Diagnostic Systems

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Liver Detoxification

The human body is continuously confronted with foreign and to some extent toxic substances. These xenobiotics include drugs, stimulants and chemicals. A great many of these foreign substances cannot be readily excreted as they are lipophilic, i.e. poorly or not at all water-soluble. Biotransformation prevents accumulation of xenobiotics by transforming lipophilic compounds into more hydrophilic ones that can be excreted from the body. Additionally biotransformational processes are involved in converting pro-drugs (inactive medication precursors) into their active forms.  

There are three phases of biotransformation. During phase I functional groups are attached, either via oxidation, reduction or hydrolysis. Whilst phase II the intermediate products from phase I are coupled to endogenous, mostly highly hydrophilic functional groups (conjugation reaction). The last phase, phase III, encompasses export of now water-soluble substances from the cells. If this process is hampered by enzymatic deficiencies, e.g. caused by polymorphisms and other genetic variations, severe complications can develop. Foreign substances accumulate in the body potentially leading to serious side-effects or diseases, as for example cancer. Consequently, also pro-drugs are not activated and are not fully functional and efficient.

Reactive oxygen species (ROS) are damaging to all kinds of biomolecules. During biotransformation, as well as by drugs and environmental toxins, ROS can be produced inside the body. The antioxidative system is engineered to eliminate ROS and protect cells before they are damaged. Among others, cancer and neurodegenerative diseases can be the consequence of impaired ROS clearing, e.g. by dysfunctional enzymes.

GenoChip Toxo

A number of polymorphisms and gene variations with relation to detoxification have been identified so far. Genotyping detects these mutations and identifies such persons who are at a higher risk for developing the above mentioned conditions.

Table 1: Genes and markers participating in detoxification

Gene

Variation

rsNumber

CYP1A1

*2A (3798T>C)

rs4646903

CYP1A2

*1C (-3860G>A)

rs2069514

*1F (-163C>A)

rs762551

CYP2C9

*2 (430C>T)

rs1799853

*3 (1075A>C)

rs1057910

CYP2C19

*2 (681G>A)

rs4244285

*3 (636G>A)

rs4986893

*17 (-806C>T)

rs12248560

CYP3A5

*2 (27289C>A)

rs28365083

*3 (6986A>G)

rs776746

CYP3A4

*1B (-392A>G)

rs2740574

CYP2B6

516G>T

rs3745274  

VKORC1

*2 (-1639G>A)

rs9923231

GSTM1

14kb Deletion

---

GSTT1

50kb Deletion

---

GSTP1

Ile105Val

rs1695

Ala114Val

rs1138272

NAT2

191G>A

rs1801279

341T>C

rs1801280

481C>T

rs1799929

590G>A

rs1799930

857G>A

rs1799931

COMT

Val158Met

rs4680

SOD2

Val16Ala

rs4880

MDR1

3435C>T

rs1045642

CYBA

242C>T

rs4673

MTHFR

677C>T

rs1801133

Factor V

1691G>A

rs6025

Factor II

20210G>A

rs1799963

 
References:
 
Coleman M. D. Human Drug Metabolism: An Introduction. 2. Edition, 2010. Hoboken, NJ, USA: Wiley-Blackwell.
 
Ernst B., Vögtli A. Moderne Pharmakokinetik: Transport durch Membranen. 1. Edition, 2010. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co KGaA.
 
Karlson P., Doenecke D., Koolman J., Fuchs G., Gerok W. Karlsons Biochemie und Pathobiochemie. 5. Edition, 2005. Stuttgart, Germany: Thieme Verlagsgruppe.

Osteoporosis

Osteoporosis is a widespread disease affecting the skeleton. It is characterised by low bone mineral density (BMD) and defects in the bone’s micro architecture resulting in lower bone strength and increased susceptibility to fractures. Osteoporosis can arise if either (1) the maximal bone density is not reached or (2) there is an exaggerated bone resorption and (3) there is reduced bone formation during the constant process of bone remodelling. However, the occurrence of fragile bone tissue is usually caused by interplay of all these mechanisms.

Hormones play an essential role in the preservation of bone mass, for example oestrogen reduction leads to increased bone resorption in postmenopausal women. Another important element is the calcium metabolism. A deficit in calcium, and vitamin D as well, might result in reduced bone formation. Calcium levels in the blood are regulated by the hormones parathyroid hormone (PTH) and calcitonin. PTH activates osteoclasts stimulating bone formation, while calcitnonin affects osteoblasts enforcing bone resorption. A further mechanism influencing bone formation and resorption is the RANKL-RANK-OPG signalling pathway. Osteoblasts produce RANKL, a ligand for the receptor activator of NF-κB (RANK) on haematopoietic stem cells. Binding of RANKL to RANK initiates differentiation of osteoclasts, maintaining their function. Additionally osteoblasts produce osteoprotegerin (OPG, also known as TNFRSF11B), a decoy receptor blocking the RANK/RANKL interaction. The RANKL/OPG ratio is one of the key factors to maintaining a normal bone turnover, leading to a healthy bone mass and strength respectively.

Apart from age and gender, several environmental factors have an impact on osteoporosis risk. These include diet, physical activity, medications and concomitant diseases. However, one of the most important risk factors is a family history of osteoporosis, underlining the relevance of genetics for its pathogenesis. It also offers an explanation to the heterogeneity of the disease, which does not solely result from differences in systemic and local regulators, but rather has its causes in receptors, signalling pathways, transcription factors, enzymes or structural proteins.

GenoChip Osteo

So far, genome-wide association studies and linkage analyses have identified a number of genes which have a significant, or at least a suggestive, association towards the clinical picture of osteoporosis

 

Table 1: Genes and markers correlated to osteoporosis

Character

Gene

Variation

rs number

Vitamin D endocrine

system

 

VDR

FokI

rs2228570

BsmI

rs1544410

CYP24A1

E143del

-

R159Q

-

E322K

-

R396W

rs114368325  

L409S

rs 6068812

A457fsX490

-

Oestrogen endocrine

system

ESR1

XbaI

rs 9340799

PvuII

rs 2234693

RANK/RANKL/OPG

signalling pathway

TNFRS11B

A-163G

rs 3102735

G-245T

rs 3134069

Bone turnover

COL1A1

Sp1

rs1800012

BGLAP

T-198C

rs1800247

C>T

rs1543294

CALCR

AluI

Rs1801197

KL

C>A

rs 577912

 
References:
 
Ralston S. H., Ultterlinden A. G. Genetics of Osteoporosis. Endocrine Rev 31 (2010), 629-662.
 
Zheng H.-F., Spector T. D., Richards J. B. Insights into the genetics of osteoporosis from recent genome-wide association studies. Expert Rev Mol Med 13 (2011), 1-12.
 
Raisz L. G. Pathogenesis of osteoporosis: concepts, conflicts, and prospects. J Clin Invest 115 (2005), 3318-3325.
 
Sandhu S. K., Hamson G. The pathogenesis, diagnosis, investigation and management of osteoporosis. J Clin Pathol 64 (2011), 1042-1050.
 
Richards J. B, Zheng H.-F., Spector T. D. Genetics of osteoporosis from genome-wide association studies: advances and challenges. Nature Rev Gen 13 (2012), 576:588

5-FU

Next to age, gender and environmental influences such as diet and comedication, there are genetic factors that can have a major impact on the catalytic activity of many enzymes. It is known that patients with decreased or totally absent function of the Dihydropyrimidin-Dehydrogenase (DPD) enzyme, are at a higher risk to develop severe to lethal side effects upon chemotherapy with 5-Fluorouracil (5-FU). Those are ranging between grades 3-4 according to the WHO.

5-FU is a cytostatic routinely used for the treatment of many solid tumors. Analysis of the DPYD gene can help to reduce the risk of 5-FU related toxicities by anticipating the catalytic function of the DPD enzyme for each individual patient. Carrier of one of the examined genetic variants of the DPYD-gene can therefore be identified prior to therapy initiation and can be treated accordingly with lower doses or alternative medications. The most common variation of the DPYD-gene is the DPD*2A (exon-14-skipping) mutation. This mutation leads to a deletion of a 165 basepair long fragment within exon 14 and thus to the loss of normal enzyme activity. Of all patients carrying one of the variants of the DPYD gene and are nevertheless treated with 5-FU, more than 40 % will experience severe side effects (grade 4 according to WHO). Therefore, in many cases the occurrence of reduction in enzymatic activity or side effects in consequence of 5-FU treatment can be explained with the exon-14-skipping mutation in the DPYD-gene.

GenoChip 5-FU

Since the DPD*2A (exon-14-skipping) mutation is not the only one in the DPYD-gene causing a reduction of enzyme activity, the GenoChip 5-FU tests for a total 13 of those gene variations.

Table 1: DPYD gene variations and their frequencies in the Caucasian population
Gene variant Allele frequency
DPYD*2A 1 %
DPYD*3 1%
DPYD*4 2 %
DPYD*5 14 %
DPYD*6 Unknown
DPYD*7 Unknown
DPYD*8 Unknown
DPYD*10 4 %
DPYD*12 Unknown
DPYD*13 1 %
DPYD M166V 8 %
DPYD A551T Unknown
DPYD D949V 0,6 %

If no explanation for the occurrence of severe side effects by exclusive determination of the exon-14-skiping mutation is found, a further molecular genetic analysis of the DPYD-gene should be given consideration.

Such a genotyping can help to optimize therapies individually and to avoid undesirable side effects. Nevertheless, individual, not genetic-based factors should always be examined to guarantee the best possible patient safety.