What is molecular genetics?

Molecular genetics is the study of abnormalities in genes at the molecular level. For example, the mutation analyses of monogenic disorders such as Beta-thalassemia and Y-chromosome microdeletion are done using molecular techniques.

Here are some of the molecular tests performed in Istanbul Memorial Hospital Reproductive Genetics Department.

Molecular Genetic Tests

Test	Sample	Duration
Beta-Thalassemia	Blood with EDTA/Amniosynthesis/CVS*	20 days
Cystic Fibrosis-34 mutation	Blood with EDTA/ Amniosynthesis /CVS*	15 days
Cystic Fibrosis-500 mutation	Blood with EDTA/ Amniosynthesis /CVS*	40 days
FMF-12 mutation	Blood with EDTA/ Amniosynthesis /CVS *	30 days
FMF-whole gene	Blood with EDTA/ Amniosynthesis /CVS *	45 days
Y-chromosome microdeletion	Blood with EDTA	10 days
Thrombophilia Panel	Blood with EDTA	5 days
QF-PCR	Amniosynthesis /CVS	3 days
DNA Identification	Sperm	**

*The analysis of mutations for single gene disorders can also be performed using chorionic villus tissue. However, chorionic villus tissue must first be cultured and then analyzed for mutation. To exclude any possibility of contamination a “maternal contamination test” is also performed. 
**It is a legal requirement that the report shall not be given to the patient. 

What are monogenic disorders?

A monogenic disorder is the result of a single mutation in a gene. There are estimated to be over 4,000 human diseases caused by single gene defects.

What is a mutation?

Mutations are changes in the DNA sequence of a gene. There are estimated to be 30,000 different genes in the human genome. Mutations in these genes are responsible for genetic diseases. They are transferable from generation to generation with a broad range of inheritability.

How are monogenic disorders inherited?

There are different mechanisms of inheritance:

• Autosomal recessive (Figure 1a)
• Autosomal dominant (Figure 1b)
• X-linked
• Y-linked
• Mitochondrial

What are the most common autosomal recessive disorders?

The most frequently diagnosed are beta-thalassemia, cystic fibrosis, sickle cell anemia and spinal muscular atrophy.

What are the most common autosomal dominant disorders?

The most common dominant diseases are myotonic dystrophy and Huntington's disease.

What are the most common X-linked disorders?

Haemophilia A, fragile X syndrome and Duchenne muscular dystrophy are some of the most frequently diagnosed X-linked diseases. 

Figure 1A: Autosomal recessive inheritance

Figure 1B: Autosomal dominant inheritance

What samples are needed from the patient for molecular testing?

Molecular techniques are applicable to a broad range of samples and can be performed at three different stages:

• Postnatal diagnosis (peripheral blood)
• Prenatal diagnosis (amniotic fluid or CVS)
• Preimplantation genetic diagnosis (embryos)

When is postnatal diagnosis advisable?

Tests for postnatal diagnosis (diagnosis after birth) can be carried out on babies (including newborn), children or adults. If there is a family history of an inherited disorder, mutation analysis can be used to identify carriers of the mutation who have a risk of transmitting the disease to their offspring. Couples referred for assisted reproduction techniques may be advised to have a screening test, such as trombophilia panel if they are experiencing recurrent pregnancy losses or Y-chromosome microdeletion analysis in the case of  severe male infertility.

What tests can be performed on prenatal samples?

Prenatal diagnosis tests are performed before birth, during pregnancy and are used to identify mutations in the fetus (unborn baby). They are advisable for couples who have a high risk of transmitting a genetically inherited disease. Tests can be carried out on chorionic villus biopsies CVS  (11th-14th gestational weeks) and amniotic fluids (15th-20th gestational weeks). There is also a rapid aneuploidy detection test called QF-PCR for which both amniotic fluids and CVS materials can be used. For more information about chromosomal analysis please visit our web page for cytogenetics.

Preimplantation Genetic Diagnosis (PGD): In PGD, diagnostic tests are carried out on embryos before they are transferred into the mother’s uterus (before pregnancy) (Figure 2). PGD offers couples the possibility of having disease-free embryos selected and transferred, and thus having healthy children and avoiding the complications of terminating a pregnancy. For detailed information please visit our website for preimplantation genetic diagnosis.

Figure 2: In PGD studies, PCR reactions are performed in a sterile cabinet.

What types of monogenic disorders can PGD detect?

PGD can be performed for any single gene disorder for which molecular testing is possible. In Memorial Hospital, we have performed PGD for 71 different monogenic diseases for 195 couples (277 cycles). Below you can find the complete list of those disorders. Additionally, we have performed PGD for HLA typing in 461 cycles (242 couples). 

Adrenoleukodystrophy Arthrogryposis-Renal dysfunction-Cholestasis (ARC) syndrome Bartter syndrome Becker muscular dystrophy Beta Thalassemia Breast Cancer Charcot Marie Tooth (CMT) Citrullinemia Co-enzyme Q deficiency Congenital adrenal hyperplasia Congenital deafness Congenital factor VII deficiency Cystic fibrosis Delta beta talassemia Donohue syndrome Duschenne Muscular Dystrophy Ehler-Danlos Syndrome (EDS) Type VIIC Epidermolysis bullosa simplex Facioscapulohumeral muscular dystrophy (FSHD) Familial Hemophagocytic lymphohistiocytosis Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis Familial Mediterranean Fever Fanconi Anemia Fragile X syndrome Fraser Syndrome Galactosemia Glucose-6-phosphate dehydrogenase (G6PD) deficiency Hereditary multiple exostoses Hunter Syndrome Huntington's disease Hyper IgM syndrome Hypohidrotic ectodermal dysplasia Hypomyelination and congenital cataract Infantile neuroaxonal dystrophy Jubert Syndrome Keratitis–ichthyosis–deafness (KID) syndrome		Krabbe Disease Lafora Disease Leber's congenital amaurosis Li–Fraumeni syndrome Limb-girdle muscular dystrophy Maple syrup urine disease metachromatic leukodystrophy MTHFR deficiency Mucolipidosis Type I  Mucopolysaccharidosis Type I (Hurler Syndrome) Mucopolysaccharidosis Type IIIA Mucopolysaccharidosis Type IIIB Mucopolysaccharidosis Type VI Myotonic Dystrophy Nemaline myopathy Neurofibromatosis Niemann–Pick disease Nonketotic hyperglycinemia Osteogenesis imperfecta Osteopetrosis Phenylketonuria Polycystic kidney disease Pompe disease Propionic acidemia Retinoblastoma Sickle Cell Disease Spastic Paraplegia Type III Spastic Paraplegia Type V Spinal Muscular Atrophy Spinocerebellar ataxia type 2 (SCA2) Tay–Sachs disease TNF receptor associated periodic syndrome (TRAPS) Tuberous sclerosis Usher Syndrome Type 1B Zelweger syndrome

•    Beta-Thalassemia

Beta-Thalassemia is autosomal recessively inherited and is the most frequent single gene disorder in Mediterranean countries. Although the incidence in Turkey as a whole is around 4-5%, in its southern regions the incidence may rise to 14-20%.  The incidence is higher in regions where consanguineous marriages are common. 

Which mutations are responsible for beta-thalassemia?

The beta-thalassemia gene resides on chromosome 11 and is responsible for the synthesis of globin which is used in the hemoglobin protein structure. Mutations in this gene decrease or inhibit the synthesis of globin molecules. This can cause the formation of abnormal hemoglobin molecules, thus causing anemia.

How can carriers be identified?

Blood tests such as peripheral blood smear and hemoglobin electrophoresis help to identify the presence of this disorder.  Next,  molecular tests identify the mutation responsible for the disease. In Istanbul Memorial Hospital, mutation analysis is done using gene sequencing analysis techniques.

•    Sickle Cell Anemia

Hemoglobin is the iron-containing oxygen-transport protein in the red blood cells. Sickle Cell anemia is a disease in which as a result of incorrectly functioning globin, the hemoglobin protein has an abnormal shape resembling a reaping hook or “sickle”. As a result, blood flow and the transport of oxygen to parts of the body are impeded.  There are different kinds of hemoglobin protein: HbF in fetuses and in newborns, HbA in adult humans and  HbS in sickle cell disease patients.

Which genetic factors are responsible for sickle cell disease?

Sickle cell diasease is an autosomal recessive disorder in which there is a mutation in the 6th codon which changes the amino acid “glutamic acid” into another aminoacid called “valine”. This causes functional and morphological damage.
In Istanbul Memorial Hospital the diagnosis of the disease is performed by analyzing the region “COD 6” using molecular techniques.

•    Cystic Fibrosis

Cystic fibrosis (also known as CF or mucoviscidosis) is a recessive autosomal inherited disease affecting mostly the lungs, pancreas, liver, and intestines. It is characterized by the abnormal transport of chloride and sodium across epithelium, leading to thick, viscous secretions. The incidence of cystic fibrosis is quite high among Caucasians.

What are the genetic factors that cause cystic fibrosis?

CF is caused by a mutation on chromosome 7, in the gene for the protein cystic fibrosis transmembrane conductance regulator (CFTR). This gene regulates the components of sweat, digestive juices, and mucus. 1,400 different mutations have been reported in the CFTR gene. Their effects are diverse, ranging from infertility problems such as Congenital Unilateral Absence of Vas Deferens (CBAVD) in males, to breathing and gastrointestinal problems.

Molecular diagnosis of the disease is made by analysis of the most common 33 mutations in addition to polyT analysis via PCR-OLA (polymerase chain reaction- oligonucleotide ligation assay). When no mutations are present among the first 33, the broader panel, which screens 500 mutations, may be applied. If there is congenital absence of the vas deferens (CBAVD) in the male partner due to cystic fibrosis mutations, the female partner should also undergo mutation analysis.  If the female partner is also a carrier of the disease, preimplantation genetic diagnosis is recommended to the couple to prevent transmitting the disease to their offspring.

PGD of rare monogenic diseases:

As well common diseases like Beta-talasemia, sickle cell and cystic fibrosis, PGD can also be performed for rare monogenic diseases. In Memorial hospital we have performed PGD for rare monogenic diseases like Ehler-Danlos Syndrome (EDS) Type VIIC (ADAMTS2), Co-enzyme Q deficiency (COQ2), Lafora Disease (EPM2A), Leber's congenital amaurosis (RPE65), Bartter Syndrome (BSND), Fraser Syndrome (FRAS1), Arthrogryposis-Renal dysfunction-Cholestasis (ARC) syndrome (VIPAR), Hypomyelination and congenital cataract (FAM126A).

Ehler Danlos Syndrome:

Ehler danlos syndrome (TYPE VIIC) which is also called dermatosparaxis, is an extremely rare autosomal recessive disease where only 10 cases are reported in the literature up to now. EDS Type VIIC is characterized by extremely fragile and sagging skin and it is caused by mutations in ADAMTS2 gene which encodes for the protein called ‘procollagen protease’. In 2012 we have performed PGD successfully for a couple who were carriers for EDS Type VIIC. The couple had a healthy baby born in 2013 and you can see her photo below with her brother suffering from the disease.

Healthy baby born after PGD for Ehler Danlos Syndrome with his brother

Coenzyme Q deficiency:

Coenzyme Q10 deficiency is a rare, autosomal recessive disorder, often presenting with neurological and/or muscle involvement. There are 6 different genes that can be responsible for Coenzyme Q deficiency. In our case mutations in COQ2 gene were responsible where only five patients have been described in literature with primary coenzyme Q10 deficiency due to mutations in COQ2. 

Our couple had 8 previous IVF cycles with the indication of obstructive azoospermia before coming to our center. In one of the cycles she got pregnant but she lost the baby 5 months after the delivery with diagnosis of CoQ deficiency. After setup work, we have achieved a successful PGD cycle with a healthy baby born on May 2013. 

•    Y- chromosome microdeletions

The Y chromosome is responsible for the male phenotype and spermatogenesis (sperm formation process). Microdeletions in the Y-chromosome may cause reduced sperm production, which ranges from oligozoospermia (reduced count) to azoospermia (complete lack of sperm). Many men with Y-chromosome microdeletion exhibit no symptoms other than infertility.

What is the significance of microdeletions in male infertility?

Microdeletions in the Y chromosome have been found at a much higher rate in infertile men than in fertile controls. Since deletions may cause azoospermia, the region is called Azoospermia Factor (AZF). There are three subregions within the AZF region (AZFa, AZFb and AZFc). The complete deletion of AZFa and AZFb causes Sertoli Cell-Only (SCO) syndrome and spermatogenic arrest. However, the effect of partial deletions in AZFc, ranges from oligospermia (decreased sperm count) to spermatogenic arrest. The most common deletion is AZFc deletion (%60). Following that come AZFb deletions, and the combined deletions in more than two or three regions; AZFb+c and AZFa+b+c (%35). Deletions in the AZFa region-only are very rare (%5).

Y-chromosome microdeletion screening has important advantages. For example,  the presence of complete AZFa and AZFb deletions has a negative prognostic value in surgical testicular sperm extraction. This information means that unnecessary testicular biopsies can be avoided.

Y-chromosome microdeletions are screened by looking at 28 different short tandem repeat STR markers, which increases the reliability of the test. Each region is screened by fluorescent multiplex PCR (polymerase chain reaction). The amplified DNA is analyzed by fragment analysis in a DNA sequencer machine.

•    Thrombophilia

Thrombophilia is an abnormality of blood coagulation that increases the risk of thrombosis (blood clots in blood vessels).

What are the advantages of testing for thrombophilia?

Mutations in some of the genes related to coagulation are thought to be associated with repeated pregnancy losses. In particular, polymorphisms in Factor II and Factor V genes are highly associated with repeated pregnancy losses. For further information, please visit clinical part.

Which mutations are analyzed in the thrombophilia panel?

• Factor V (Leiden)
• Factor II (Prothrombin 20210A)
• MTHFR C677T
• MTHFR A1298C
• PAI 4G/5G polymorphism  

•    Factor V:  Factor V is one of the most important proteins that regulate the blood coagulation system. Factor V is necessary to the function of an enzyme called thrombin. Thrombin converts fibrinogen to fibrin, to clot the blood. Activated protein C (APC) is a natural anticoagulant that limits the extent of the clot by cleaving and degrading factor V.
Mutation of this gene is located in exon 10. It changes a protein's amino acid from arginine to glutamine. This mutation results in a factor V variant which cannot be easily degraded by APC. This in turn results in dense clot formations in the blood. Factor V is the most common polymorphism responsible for thrombosis. Risk is further increased in the presence of accompanying Factor II mutation and some environmental conditions such as usage of birth control pills and smoking.

•    Factor II (Prothrombin 20210A): This is the second most common polymorphism responsible for thrombosis. It increases the concentration of protein prothrombin in the blood.

•    MTHFR polymorphisms: MTHFR enzyme plays a role in the synthesis of protein and methylation reactions. Genetic variation in this gene influences susceptibility to vascular diseases, neural tube defects, early onset heart disease and venous thrombosis.

        MTHFR C677T: Nucleotide change at position 677 results in thermo labile enzymes with reduced activity. High homocystein and low folate concentration is observed in homozygous carriers. Enzyme activity is around 30% compared to the wilde-type.
        MTHFR A1298C: MTHFR A1298C mutation affects the concentration of homocysteine and plasma folate but the negative effect on the enzyme is lower compared to MTHFR C677T polymorphism. The enzyme activity is approximately 65%.

•    PAI 4G/5G Ploymorphism: Plasminogen activator inhibitor 1 (PAI-1) 4G/5G, interferes with fibrin cross-linking and may therefore contribute to early pregnancy loss. According to studies, in combination with other risk factors, the 4G allele is an additional risk factor for the occurrence of deep venous thrombosis and myocardial infarction at a younger age.

•    QF-PCR

QF-PCR analysis is the fast detection of aneuploidy in amniotic fluids or CVS tissues. In this test the sample is analyzed for the chromosomes 13,18, 21 and X,Y to exclude the most common chromosomal abnormalities that are seen in miscarriages and in newborns. These are Patau Syndrome (trisomy 13), Edwards Syndrome (trisomy 18), Down syndrome (trisomy 21) and Turner Syndrome (45,X0). Since this test can give information on only a limited number of chromosomes, cytogenetic tests including long term culture should be completed to have the complete karyotype of the sample.

When is QF-PCR advisable?

•    If one or more of the following have been diagnosed during pregnancy:

• Abnormal ultrasound findings
• Abnormal trimester test
• Abnormal triple test

•    If either partner

• is the carrier of a chromosomal rearrangement
• has gonadal mosaicism

•    If the female partner

• is of advanced maternal age
• has a history of a chromosomally abnormal fetus