Molecular genetics

Cystic fibrosis

Cystic fibrosis (CF) is a most common fatal autosomal recessive illness in Caucasian population with approximate frequency 1:2500 – 3000 newborns. Illness affects especially lungs and pancreas and often causes fertility problems. The problem is caused by disorder of transport of chloride ions over cell membrane, increasing its concentration in sweat and the amount of mucus in respiratory and gastrointestinal tract and often obstruction in male reproductive tract is also present.

Illness occurs in case, where both alleles of CFTR gene, located in chromosome 7 in area 7q31.2, have mutated (affected homozygote). 4% of Caucasian population (1 person out of 25 people) transmits mutated allele – these are heterozygote which does not carry signs of illness, but transmit one mutated (damaged) allele of CFTR gene (pic. 1, normal gene is blue, mutated is labelled with red colour).

(Pic. 1) Inheritance of cystic fibrosis

 

Over 95% of males with cystic fibrosis are infertile, because their vas deferens is not developed – this phenotype is known as congenital bilateral absence of vas deferens (CBAVD). On the other hand some infertile males without cystic fibrosis have CBAVD that associates with specific mutated alleles of CFTR gene.

In CFTR gene until today more that 900 various mutations have been identified. Many of those occur in low frequency, or are specific for some population groups or families. Illness is diagnosed using molecularly-genetic methods from blood, amniotic fluid or choroinic villi. Our clinic examines 30 mutations, thus covering 93% of all mutations of entire mutation spectrum occurring in Czech population.

Mutation of F5 gene and gene for prothrombin

Blood clotting is realized by cascade of individual steps, which are controlled by activity of plasmatic proteins. Their imbalance leads to various syndromes, for example risk of thrombosis. Thrombosis risk is increased by various hereditary factors, for example surgical operation, smoking, overweight, pregnancy, using hormonal contraception or HRT in perimenopausal women. The most important hereditary factor increasing risk of thrombosis is mutation of gene F5 for plasmatic factor FV (Leiden mutation), in gene coding prothrombin (mutation G20210A) and less often in genes for other plasmatic proteins.

Mutated allele of factor V (Leiden mutation or FV G1691A) occurs in about 2,5% of Caucasian population. Gene F5 for factor V is found in chromosome 1 in area 1q32. Illness is manifested under principles of autozomally intermediary heredity (pic. 2, where normal gene is labelled blue and mutated red.)

(Pic. 2) Inherited thrombosis tendencies

 

Mutation G20210A in gene for prothrombin is second most important hereditary risk factor for conception of thrombosis in Caucasian population with occurring frequency 2,4%. The type of inherited illness is the same as in previous case. Heterozygote individuals (females and males) – carriers of mutated allele gene coding prothrombin have in comparison with healthy individuals 3 times higher risk of thrombosis of deep veins of legs and 20 times higher risk of thrombosis of cerebral veins. This risk substantially increases with using hormonal contraception (up to 150 times), smokers are in a threat of a heart.

Approximately 15 to 40% of female carriers – carry both stated mutations (G1691A and G20210A). These women have significantly increased gynecological complications – repeated reproductive losses (spontaneous miscarriages, still born children) and others (severe pre-eclampsia, in utero retardation of fetal growth – IUGR, thrombosis of deep veins after ovarian stimulation).

Presence of mutation is found by blood examination using modern methods of molecular genetics. Females from risk groups require intensive treatment in preparation for pregnancy and also during the pregnancy and birth.

Chromosome Y

Chromosome Y is the smallest human chromosome. Relatively small number of functional genes are found in it, most of those have its own homolog – same functional copy on X chromosome. Only some genes are specific for chromosome Y: gene SRY and gene family DAZ (deleted in azoospermia) in area called AZF (azoospermia factor). Absence of these genes has been found in males with severe form of oligospermia (low number of sperm in ejaculate) and in men with azoospermia (absence of sperms in ejaculate).

Picture 3 is schematically demonstrating Y chromosome where most important genes and areas are highlighted.

(Pic. 3) Y chromosome scheme

 

Gene SRY is found on the distal part of short arm of Y chromosome. Its product functions as a factor regulating the development of undifferentiated gonad into male genital gland – testis. Occasionally (with frequency 1:10 000 newborn males) gene SRY transfers onto X chromosome during pairing of gonosomes X and Y resulting in males with karyotype 46,XX. Such men have muscular habitus, but due to shortage of other genes necessary for spermatogenesis they are sterile. Mutation or deletion of gene SRY has been found in females with karotype 46,XY and infertility.

Approximately 10% of males with reproductive disorders (significant decrease in number and quality of sperm or its absence) have microdeletion in AREA AZF. Genes found in this area are responsible for creation, evolution and maturing of sperm. Its presence is demonstrated in various forms from low quality sperm (reduced quantity, abnormal motility or morphology) to its total absence. Not all cases of infertility are caused by microdeletion of DAZ genes in area AZF. It has been found that spermatogenesis disorders are caused by spot mutations in other genes, for example CD1Y or USP9Y.

(Pic. 4) Deletion of long limb of Y chromosome in karyotype of male with oligospermia.

 

Using molecular-genetic methods, deletion of entire part of AZF has been found. Similar methods are used in diagnosing microdeletions of Y chromosome, which under microscope appear to be normal.

 

Modern micro-sugical and micromanipulative methods used in our clinic enable bypassing biological and genetic barriers of sterility and achieving pregnancy in cases where natural conception is not possible. If sperm carries microdeletion on Y chromosome, than it carries this genetic defect to all male offspring with 100% probability. That means all male children will have the same the problem as their fathers.

Picture 5 Picture 5 schematically demonstrates the transfer of microdeletion on Y chromosome under holandric inheritance (transfer from father to son, microdeletion of chromosome is represented by red sign):

(Pic. 5) Scheme demonstrating microdeletion in AZF area on Y chromosome