Genetics

Pre-implantation Genetic Diagnosis

Homapage / Pre-implantation Genetic Diagnosis

The PGT method was applied for the first time in the world in 1989 for a hereditary disease using single cell PCR (polymerase chain reaction) technique in England. In Turkey, PGT method was first used in couples with advanced maternal age and recurrent IVF failure in 1997 by Dr.Kahraman and her colleagues. With the genetic diagnosis and HLA typing studies in the embryo developed in the USA in 2000, PGT has not only been a diagnostic method, but has also made it possible for families to have a healthy baby, while it can also be treated with HLA compatible stem cells taken from the siblings of sick children. Dr. Kahraman and his team became the first practitioners in our country to diagnose single gene diseases and HLA tissue determination in embryos in 2003. 

Preimplantation Genetic Test (PGT)

Genetic procedures before embryo transfer are called Preimplantation genetic testing in order to obtain a healthy embryo and a healthy pregnancy in couples undergoing IVF treatment. The PGT procedure can be summarized under 3 subtitles according to the characteristics of the candidate couple.


PGT-M (Preimplantation Genetic Test- Single gene (Monogenic) diseases):
In embryos of couples with the disease transmitted with a single gene with a familial genetic transition, PGT-M can select healthy embryos that do not carry the mutation that causes genetic disease. In this test process, a detailed map of the gene region carrying the mutation causing the disease is determined first, then the mutation region and its environment are identified. In the first step of the test, DNA replication of biopsy samples taken from embryos obtained by IVF treatment is performed. Then single embryos that are healthy in terms of single gene without mutation are selected. Then, PGT-A process can be performed to determine the chromosome numbers of healthy embryos. Thus, embryos that are healthy in terms of a single gene and carrying the correct number of chromosomes in terms of the number of chromosomes can be selected. (For detailed information, you can examine the PGT-M page.Link…)

PGT-SR (Preimplantation Genetic Test- Structural Rearrangements): Abnormalities in the structures of chromosomes that can be transferred from parents to their children in families are called translocations. There are two different types of translocation where two or more parts of chromosome are replaced by each other (reciprocal) or two chromosome parts are added end to end in a single chromosome appearance (robertsonian). When it is balanced, there is no excess in the number or structure of chromosomes and these individuals can continue their lives in a healthy way. However, when they want to have a baby, they can produce eggs or sperm that are unstable translocation carriers. If one of the sperm or eggs is in this unstable translocation, pregnancy may not occur, if pregnancy occurs, it may end with a miscarriage or the baby will be born alive. A child with an unbalanced translocation may experience loss of learning ability, developmental delay, or different types of health problems. Their severity depends on the size and location of the lost region of the chromosome. Because some parts of the chromosomes are more important than others. Depending on the size of the interchanging chromosome fragments in which the translocation is observed or the region on which the chromosome is located, analysis can be performed by Fluorescent In Situ Hybridization (FISH) analysis or Next Generation Sequencing (NGS). Since the translocation regions are examined in FISH analysis, NGS (24 types, to analyze 46 chromosomes) analysis can also be performed in the same embryos. (For detailed information, you can examine the PGT-SR page. Link ...)

PGT-A (Preimplantation Genetic Test- Aneuploidy): The fact that the embryo has the correct number and sequence of chromosomes is essential for healthy baby development. If the embryo is missing or carries a large number of chromosomes, it is an unhealthy embryo, which is called aneuploidy. An aneuploid embryo transferred without knowing the genetic status will not create any pregnancy with good probability, the worst case is the termination of pregnancy with early or advanced abortion. However, the worst possible scenario is that babies with various health problems are born due to the presence of excess chromosomes. For example, individuals carrying 3 copies from chromosome 21 have down syndrome. Today, the most advanced technology, the next generation sequencing (Next Generation Sequencing - NGS), the presence of 24 types of chromosomes and 46 chromosomes in embryos can be examined. (For detailed information, you can examine the PGT-A page. Link….)

Single gene diseases and HLA tissue typing

In couples who have a high risk of transmitting a genetic disease to their children, it is possible to understand whether pregnancy is healthy or not by prenatal diagnosis methods. Unfortunately, many couples have to bear the psychological, physical and financial burden of termination of pregnancy numerous times until they have healthy children. PGT for single gene diseases is based on the pre-selection of embryos in the disease-bearing couples and the transfer of normal or healthy ones. Thus, it is aimed for couples to have healthy children and to eliminate the necessity that may lead to unwanted complications such as termination of pregnancy. For this purpose, couples, who are the only carriers of genetic disease that apply to our center, are included in the IVF treatment program. In this process called IVF (In Vitro Fertilization), egg cells from the mother and sperm cells from the father are fertilized in a laboratory environment. Following fertilization, polar cell, blastomer cell, or trophectoderm tissue are taken from embryos and the genetic region carrying the disease is reproduced by single cell PCR method.

Preimplantation Genetic Diagnosis can be performed for each genetic disease whose mutation is determined in Istanbul Memorial Hospital Genetic Diagnosis Center.

Allogeneic Stem Cell transplantation is the only treatment for many severe blood diseases such as beta thalassemia, sickle cell anemia and Fanconi Anemia. The best treatment result can be obtained as a result of transplantation from a fully HLA compatible sibling donor. If the transplantation is not made from the HLA compatible sibling, high morbidity occurs and the chance of survival decreases.

Blood from the umbilical cord of the HLA compatible sibling has been reported to be an excellent source of stem cells. Therefore, the demand for Preimplantation HLA Typing is gradually increasing for families who are genetically carriers of blood disease and who have brought the sick child to the world, so that they can have the treatment of both healthy and sick siblings. Istanbul Memorial Hospital IVF and Genetic Diagnosis Center is one of the first and few centers in the world to perform Preimplantation HLA Typing.


Single gene diseases

Single gene diseases are diseases caused by mutations or malformations that occur in a single gene. Approximately 4000 single gene diseases have been identified to date. They may show autosomal dominant, autosomal recessive (Figure 3), X chromosome-dependent dominant and recessive, Y chromosome-dependent and mitochondrial inheritance.




Figure 1: It is expected that 25% of embryos created in PGT studies applied for autosomal recessive diseases will be normal, 50% will be carrier and 25% will be patient (A). In autosomal dominant diseases, one of the couples is mostly affected by the disease, and the probability of passing this disease to his child is 50% (B).

Due to the frequent consanguineous marriages in our country, it is very important to diagnose autosomal recessive diseases such as beta-thalassemia, which is known as Mediterranean anemia, at the embryo stage. This method can be applied for every single gene disease in which the relevant chromosome region is defined. These include all single gene diseases that can be analyzed by postnatal and / or prenatal diagnosis.

The number of diseases studied in our center since 2000 (Table 1) has gradually increased and continues to increase.

Which single gene diseases can be diagnosed with PGT?

Preimplantation Genetic Diagnosis can be performed for each genetic disease whose mutation is determined in Istanbul Memorial Hospital Genetic Diagnosis Center. To date, at the Memorial Hospital IVF Center, PGT has been applied in 196 families for 71 different single gene diseases in 277 PGD cycles. Below you can see the full list of single gene diseases with PGT application. In addition, HLA typing was performed in 461 cycles of 242 families.

Table 1: List of diseases that PGT has been applied in our center so far.

Adrenoleukodystrophy

Congenital factor 7 deficiency

Familial Mediterranean fever

Congenital deafness

Familial Hemophagocytic lymphohistiocytosis (HLH)

Krabbe disease

Familial Hypomagnesaemia (with Hypercalciuria and Nephrocalcinosis)

Lafora disease

Maple syrup urine disease

Leber's congenital amorosis

ARC syndrome (Arthrogryposis-Renal dysfunction-Cholestasis)

Li-fraumeni syndrome

Bartter's syndrome

Limb Girdle Muscular Dystrophy (DMD)

Becker muscular dystrophy (BMD)

Breast cancer

Beta thalassemia

Metachromatic leukodystrophy

Osteogenesis Imperfecta

Myotonic dystrophy

Charcot Marie tooth (CMT) syndrome

MTHFR deficiency

Delta-Beta thalassemia

Mucolipidosis

Donohue syndrome (Leprechaunism)

Mucopolysaccharidosis Type 1 (Hurler Syndrome)

Duchenne muscular dystrophy

Mucopolysaccharidosis Type 3A (Sanfilippo syndrome)

Ehler-Danlos Syndrome (EDS) Type VIIC

Mucopolysaccharidosis Type 3B (Sanfilippo syndrome)

Epidermolysis bullosa

Mucopolysaccharidosis Type 6

Facioscapulohumeral muscular dystrophy (FSHD)

Niemann-Pick disease

Fanconi anemia

Nemalin myopathy

Phenylketonuria

Nonketotic Hyperglycinemia

Fragile X syndrome

Neurofibromatosis

Fraser Syndrome

Sickle cell anemia

Galactosemia

Osteopetrozis (Marble bone disease)

Glucose 6 phosphate dehydrogenase (G6PD) deficiency

Polycystic kidney disease

Hereditary multiple exocytosis

Pompe's disease

Hyper IgM Syndrome

Propionic acidemia

Hypohydrotic Ectodermal Dysplasia

Retinoblastoma

Hypomyelination and congenital cataract

Citrullinaemia

Hunter Syndrome

Spastic paraplegia Type 3

Huntington disease

Spastic paraplegia Type 5

Infantile Neuroxonal Dystrophy

Spinal muscular atrophy (SMA)

Joubert Syndrome

Spinocerebellar Ataxia Type 2 (SCA2)

Keratitis-ichthyosis-deafness (KID) syndrome

Tay-Sachs disease

Cystic fibrosis

Tuberous sclerosis

Coenzyme Q deficiency

Tumor Necrosis Factor Receptor Associated Periodic Syndrome (TRAPS)

Congenital adrenal hyperplasia

Usher syndrome Type 3B

 

Zellweger's syndrome


Thalassemia, cystic fibrosis, alpha mannosidosis diseases first time studied by Memorial Hospital IVF and Genetics team in Turkey, and the Bartter syndrome has been studied by Memorial Hospital IVF and Genetics team first time in the world. To date, PGT method has been used in congenital diseases such as cystic fibrosis, thalassemia and sickle cell anemia. However, it is also used in late-onset diseases such as Huntington's and Alzheimer's Disease, and inherited predisposition diseases that increase the risk of developing cancer such as neurofibromatosis, retinoblastoma and Li Fraumeni, which are caused by mutations in tumor suppressor genes.

Mini Dictionary

Gene: It is the smallest unit that contains the regulatory and functional features that allow the genetic properties on the DNA molecule to be transferred in cells for generations.

Allele: Each of the different forms of genes is called allele. It establishes the genotype by creating two separate allele genes that are transferred from the mother and father.

Autosomal recessive: Heredity pattern is recessive. Both of the alleles that produce the gene must be mutants for the disease to occur. Generally, parents are carriers and the probability of this disease in their children is 25% (Figure 3A). Mediterranean anemia, sickle cell anemia, spinal muscular dystrophy and cystic fibrosis can be counted among these diseases.

Autosomal dominant: Heredity pattern is dominant. In these diseases, it is sufficient for only one of the alleles forming the gene to be mutant. In such diseases, one of the parents was affected by the disease. The probability of occurrence in children is 50% (Figure 3B). Huntington, myotonic dystrophy can be counted among these diseases.

Complex multifactorial and multi-genetic diseases: Diseases in which many genes affect the occurrence of the disease and lifestyle and environmental factors are also affected. These include asthma, autoimmune diseases, cancer, hypertension and diabetes.

HLA (Human Leukocyte Antigen): Many genes related to the human immune system are encoded in a region on the p arm of chromosome 6. HLA compliance of donors and recipients is vital in tissue and organ transplantations.

PCR (Polymerase Chain Reaction): It is a sensitive method for logarithmically replicating the DNA molecule using a special polymerase enzyme derived from heat resistant bacteria. Each PCR cycle consists of phases of high-temperature DNA molecule chains separation (denaturation), binding of primers to the matched regions (annealing), adding base sequences of the polymerase enzyme (synthesis) and extension of the synthesized molecule. The synthesized DNA fragment is used as a template in the reaction medium in the next cycle, thereby doubling the number of molecules multiplied in each cycle. Millions of DNA molecules can be obtained after 30 or 40 cycles.

Single cell PCR (polymerase chain reaction) Method

The blastomer cells taken by biopsy are transferred to PCR tubes containing lysis solution. A series of enzymatic reactions, called Single Cell PCR, are applied to the pure DNA molecule released in this way by using specially designed primers to reproduce the genetic area of the disease in millions of copies, and following this process, healthy or sick embryos can be distinguished by mutation analysis.

Since this method is designed to be sensitive enough to make millions of copies from a single chain of genetic material, it should be studied meticulously against any external contamination. Our laboratory follows the quality standards of the European Society for Human Reproduction and Embryology (ESHRE) and the Preimplantation Genetic Diagnosis International Community (PGDIS).

HLA Tissue Determination

In addition to being a diagnostic method for single gene diseases, PGT procedure appears as a treatment method when performed with HLA compatible embryo selection. The first HLA typing study was combined with a PGT study for Fanconi anemia (Verlinsky et al., 2001), and successful treatment was provided to the sick sibling after transplantation of stem cells taken from the umbilical cord after pregnancy. For a number of diseases such as leukemia and aplastic anemia, where there is no mutation in its etiology and HLA compatible stem cell transplantation is required for treatment, only HLA typing is performed.

The fact that there is a very low chance of finding an HLA-compatible embryo (25%) and if the mutation analysis is to be made, this probability decreases up to 18% is the biggest factor limiting the success in treatment. For this reason, well-designed stimulation protocols should be applied for obtaining good quality and sufficient biopsy embryos.

Despite the low probability of finding suitable embryos for embryo transfer, the data obtained demonstrate the feasibility and utility of the PGT-HLA process. Patients who applied for HLA typing achieved very high pregnancy rates when a healthy and HLA compatible embryo was available for transfer. Healthy and HLA-compliant babies born as a result of studies by Dr. Kahraman and her team have created hope for the survival of sick siblings. Treatment was provided in sick siblings with the obtained cord blood or bone marrow stem cells. Among these, common cases such as Mediterranean Anemia have been successful, as well as rare diseases such as Wiscott Aldrich Syndrome, Congenital hypoplastic anemia mucopolysaccharidosis.

Table 2: List of applied HLA typing diseases in our embryos

Name of Diseases

Beta Talasemie

ALL (Acute Lymphoid Leukemia)

AML (Acute Myeloid Leukemia)

Diamond Blackfan Anemia

Alpha Mannosidosis

Gaucher Disease

Wiskott-Aldrich Syndrome

Fanconi Anemia

Hurler Syndrome

Histiocytosis

X-ALD (Adrenoleukodystrophy)

Burkitt Lymphoma

KML (Chronic Myeloid Leukemia)

MDS (Myelodysplastic syndrome)

Aplastic Lymphoma

Glanzman's Disease

Anaplastic Large Cell Lymphoma

Hyper IgM Syndrome

Sickle Cell Anemia

Thrombocytopenia

Hyper IgD

HLA Preliminary Study, Selecting and Transferring Healthy and HLA Tissue-Compatible Embryos to the Mother

Before embarking on genetic studies in embryos, mutations and HLA haplotypes in the family should be detected. When families apply to our center for this purpose, blood is collected from the mother, father and sick child, and DNA from each individual is isolated. The isolated DNA is used to detect the HLA haplotypes of the mother, father and sick child of the mutation that causes the disease in the family. HLA haplotypes of family members are made using STR markers (repeat sites on the DNA molecule) on the HLA gene complex. For this purpose, approximately 30 separate HLA STR markers are scanned and among them, at least 12 STR markers are selected and used in genetic study in embryos (Figure 4).


Figure 4: Molecular genetic analysis scheme in embryos

Auxiliary PGD Applications in Infertility Treatment

Numerical and Structural Chromosome Disorders

FISH technique

FISH technique is mostly used for the determination of aneuploidy (numerical chromosome disorders) and translocations (structural chromosome disorders). This technique consists of glass plate fixation of biopsied cells, obtaining the cell nucleus, denaturation after fluorescently labeled probe application, hybridization, washing for the removal of non-binding probes after hybridization, counterstain application and analysis steps. In our center, panels used in 8,13,14,15,16,17,19,18,20,21,22, X and Y chromosomes and based on the detection of the most common comorbid anomalies in spontaneous abortions are used. With these panels, a large part of chromosomal anomalies that can occur in embryos can be detected.

As an alternative to the FISH method, where a limited number of chromosomes are examined, the comparative genomic hybridization (CGH, comparative genomic hybridization) method makes it possible to examine all chromosomes in embryos. However, the technique has a significant disadvantage that the embryo has to be frozen because of the long process (about 4-5 days). If fresh embryo transfer will be performed, CGH applications to be performed after polar cell biopsy, whose maternal additives can only be determined, can be preferred.

In recent years, with the development of the array-CGH method, which is based on the same principle as CGH, but is an array-based system, the processing time has been reduced to 48 hours and the resolution has been increased compared to the previous method. Routine use has been very limited due to the fact that the technique is very expensive, optimization takes time, interpretation of the results, difficulties in analysis, specially trained personnel recuirement and appropriate laboratory infrastructure. Therefore, detection of numerical and structural anomalies with FISH technique still maintains the advantage of being the most common, economical and practical method.

Aneuploidy Screaning

The PGT method has been used until today to reveal the chromosomal disorders that cause pregnancy loss and infertility and to increase the chance of success in IVF treatment. Aneuploidy screening in embryos is a method used to increase the success in treatment and decrease pregnancy losses in couples with indications such as recurrent miscarriages, severe male factor, especially in cases where the expectant mother is older.

All diploid human cells consist of 22 autosomes and 1 gonosome and contain 23 pairs of chromosomes. The numerical disorder in this chromosome establishment is called aneuploidy, and cells with an excess (for example trisomy) or incomplete (monosomy) number of chromosomes are called aneuploid cells.

 Normal blastomere
Monosomy 21, Trisomy 22
 Complex Aneuploid

Figure 5: Normal (A) and Abnormal (B, C) blastomer FISH images: Analyzed chromosomes; 13 (red), 16 (light blue), 18 (blue), 21 (green), 22 (yellow). FISH images evaluated as "Normal" (A), "Monosomy 21, Trisomy 22" (B), and "complex aneuploid" (C). (Images were taken in Istanbul Memorial Hospital, IVF and Genetics Laboratory).

After the hormonal, uterine, and immunological factors were eliminated, PGDstudies in couples with repeated implantation failures (TIB) in 3 or more trials were found to have a high rate of chromosomal disorders in PGT studies. A significant part of these disorders are complex, that is, numerical anomalies containing 3 or more chromosomes (Figure 5C).

With the PGT procedure applied in our center since 2000, a higher rate of pregnancy was obtained compared to the patients with the same indication and without PGT.
 

 Translocation Carriage

While the incidence of chromosomal translocations in newborns is 0.2%, this rate increases to 2.5% in couples with repeated failed attempts and to 9.2% in couples with recurrent miscarriages.

Robertsonian translocations are formed by the joining of the p arms of two acrocentric chromosomes (13,14,15,21,22) (Figure 6A). Balanced reciprocal translocations occur mostly in the form of displacement of the end regions of the chromosomes, without loss of parts between two non-homologous chromosomes (Figure 6B).


Figure 6: A karyotype view of the Robertsonian translocation carrier: 45XY, der (13; 14) (q10; q10) (A). Karyotype view of the reciprocal translocation carrier: 46, XY, t (7; 13) (q22; q12) (B) (Images were taken in Istanbul Memorial Hospital, IVF and Genetics Laboratory).

Figure 7: An example of a set up run before the PGT procedure. Confirmation of fracture sites by FISH in translocation 46, XY, t (1; 16 (q10; q10) occurring between chromosomes 1 and 16. Probes used: CEP 1 (spectrum orange), CEP 16 (spectrum aqua), Tel 16p (spectrum green), Tel 16q (spectrum orange) (Vysis, Abbott) (A). Robertsonian 13;14 carriers was identified by FISH study with LSI chromosome 13 (green) and Tel 14 chromosome (red) probes in sperms of translocation carrier 45,XY,der(13;14)(q10;q10). A normal or balanced sperm is seen at the top and an unbalanced (nullism 14) sperm at the bottom (B) (Images were taken at the Istanbul Memorial Hospital, IVF and Genetics Laboratory).

The Importance of Pretreatment Study Before PGT in Translocation Carrier

Before the PGT studies to be carried out for translocation carriers, a preliminary preparation (set-up) must be completed. At this stage, in couples with translocation carriage detected by cytogenetic analysis, verification of fracture points by FISH analysis is very important in determining the probes to be used in the PGT study (Figure 7A). For this, it is necessary to collect blood from the spouses and examine the chromosomes and confirm the fracture points on the chromosomes with the FISH method. Similarly, before PGT, if the father is a translocation carrier from couples, it is possible to determine the normal or balanced / unbalanced gamet ratio in sperm cells by FISH analysis to be performed in sperm. Sperm FISH is particularly useful for guiding the balanced / unbalanced embryo ratio and guiding treatment in embryos of reciprocal translocation carriers (Figure 7B).

Despite the low chance of finding a normal or balanced embryo, pregnancy rates obtained in the treatments that result in transfer are quite high. PGT increases the pregnancy rate and decreases pregnancy losses in this patient group.

High abn

ormalities were detected in embryos of patients who underwent PGT due to Robertsonian or Reciprocal translocation carriage at the Istanbul Memorial Hospital IVF and Genetic Center. By eliminating abnormal embryos with PGT method and transferring normal or balanced embryos, quite high pregnancy rates were obtained and these patients were prevented from recurring pregnancy losses.

Microarray Analysis

Array-Comparative Genomic Hybridization (a-CGH) is the newest molecular method that detects changes in the amount of DNA. With this technique, all of the chromosomes in embryos can be examined. This method makes it possible to examine chromosomes structurally as well as numerically. Thus, deletions or duplications on chromosomes and abnormalities in the arms of chromosomes caused by translocation carriage can be detected. As a result of the short processing time and the analysis process being done more automatically with computer programs, data related to chromosomes can be obtained in as short as 12-24 hours. In this way, the results are obtained before the transfer day without the need to freeze the embryos. The a-CGH method is the most reliable method for choosing the right embryo, so the a-CGH method is used in the Istanbul Memorial Hospital IVF and Genetics Laboratory. (Click for more details)

 
SAYFA BAŞINA DÖN