Location:
Faculty of Medicine in Rijeka
Department of Medical Biology and Genetics (main building, second floor)
Braće Branchetta 20, 51 000 Rijeka
Contact:
Phone: +385 (0)51 651 131
Fax: +385 (0)51 678 89
E-mail: medgen.medri©uniri.hr
Service description
1. GENETIC TESTING FOR THE ANGIOTENSIN-CONVERTING ENZYME (ACE)
ACE activity
The renin-angiotensin system is an important regulator of blood pressure and electrolyte homeostasis. The angiotensin-converting enzyme catalyses the hydrolysis of angiotensin I into angiotensin II, which is a powerful vasoconstrictor. The enzyme also inactivates bradykinin, which is a potent vasodilator.
Genetic predisposition
Serum and tissue ACE concentration/levels varies depending on the insertion/deletion (I/D) polymorphism, which is responsible for 14–50% of the interindividual variability in enzymatic activity. Deletion of part of intron 16 on one or both alleles (I/D or D/D genotype) is responsible for elevated blood enzyme concentration/levels. The ACE I/D polymorphism is associated with hypertension, coronary heart disease, stroke, diabetic nephropathy, etc. Studies have shown that homozygous ACE D/D carriers have twice the risk of myocardial infarction or stroke compared with the I/I genotype. Furthermore, the DD genotype is also a marker of poor prognosis after a stroke. Therefore, the ACE I/D genotype has both prognostic and pharmacological significance in ACE inhibitor therapy. According to pharmacogenetic tests, patients with hypertension and D/D genotype are less responsive to ACE inhibitor therapy than patients with I/I genotype.
Detection/Analysis method:
PCR, gel electrophoresis
Test results:
Genotype: – I/D heterozygote – D/D homozygote – I/I homozygote
- SAMPLE REQUIRED FOR THE TEST: VENOUS BLOOD (EDTA anticoagulant)
- PATIENT ADMISSION: EVERY DAY FROM 9 AM TO 12 PM
- DURATION OF THE TEST: 5–10 DAYS
- SERVICE COST: HRK 306.96 (+ HRK 220.78 for DNA isolation) or internal referral of the CHC Rijeka
2. GENETIC TESTING FOR HEREDITARY HEMOCHROMATOSIS (HFE GENE)
Hemochromatosis
Hemochromatosis is an iron metabolism disorder in which increased iron absorption leads to its excessive deposition in parenchymal organs, primarily the liver, pancreas, heart and pituitary gland. Clinical manifestations of hemochromatosis include hepatomegaly, skin pigmentation, diabetes, heart disease, arthritis and hypogonadism. Symptoms of the disease appear between the ages of 40 and 60, and it starts with weakness, exhaustion, weight loss, skin discolouration, abdominal pain, decreased libido and diabetes. Biochemical findings that indicate excessive iron accumulation in the liver are primarily elevated serum ferritin levels and increased transferrin saturation. Along with the appropriate clinical presentation and biochemical parameters, hemochromatosis is confirmed by a molecular genetic test for HFE gene mutation.
Genetics of hemochromatosis
There are several types of genetic hemochromatosis. The most common one is hereditary hemochromatosis type 1, in which an HFE gene mutation for hemochromatosis is located on the short arm of chromosome 6 (6p21.3). Two mutations are most commonly associated with the disease, in which the amino acid cysteine is replaced with tyrosine at position 282 (C282Y) or histidine is replaced with asparagine at position 63 (H63D), which leads to the formation of altered hemochromatosis protein. In approximately 87% of cases, patients with hereditary hemochromatosis are homozygous for the C282Y mutation on both alleles or compound heterozygotes for the C282Y mutation on one allele and for the H63D mutation on the other allele. There is approximately 1% of homozygotes for the H63D mutation on both alleles.
Detection/Analysis method:
PCR-RFLP
Test results:
| Mutation | Genotype | ||
| C282Y | C282Y/C282Y | C282Y/wt | wt/wt |
| H63D | H63D/H63D | H63D/wt | wt/wt |
- SAMPLE REQUIRED FOR THE TEST: VENOUS BLOOD (EDTA anticoagulant)
- PATIENT ADMISSION: EVERY DAY FROM 9 AM TO 12 PM
- DURATION OF THE TEST: 5–10 DAYS
- SERVICE COST: HRK 639.46 (+ HRK 220.78 for DNA isolation) or internal referral of the CHC Rijeka
3. GENETIC TESTING FOR PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1)
PAI-1 activity
Plasminogen activator inhibitor-1 is a glycoprotein that regulates the fibrinolytic system, primarily inhibiting tissue and urokinase plasminogen activator (tPA and uPA). The main function of PAI-1 is to reduce fibrinolysis, which leads to fibrin accumulation, while an increase in plasma PAI-1 affects the normal fibrin degradation and promotes thrombosis. According to recent studies, an increase in PAI-1, except it leads to hypofibrinolysis, reduces the activity of matrix metalloproteinases (MMP) and cell adhesion.
Genetic predisposition
In the promoter region of the PAI-1 gene, the specific 4G/5G polymorphism was detected that affects the PAI-1 expression. Studies have shown that 4G/4G homozygotes have a 25% higher PAI-1 plasma concentration/levels than persons with the 5G/5G genotype. According to previous studies, the 4G allele represents an additional risk factor for deep vein thrombosis and myocardial infarction at a younger age in combination with other genetic and environmental risk factors. Studies are being conducted on the link between the 4G/5G polymorphism and other multifactorial disorders.
Detection/Analysis method:
PCR-RFLP
Test results:
Genotype: – 4G/5G heterozygote – 4G/4G homozygote – 5G/5G homozygote
- SAMPLE REQUIRED FOR THE TEST: VENOUS BLOOD (EDTA anticoagulant)
- PATIENT ADMISSION: EVERY DAY FROM 9 AM TO 12 PM
- DURATION OF THE TEST: 5–10 DAYS
- SERVICE COST: HRK 343.14 (+ HRK 220.78 for DNA isolation) or internal referral of the CHC Rijeka
4. GENETIC TESTING FOR CONGENITAL SENSORINEURAL HEARING LOSS
(connexin 26 – GJB2 and connexin 30 – GJB6)
Congenital sensorineural hearing loss
Hearing loss is the most common sensory disorder in which early, congenital hearing loss adversely affects speech development and cognitive processes. The incidence of congenital hearing loss is 1–4/1000 newborns in different populations. In about half of cases, hearing loss is hereditary, of which one-third occurs as part of different syndromes (syndromic hearing loss), whereas in two-thirds of cases, hearing loss is the only symptom (nonsyndromic hearing loss).
The most common mutation, significant for autosomal recessive congenital sensorineural hearing loss (nonsyndromic hearing loss and deafness, DFNB1), is a guanine nucleotide deletion at position 35 (35delG) in the GJB2 gene for connexin 26. The coding gene sequence consists of 681 bp and is translated into a protein of 226 amino acids. The gene is located on chromosome 13q11-12. A mutation in the GJB6 gene for connexin 30 (delGJB6-D13S1830) located on chromosome 13q12 is also significant for DFNB1.
Detection/Analysis method:
PCR, gel electrophoresis
Test results:
| Mutation | Genotype | ||
| 35delG | +/+ | +/- | -/- |
| delGJB6-D13S1830 | +/+ | +/- | -/- |
– mutation not present
+ mutation present
- SAMPLE REQUIRED FOR THE MOLECULAR GENETIC TEST: 6 ml of VENOUS BLOOD (EDTA anticoagulant)
- DURATION OF THE TEST: 5–10 DAYS
- SERVICE COST: HRK 387.92 (+ HRK 220.78 for DNA isolation) or internal referral of the CHC Rijeka
5. Y CHROMOSOME MICRODELETION TEST
Indications for testing:
- abnormal semen analysis results (oligozoospermia or azoospermia)
- preparing for in vitro fertilisation (intracytoplasmic sperm injection – ICSI)
What does the test detect?
The test detects the presence or absence of certain genetic markers or gene parts (microdeletions) in the AZF (azoospermia factor) regions of the Y chromosome responsible for spermatogenesis, which represent a possible cause of infertility in 5–14% of infertile men. The presence of Y chromosome microdeletions in the DNA sample is detected by polymerase chain reaction (PCR). The presence of seven genetic markers (STS) is analysed: sY14, sY84, sY86, sY127, sY134, sY254 and sY255 (Simoni et al. EMQN, 2001). Repeated negative PCR test results indicate the presence of microdeletions.
What to do in case of confirmed microdeletion?
If microdeletions are detected in one of the AZF regions (AZF a, b or c), the molecular genetic test can be extended by testing additional genetic markers to identify the limits of microdeletions and thus the number of lost genes. Men with confirmed Y chromosome microdeletions are recommended genetic counselling before undergoing testicular sperm extraction (TESE) and in vitro fertilisation since microdeletions are transmitted to 100% of male offspring.
Detection/Analysis method:
PCR, gel electrophoresis
- SAMPLE REQUIRED FOR THE TEST: VENOUS BLOOD (EDTA anticoagulant)
- PATIENT ADMISSION: EVERY DAY FROM 9 AM TO 12 PM
- DURATION OF THE TEST: 2–3 WEEKS
- SERVICE COST: HRK 396.80 (+ HRK 220.78 for DNA isolation) or internal referral of the CHC Rijeka
6. GENETIC TESTING FOR MTHFR GENE POLYMORPHISMS
MTHFR (N5,N10-methylenetetrahydrofolate reductase) is a key enzyme in reducing N5,N10-methylenetetrahydrofolate to 5-methyl-THF, which is the main substrate in folate and homocysteine metabolism. The MTHFR gene’s two most important polymorphisms are C677T and A1298C. The MTHFR C677T polymorphism is caused by a base substitution of cytosine for thymine in the fourth exon, leading to an amino acid substitution of alanine for valine (A222V). The polymorphism is located in the catalytic domain of the protein, causing its reduced activity and affecting its thermolability. The enzyme with reduced activity binds folates more poorly, which significantly reduces the transfer of methyl groups and can lead to various disorders. The second functional polymorphism MTHFR A1298C is caused by the substitution of adenine for cytosine, which leads to the substitution of the amino acid glutamine with alanine (G429A). It is located in the regulatory domain of the protein. Its influence on the enzymatic activity is less pronounced than the influence of the MTHFR C677T polymorphism.
Detection/Analysis method:
PCR-RFLP
Test results:
| Genotype | Polymorphism – Genotype | |
| MTHFR C677T | MTHFR A1298C | |
| homozygote | CC | AA |
| heterozygote | CT | AC |
| homozygote | TT | CC |
- SAMPLE REQUIRED FOR THE TEST: VENOUS BLOOD (EDTA anticoagulant)
- PATIENT ADMISSION: EVERY DAY FROM 9 AM TO 12 PM
- DURATION OF THE TEST: 5–10 DAYS
- SERVICE COST: HRK 352.72 (+ HRK 220.78 for DNA isolation) or internal referral of the CHC Rijeka
7. EXON GENOMIC DIAGNOSTIC TESTING
Exon genomic diagnostic testing is conducted using the next-generation sequencing (NGS) technique and is used in clinical diagnostics to detect pathological variants in patients suspected of a genetic predisposition for a particular disease. NGS is used to diagnose medical disorders such as neurological diseases, cardiac diseases, developmental and intellectual disabilities, autism, behavioural disorders, multiple congenital anomalies and others. For diagnostic purposes, the NGS method is used for clinical exome sequencing, or targeted panels are used to diagnose diseases that have a similar clinical presentation and are caused by mutations in different genes. The NGS method is implemented in cooperation with the Clinical Institute of Genomic Medicine, University Medical Centre Ljubljana.
- SAMPLE REQUIRED FOR THE TEST: VENOUS BLOOD (EDTA anticoagulant)
- PATIENT ADMISSION: EVERY DAY FROM 9 AM TO 12 PM
- DURATION OF THE TEST: 6–9 MONTHS
- SERVICE COST: HRK 17,079.86 (+ HRK 220.78 for DNA isolation) or internal referral of the CHC Rijeka
8. DNA ISOLATION
Genetic testing is preceded by genomic DNA isolation. Genomic DNA is isolated from the patient’s peripheral blood (or other tissue) using isolation kits (Qiagen FlexiGene DNA Kit – QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions.
- SERVICE COST: HRK 220.78 or internal referral of the CHC Rijeka