GenoID

Genetic factors of male infertility

• Y chromosome microdeletion (AZF-a, -b and -c)
• Chromosomal sex (X-Y aneuploidias)
• Cystic fibrosis (5 mutations)

The cause of male factor origin infertility associated with oligo-azoospermia is unknown in 50% of cases. These “idiopathic” cases are commonly related to genetic disorders.

One of the most common causes is the presence of a genetic lesion in the azoospermia factor (AZF) region of the Y chromosome. Microdeletion of the AZF region can be detected in about 1 in 10 men with oligo-azoospermia. Microdeletion destroys Y chromosome regions that play an important role in the regulation of spermatogenesis (DAZ, RMBY). Testing is based on the amplification by PCR of known sequences in the AZF A, B and C regions. The failure of amplification is consistent with a microdeletion affecting the tested region. This warrants the involvement of the patient in an in vitro fertilisation programme based on the ICSI technique.

Mutations in the gene for cystic fibrosis (CF) also comprise a relatively common cause of male infertility. Certain mild defects in the CF gene, rather than causing typical cystic fibrosis, result in bilateral obstruction of vas deferens thereby leading to infertility. This may arise from either of two situations – one copy of the CF gene carries a classical, or “severe”, mutation while the other copy contains a “mild” mutation, or both copies carry a “mild” mutation. The best known of the “severe” mutations is the ΔF508, but the R117H, G542X and N1303K mutations are also common. The 5T variant of the IVS8 5T/7T/9T polymorphism is considered a “mild” mutation, which leads to the production of a significantly reduced amount of functional gene product.

Chromosomal factors have a causative role in about 5% of infertile men. This proportion rises to 15% among azoospermic men, with the numerical (63%) or structural abnormalities of the sex chromosomes comprising the majority of cases. Klinefelter syndrome (47,XXY) is the most common sex chromosome aneuploidy,  , followed by the 47,XYY and 45,X karyotypes and their mosaic forms. Atresia of germ cells leads to azoospermia. Karyotype analysis should be part of the diagnostic workup of every men with oligo- or azoospermia. The molecular genetic test to detect sex chromosome aneuploidy can be used as a faster alternative to the time-consuming cytogenetic analysis, or as a pre-screen.

Indications for testing:

• part of the diagnostic workup of male infertility associated with oligo- and azoospermia
• before the use of assisted reproduction techniques
• other andrological indications

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Genetic testing for susceptibility to deep venous thrombosis

• Leiden mutation factor V. (can also be requested from gynaecological sample)
• Faktor II (prothrombin) G20210A mutation
• MTHFR C677T mutation

Deep venous thrombosis (DVT) and thromboembolism are common diseases in developed countries. Many risk factors are known, including prolonged bedrest, major surgery, trauma, malignancy, and smoking. Natural estrogens and their synthetic derivatives elevate the plasma levels of certain coagulation factors, thus, the risk of DVT is significantly increased during pregnancy, childbirth, oral contraceptive use. Being present in 50% of thromboembolic diseases, genetic causes are also important risk factors. About 20-30% of patients with DVT carry the Leiden mutation, and about 80% of familial thrombophilia is associated with this genetic defect. (The rest of the genetic factors, which are together involved in about 15% of inherited thrombotic cases, include mutations in the antithrombin, protein C, protein S genes.) Increased plasma homocystein levels also increase the risk of cardiovascular diseases, including DVT. The most common inherited causative factors of homocystein level elevation are a point mutation of the gene for the 5,10-methylene tetrahydrofolate reductase (MTHFR) enzyme. The most common mutation of the prothrombin (coagulation factor II) gene represents a mild risk factor of thrombosis by causing an increased prothrombin activity. As part of the diagnostic workup of thrombophilia, genetic testing may help set up the correct diagnosis; it allows the identification of a high-risk group for whom close surveillance, lifestyle change, perhaps preventive anticoagulant therapy are necessary; finally, it may indicate the need for family testing.

Indications for testing:

• venous thromboembolism (thrombophlibitis, pulmonary embolism)
• multiple thrombosis or thromboembolism in the family history
• thrombosis in an unusual location (mesentery, brain, retina)
• prior to pregnancy, oral contraceptive use, major surgery if there is a personal or family history of thrombosis
• recurrent pregnancy loss of unknown cause
• arteriosclerosis developing at a young age

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Polymorphisms in the ACE and angiotensinogen genes

The angiotensin-converting enzyme (ACE) converts the angiotensin I protein into angiotensin II, which in turn exerts a strong prompt and long-term effect on the cardiovascular system. The plasma ACE level is typical for an individual but may vary in the population. About half of this variation is determined by an insertion/deletion polymorphism in the ACE gene, which means the presence (insertion, I) or absence (deletion, D) of a 287 base-pair fragment in intron 16, resulting three possible genotypes: II, ID, or DD. In accordance to the higher plasma ACE levels associated with the D variant, the mean plasma ACE level of individuals with the DD genotype is approximately twice as high as the ACE level of individuals carrying the II genotype, while the ID genotype represents an intermediate ACE level. The presence of the D variant has been shown to be associated with an increased risk for cardiovascular diseases. The DD genotype is significantly overrepresented among patients with myocardial infarction or stroke. The polymorphism also plays a role in the response to ACE-inhibitors used in the treatment of hypertension – patients with the II genotype tend to respond better. It is noteworthy that the II genotype is associated with higher endurance performance.
The protein encoded by the angiotensinogen gene is cleaved by renin produced when blood pressure drops to result in angiotensin I, which in turn is converted into angiotensin II by further enzymatic action. Angiotensin II is a powerful vasoconstrictor, causes salt retention by stimulating aldosterone secretion, and elevates blood pressure. A certain position in the gene (M235T) encodes for methionine at both alleles in 42%, methionine in one copy and threonine in the other copy in 46%, and threonine at both alleles in 12% of the Caucasian population, respectively. The presence of the T allele, presumably via an increased angiotensinogen production, has been shown to confer a slightly higher risk than the M allele to hypertension; furthermore, some authors describe a two-fold risk to develop coronary heart disease in the presence of the T allele.. Because of its involvement in the development of hypertension during pregnancy, the T allele is associated with an increased risk for preeclampsia.

Indications for testing:

• increased risk for cardiovascular disease (presence of classical risk factors, positive family history)
• stroke at a young age (ACE)
• before treatment of hypertension (ACE)
• to judge development potential in endurance sports athletes (ACE)

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Genetic testing for hereditary haemochromatosis

• Haemochromatosis panel
(HFE C282Y, HFE H63D)

Hereditary haemochromatosis (HH) is a recessive inherited autosomal disorder of iron metabolism, characterized by continuous absorption of iron in the upper intestinal tract despite fully saturated iron stores. Progressive accumulation of iron in various organs causes progressive development of clinical symptoms with increasing age, finally leading to irreversible tissue damage. The main target organs are the liver (cirrhosis, hepatocellular carcinoma), pancreas (diabetes), joints (arthralgia, arthritis), heart (cardiomyopathy) and hypophysis (hypogonadism). The disease is fairly common in the Caucasian population (1 in 300), with a carrier frequency between 1 in 8 to 1 in 10. The gene encoding the HFE protein that is responsible for the disorder harbours two mutations that are associated with HH: C282Y and H63D. 85% of patients are homozygous for the C282Y mutation, while the H63D mutation can be considered as a predisposing factor when combined with other genetic (C282Y heterozygosity) or environmental factors. Early detection of the disease and treatment by regular phlebotomy offer a normal life expectancy for the patient. HH is the most common genetic disease in Europe and its treatment is simple and efficient.

Indications for testing:

• increased transferrin saturation (>50%) or serum ferritin concentration (>400 g/l in men, >200 g/l in women)
• the clinical diagnosis of haemochromatosis
• clinically diagnosed haemochromatosis or family history of verified C282Y mutation (combined with genetic counselling)

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Genetic testing for susceptibility to breast and ovarian cancer

• BRCA1(185delAG, 300T>G, 5382insC)
• BRCA2 (6174delT, 9326insA)

Breast cancer is one of the most common malignancies in women, showing 10% lifetime risk.. Approximately 6% of cases can be attributed to an inherited genetic defect (mutation), while this proportion is about 12% for ovarian cancer. Besides breast and ovarian cancer, the susceptibility also includes, to a lesser extent, cancers of other organs. Inherited breast cancer should be suspected if the cancer develops at a markedly young age (below 40), if it affects both breasts, if it develops in a man, if the breast or ovarian cancer affects multiple family members in several generations. About half and one third of the mutations conferring susceptibility are located in the BRCA1 and BRCA2 gene, respectively. Carriers of a mutation in one of the two genes have a 40 to 80% risk of developing breast cancer, and 10 to 60% risk of developing ovarian cancer by the age of 75.

Indications for testing:

• bilateral breast cancer
• multiple primary breast cancer or breast AND ovarian cancer
• male breast cancer
• one of the above in a first-degree relative
• breast cancer developing below the age of 40
• one or several relatives with breast AND ovarian cancer within the same lineage
• relative with a confirmed mutation in the BRCA1 or BRCA2 gene

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Paternity and relationship testing

Testing starts with the isolation of DNA from the submitted samples. The DNA samples are then subjected to examination of 15 selected sites that show variability in length in the population.  These 15 DNA fragments, with 2 copies each, make up a length pattern (profile) that is virtually unique to every person. In parent-child relationships, however, one copy of each fragment is identical by inheritance. In paternity testing, testing the mother helps to identify the paternal copies thus making testing more accurate. For the statistical calculations of the probability of paternity the frequencies of each length variation in the relevant population are considered. In most trio cases the probability of paternity either reaches 99.99% or is virtually zero. In motherless cases the number is, rarely, slightly lower.

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