A comparative analysis of the eff ectiveness of cytogenetic and molecular genetic methods in the detection of Down syndrome

Th e goal of this study was to examine the eff ectiveness of  STR markers application (DS, DS, DS, DS, DS and IFNAR) in molecular genetic diagnostics of Down syndrome (DS) and to compare it with cytogenetic method. Testing was performed on  children, with the previously cytogenetically confi rmed Down syndrome. DNA isolated from the buccal swab was used. Previously mentioned loci located on chromosome  were simultaneously amplifi ed using quantitative fl uorescence PCR (QF PCR). Using this method,  previously cytogenetically diagnosed DS with standard type of trisomy  were confi rmed. Furthermore, six of eight children with mosaic type of DS were detected. Two false negative results for mosaic type of DS were obtained. Finally, fi ve children with the translocation type of Down syndrome were also confi rmed with this molecular test. In conclusion, molecular genetic analysis of STR loci is fast, cheap and simple method that could be used in detection of DS. Regarding possible false results detected for certain number of mosaic types, cytogenetic analysis should be used as a confi rmatory test. ©  Association of Basic Medical Sciences of FB&H. All rights reserved


INTRODUCTION
Down syndrome (DS) is the most common disorder that prevents normal physical and mental development of children.Th e higher incidence of DS is detected within group of children whose mothers were older than  at the time of conception [].It occurs as a result of chromosomal aberrations.Th e  of all detected DS types is typical chromosome trisomy , while  of detected DS could be described as "partial trisomy".It is caused by chromosome  translocation to another chromosome, usually some of acrocentric type.Th e lowest number of persons with DS have mosaic type that occurs in - of the cases [].Basic cytogenetically screening ("karyotyping") is accepted as standard diagnostic procedure for DS detection, as prenatal and postnatal diagnostics.Karyotyping is based on analysis of numerical and structural changes of all observed chromosomes.However, this method could not be used for detection of microdeletion and microduplication.In addition, karyotyping it time consuming method which requires signifi cant time period for sampling, preparation and analysis of metaphase chromosomes.Th erefore, last few years, with more or less success, various molecular-genetic analysis were introduced as alternative method.Detailed examination and extensive application of the microsatellite regions, especially short repetitive DNA sequences (Short Tandem Repeat -STR) promote this markers as possible solution as fast, accurate, cheap and simple genetic tool that could be used in DS detection [].Short tandem repeats (STRs) are easily typed, ubiquitous and polymorphic loci with high mutation rates.Th e tendency of the mass application of STR markers has clearly defi ned them as the molecular polymorphisms which are widely used in population, forensic and medical genetics.STRs are short sequences of DNA, normally of length - base pairs (according to some sources -) that are repeated numerous times at the particular locus.Number of repetitions varies from person to person.Th e real value of the application of these markers lies in the simplicity and rapidity of the process and the possibility of simultaneously testing of a large number of STR markers in the so-called multiplex STR systems, enabling an extremely high degree of individualization in identifying biological evi-dence.Also, these sequences, in addition to its wide application in forensic DNA analysis, have become very attractive as a subject of genetic research from a medical point of view, because it could be performed on the bucal swab sample and it could be associated with certain genetic disorders [].For that reason, we have decided to test  previously described STR loci, located on the chromosome  as possible DS diagnostic method.Additional intention of this study was optimization of the existing protocols and detection of all advantages and disadvantages of STR loci application as possible fast and routine screening method in molecular diagnostics of Down syndrome.

Sample
This study included  individuals from the Bosnia and Herzegovina.All tested individuals were voluntary donors which parents gave consent in compliance with ethical norms set by international conventions.These  individuals were selected according the fact that, based on the phenotype, DS was suspected.Molecular genetic testing was performed after cytogenetic confirmation of Down syndrome.

Procedures
All the samples which underwent cytogenetic analysis were processed according to standardized protocols for processing and analysis of peripheral blood samples.Karyotype description is done according to the ISCN nomenclature (International Standard Committee on Human Cytogenetics Nomenclature) [].In order to perform molecular-genetic analysis the buccal swab samples were taken.Optimized salting out procedure by Miller []

RESULTS
Based on cytogenetic fi ndings,  karyotypes of  children have a regular type of trisomy, fi ve have translocation and eight a mosaic type DS (Figure .).For all of these children analysis of six STR loci on chromosome  was performed in order to determine the effectiveness of molecular genetic method in the detection of Down syndrome.
In our study of  samples, molecular confirmation of trisomy of chromosome  was achieved in all children with a standard type of Down syndrome.We did not obtain electro-

DISCUSSION
Th e expected evidence of the trisomy is the presence of three alleles with the peak ratio :: and two alleles in an approximate peak ratio of : [].
For more reliable diagnosis it is necessary to use at least three diff erent STR markers located on the same chromosome [].One of the reasons for the use of a large number of STR markers for each chromosome lies in the fact that sometimes some of the markers are not informative because of homozygosity of parents or in cases where the parents have the same alleles [, ].However, an excessive number of markers could lead to false positive results in the diagnosis.For these reasons, in this study  markers were used.
According to Rahil et al. detection of two alleles, with the peaks in a : ratio, requires special caution especially for dinucleotide markers.Also, the size of the peaks can vary from one to another allele and also a small amplified PCR fragments can appear [, ].One of the often present products are starters, small peaks of several bases.Height of the starter for standard, tetranucleotide repeated sequences is usually below  of the height of the corresponding peak in the standard amplifi cation conditions.Forming of a starter depends on the length of the markers, sequence of STRs and the speed of DNA polymerase action [].Th is study used tetranucleotide markers, so there was no problems in obtaining readings from the two alleles, which peaks are in a : ratio (except in three samples at two markers that had almost equal peaks).It has been shown for all six markers to be suitable for the identifi cation of trisomy , regardless the type of DS.In the  buccal swab samples, for  of them the trisomy  was molecularly confirmed.In the case of two children with mosaic type molecular analysis did not confirm the presence of the DS.One child had   and another  of cells with trisomy .Other samples, in which the electrophoresis confi rmed Down syndrome, had a higher percentage of mitosis in respect to normal karyotype.In the study conducted by Yoon et al. [] on  samples with trisomy , only one sample had mosaicism of DS.For this sample the researchers obtained negative result by using quantitative fl uorescent polymerase chain reactions (QF PCR) multiplex.
Th is was explained by the small number of the cells with an excess of chromosomes .Th ey concluded that the larger number of samples is needed to determine the degree of QF PCR test reliability, when it comes to the samples with a low percentage of mosaicism [, ].However, cases of mosaicism which are analyzed to date using the PCR-STR is defi nitely insuffi cient to make a fi nal conclusion of the necessary number of the cells and the method sensitivity [].In recent studies, analysis of STR markers proved to be fast, simple and extremely sensitive method (only  ng of genomic DNA is needed) for detecting trisomy and requires less sample for analysis in relation to cytogenetic methods [].STR loci most commonly used in molecular diagnostics detect most frequent trisomies (, , , X and Y).Th e major-ity of structural and numerical changes occur on these chromosomes, but also changes occur on other chromosomes, for which additional STR markers, which would increase the time of analysis, as well as the cost of chemicals, are needed.Th us, the lack of this type of analysis is strict specifi city of applied analysis to only certain (targeted) type of aberrations.
Although cytogenetic analysis cannot detect microdeletions and microduplications on chromosomes and the analysis itself takes longer than the analysis of STR markers, it is still the gold standard in diagnosis.Therefore it should be used together with the molecular analyzes, which could be recognized as preliminary test for trisomy as well as confirmation of cytogenetic analysis [].

CONCLUSION
Based on the results, the application of the STR markers in the identifi cation of DS presents a simple, fast and inexpensive molecular technique.However, with the application of molecular method itself, there is a reduced chance for detection of mosaicism.Th erefore, in the initial stage it may provide auxiliary, but also strong additional confi rmation to standard cytogenetic assays.Combined use of these methods would signifi cantly contribute to improving the quality of work of cytogenetic laboratories in the diagnosis of chromosomopathy.

DECLARATION OF INTEREST
was used for DNA extraction.Six STR loci (DS, DS, DS, DS, DS and IFNAR) located on chromosome  (Table ) were simultaneously amplifi ed using quantitative fl uorescence PCR (QF PCR).Th e total volume of polymerase chain reaction (PCR) was  μl.It contained . μl xQiagen Multiplex PCR Master Mix and . μl Primer Pair Mix- Assay (Assay  consists of a primers system, . μl Forward and . μl Reverse concentration of . mM, and  μl of deionized water) with  μl of DNA sample.Th e PCR reaction consisted of  cycles, after heating for  minutes at  o C, followed by denaturation at  o C for  seconds, hybridization for : minutes at  o C and elongation for : minutes at  o C. Th e last cycle was extended for  minutes at  o C. PCR reaction was performed on AB Gene Amp PCR System  Th ermal Cycler (Applied Biosystems).Following amplifi cation, detection of results is carried with the DNA genetic analyzer ABI .
phoregram in case of two samples which would confi rm the cytogenetic diagnosis of Down syndrome.Both samples were from children with the mosaic type of Down syndrome (Figure .).For the remaining fi ve samples, with the mosaic type of Down syn-

FIGURE 1 .
FIGURE 1. Frequency of Down syndrome by type syndrome and sex of the child

FIGURE 2 .FIGURE 2 .
FIGURE 2. Electrophoregram of a child with mosaic type, which did not confi rm the cytogenetic diagnosis of DS.

TABLE 1 .
Characteristics of STR markers separated by capillary electrophoresis */ ** size of the fragment and primers[7]

TABLE 2 .
Polymorphism and ratio of peak areas in trisomy 21 samples