla oli asteittaista. Toisaalta kaikkien tutkittujen geenien muuttuneen entsyymiaktiivisuuden variantit olivat yleisiä ja esiintyivät tietyillä maantieteellisillä alueilla hyvin korkeilla frekvensseillä. Variaation analysointi suomalaisessa väestössä myös osoitti, että väestödemografiset tekijät voivat vaikuttaa merkittävästi farmakogeneettiseen variaatioon. Tutkimuksen tulokset korostavat yksilöllisen lääkehoidon merkitystä. Tutkittujen geenien variaatio voidaan ottaa huomioon esimerkiksi joidenkin syöpälääkkeiden, veren hyytymistä ehkäisevien lääkkeiden ja protonipumpun estäjien annostelussa optimaalisen hoitovasteen saavuttamiseksi. Farmakogenetiikka osoittautui merkittäväksi tekijäksi myös oikeuslääketieteellisessä kuolinsyynselvityksessä. Geneettisen variaation vaikutus trisyklisen masennuslääkkeen amitriptyliinin metaboliaan havaittiin oikeuslääketieteellisessä näytemateriaalissa. Lisäksi kuolemaan johtaneen doksepiini-myrkytyksen yhtenä vaikuttavana tekijänä todettiin olleen geneettinen variaatio CYP2D6-entsyymissä

Onko farmakogeneettisestä tiedosta apua lasten yksilöllisten lääkeannosten määritykseen kasvun ja kehityksen aikana?

Teema : lastenlääkkeet. English summar

Comparative evaluation of the My5-FU™ immunoassay and LC-MS/MS in monitoring the 5-fluorouracil plasma levels in cancer patients

Background: Chemotherapies of solid tumors commonly include 5-fluorouracil (5-FU). With standard doses of 5-FU, substantial inter-patient variability has been observed in exposure levels and treatment response. Recently, improved outcomes in colorectal cancer patients due to pharmacokinetically guided 5-FU dosing were reported. We aimed at establishing a rapid and sensitive method for monitoring 5-FU plasma levels in cancer patients in our routine clinical practice. Methods: Performance of the Saladax My5-FU™ immunoassay was evaluated on the Roche Cobas® Integra 800 analyzer. Subsequently, 5-FU concentrations of 247 clinical plasma samples obtained with this assay were compared to the results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and other commonly used clinical analyzers (Olympus AU400, Roche Cobas c6000, and Thermo Fisher CDx90). Results: The My-FU assay was successfully validated on the Cobas Integra 800 analyzer in terms of linearity, precision, accuracy, recovery, interference, sample carryover, and dilution integrity. Method comparison between the Cobas Integra 800 and LC-MS/MS revealed a proportional bias of 7% towards higher values measured with the My5-FU assay. However, when the Cobas Integra 800 was compared to three other clinical analyzers in addition to LC-MS/MS including 50 samples representing the typical clinical range of 5-FU plasma concentrations, only a small proportional bias (≤1.6%) and a constant bias below the limit of detection was observed. Conclusions: The My5-FU assay demonstrated robust and highly comparable performance on different analyzers. Therefore, the assay is suitable for monitoring 5-FU plasma levels in routine clinical practice and may contribute to improved efficacy and safety of commonly used 5-FU-based chemotherapie

Comparative evaluation of the My5-FU™ immunoassay and LC-MS/MS in monitoring the 5-fluorouracil plasma levels in cancer patients

BACKGROUND: Chemotherapies of solid tumors commonly include 5-fluorouracil (5-FU). With standard doses of 5-FU, substantial inter-patient variability has been observed in exposure levels and treatment response. Recently, improved outcomes in colorectal cancer patients due to pharmacokinetically guided 5-FU dosing were reported. We aimed at establishing a rapid and sensitive method for monitoring 5-FU plasma levels in cancer patients in our routine clinical practice. METHODS: Performance of the Saladax My5-FU™ immunoassay was evaluated on the Roche Cobas® Integra 800 analyzer. Subsequently, 5-FU concentrations of 247 clinical plasma samples obtained with this assay were compared to the results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and other commonly used clinical analyzers (Olympus AU400, Roche Cobas c6000, and Thermo Fisher CDx90). RESULTS: The My-FU assay was successfully validated on the Cobas Integra 800 analyzer in terms of linearity, precision, accuracy, recovery, interference, sample carryover, and dilution integrity. Method comparison between the Cobas Integra 800 and LC-MS/MS revealed a proportional bias of 7% towards higher values measured with the My5-FU assay. However, when the Cobas Integra 800 was compared to three other clinical analyzers in addition to LC-MS/MS including 50 samples representing the typical clinical range of 5-FU plasma concentrations, only a small proportional bias (≤1.6%) and a constant bias below the limit of detection was observed. CONCLUSIONS: The My5-FU assay demonstrated robust and highly comparable performance on different analyzers. Therefore, the assay is suitable for monitoring 5-FU plasma levels in routine clinical practice and may contribute to improved efficacy and safety of commonly used 5-FU-based chemotherapies

Diagnostic yield of genetic testing in a heterogeneous cohort of 1376 HCM patients

Background Genetic testing in hypertrophic cardiomyopathy (HCM) is a published guideline-based recommendation. The diagnostic yield of genetic testing and corresponding HCM-associated genes have been largely documented by single center studies and carefully selected patient cohorts. Our goal was to evaluate the diagnostic yield of genetic testing in a heterogeneous cohort of patients with a clinical suspicion of HCM, referred for genetic testing from multiple centers around the world. Methods A retrospective review of patients with a suspected clinical diagnosis of HCM referred for genetic testing at Blueprint Genetics was undertaken. The analysis included syndromic, myopathic and metabolic etiologies. Genetic test results and variant classifications were extracted from the database. Variants classified as pathogenic (P) or likely pathogenic (LP) were considered diagnostic. Results A total of 1376 samples were analyzed. Three hundred and sixty-nine tests were diagnostic (26.8%); 373 P or LP variants were identified. Only one copy number variant was identified. The majority of diagnostic variants involved genes encoding the sarcomere (85.0%) followed by 4.3% of diagnostic variants identified in the RASopathy genes. Two percent of diagnostic variants were in genes associated with a cardiomyopathy other than HCM or an inherited arrhythmia. Clinical variables that increased the likelihood of identifying a diagnostic variant included: an earlier age at diagnosis (p <0.0001), a higher maximum wall thickness (MWT) (p <0.0001), a positive family history (p <0.0001), the absence of hypertension (p = 0.0002), and the presence of an implantable cardioverter-defibrillator (ICD) (p = 0.0004). Conclusion The diagnostic yield of genetic testing in this heterogeneous cohort of patients with a clinical suspicion of HCM is lower than what has been reported in well-characterized patient cohorts. We report the highest yield of diagnostic variants in the RASopathy genes identified in a laboratory cohort of HCM patients to date. The spectrum of genes implicated in this unselected cohort highlights the importance of pre-and post-test counseling when offering genetic testing to the broad HCM population.Peer reviewe

GRINL1A Complex Transcription Unit Containing GCOM1, MYZAP, and POLR2M Genes Associates with Fully Penetrant Recessive Dilated Cardiomyopathy

Background: Familial dilated cardiomyopathy (DCM) is a monogenic disorder typically inherited in an autosomal dominant pattern. We have identified two Finnish families with familial cardiomyopathy that is not explained by a variant in any previously known cardiomyopathy gene. We describe the cardiac phenotype related to homozygous truncating GCOM1 variants.Methods and Results: This study included two probands and their relatives. All the participants are of Finnish ethnicity. Whole-exome sequencing was used to test the probands; bi-directional Sanger sequencing was used to identify the GCOM1 variants in probands' family members. Clinical evaluation was performed, medical records and death certificates were obtained. Immunohistochemical analysis of myocardial samples was conducted. A homozygous GCOM1 variant was identified altogether in six individuals, all considered to be affected. None of the nine heterozygous family members fulfilled any cardiomyopathy criteria. Heart failure was the leading clinical feature, and the patients may have had a tendency for atrial arrhythmias.Conclusions: This study demonstrates the significance of GCOM1 variants as a cause of human cardiomyopathy and highlights the importance of searching for new candidate genes when targeted gene panels do not yield a positive outcome.Peer reviewe

Parameters for characterising indoor space connected to ill health symptoms?

Proceeding volume: 33Suuren toimistokiinteistön työtiloista (11 kpl) mitattiin sisäilman mikrobilaskeumien lajistoa ja resistenssejä homeeenesto kemikaaleille, toksiineja tuottavia mikrobeja pölyissä ja rakenneavausnäytteissä; sisäpölyn toksisuutta ja sisäilmasta tiivistetyn veden toksisuutta, sekä monitoroitiin sisäilman 24/7 hengitettäviä hiukkasia (PM2.5, PM10), ilman painetta, lämpötilaa, suhteellista kosteutta sekä formaldehydiä, hiilidioksidia ja VOC aineita. Tavoitteena oli saada mittaustietoa niistä tekijöistä jotka ovat yhteydessä hyvinvointihaittaan. Tulokset osoittivat, että jokainen tutkittu, ongelmainen huone oli yksilöllinen mikrobiston, toksisuuksien, kemiallisten ja fysikaalisten parametrien suhteen, ja että useat 24/7 mitatut parametrit osoittivat viikkorytmiin, vuorokauden aikaan, tunti- tai jopa minuuttirytmiin kytkettyä syklisyyttä

Prevalence of RPGR-Mediated Retinal Dystrophy in an Unselected Cohort of Over 5000 Patients

Purpose: Comprehensive genetic testing for inherited retinal dystrophy (IRD) is challenged by difficult-to-sequence genomic regions, which are often mutational hotspots, such as RPGR ORF15. The purpose of this study was to evaluate the diagnostic contribution of RPGR variants in an unselected IRD patient cohort referred for testing in a clinical diagnostic laboratory. Methods: A total of 5201 consecutive patients were analyzed with a clinically validated next-generation sequencing (NGS)-based assay, including the difficult-to-sequence RPGR ORF15 region. Copy number variant (CNV) detection from NGS data was included. Variant interpretation was performed per the American College of Medical Genetics and Genomics guidelines. Results: A confirmed molecular diagnosis in RPGR was found in 4.5% of patients, 24.0% of whom were females. Variants in ORF15 accounted for 74% of the diagnoses; 29% of the diagnostic variants were in the most difficult-to-sequence central region of ORF15 (c.2470-3230). Truncating variants made up the majority (91%) of the diagnostic variants. CNVs explained 2% of the diagnostic cases, of which 80% were one- or two-exon deletions outside of ORF15. Conclusions: Our findings indicate that high-throughput, clinically validated NGS-based testing covering the difficult-to-sequence region of ORF15, in combination with high-resolution CNV detection, can help to maximize the diagnostic yield for patients with IRD. Translational Relevance: These results demonstrate an accurate and scalable method for the detection of RPGR-related variants, including the difficult-to-sequence ORF15 hotspot, which is relevant given current and emerging therapeutic opportunities.Peer reviewe

Increased HSF1 expression predicts shorter disease-specific survival of prostate cancer patients following radical prostatectomy

Prostate cancer is a highly heterogeneous disease and the clinical outcome is varying. While current prognostic tools are regarded insufficient, there is a critical need for markers that would aid prognostication and patient risk-stratification. Heat shock transcription factor 1 (HSF1) is crucial for cellular homeostasis, but also a driver of oncogenesis. The clinical relevance of HSF1 in prostate cancer is, however, unknown. Here, we identified HSF1 as a potential biomarker in mRNA expression datasets on prostate cancer. Clinical validation was performed on tissue microarrays from independent cohorts: one constructed from radical prostatectomies from 478 patients with long term follow-up, and another comprising of regionally advanced to distant metastatic samples. Associations with clinical variables and disease outcomes were investigated. Increased nuclear HSF1 expression correlated with disease advancement and aggressiveness and was, independently from established clinicopathological variables, predictive of both early initiation of secondary therapy and poor disease-specific survival. In a joint model with the clinical Cancer of the Prostate Risk Assessment post-Surgical (CAPRA-S) score, nuclear HSF1 remained a predictive factor of shortened disease-specific survival. The results suggest that nuclear HSF1 expression could serve as a novel prognostic marker for patient risk-stratification on disease progression and survival after radical prostatectomy.</p