Characterization and haplotype study of 6 novel STR markers related to the KCNQ1 gene in heterogeneous cardiovascular disorders in the Iranian population

The KCNQ1 gene has a significant role in long QT syndrome, Jervell and Lange-Nielsen syndrome, familial atrial fibrillation, and short QT syndrome. Analyzing such heterogeneous disorders, six novel short tandem repeat (STR) markers around the KCNQ1 gene were found and evaluated in a healthy population, and other statistical traits of the markers were detected.Materials and methods: Using Tandem Repeats Finder (TRF) and Sequence-Based Estimation of Repeat Variability (SERV) software, STR markers were detected with valid tetra- and pentanucleotide repeats. The markers were investigated for a total of 60 unrelated Iranian healthy individuals and analyzed using GenAlEx 6.502 and Cervus 3.0.7.Results: A total of 77 haplotypes was detected, of which 25 haplotypes were unique and the others occurred at least two times. The number of haplotypes per locus ranged from 7 to 18 with the highest frequency of 69.2%, and the observed heterozygosity was calculated as 0.589. The power of discrimination ranged from 0.70 to 0.96. Five of the markers meet Hardy–Weinberg equilibrium.Conclusion: A novel panel of STR markers was described with high allele heterozygosity and segregation in every locus, which may lead to faster and more credible recognition of the disease-inducing KCNQ1 gene and allow it to be used for human identity testing and prenatal diagnosis.


Introduction
The KCNQ1 gene is related to a huge family of genes that encode heart potassium channel protein, the most diverse group of ion channels, that is responsible for the repolarization phase of the cardiac action potential in the voltage dependence of activation (1). The gene product is assumed to be capable of forming a heteromultimer with the other potassium channel protein, mink (encoded by KCNE1). Loss of function mutations in the KCNQ1 gene, which induce type 1 long QT syndrome (LQTS1, MIM#192500) (2), the most common type of LQTS, cause delayed rectifier potassium current (IKs) in the cardiomyocytes (3) and inner ear (4).
In addition to LQTS, mutations in this gene are also associated with other forms of inherited arrhythmias such as Jervell and Lange-Nielsen syndrome (JLNS), familial atrial fibrillation (AF), and short QT syndrome (SQTS) (5). The gene is located in a region of chromosome 11 that contains a large number of contiguous genes, consisting of 16 coding exons spanning approximately 400 kb (6). Hundreds of different mutations with variable effects on Kv7.1 function have been reported (7). This reveals the considerable clinical importance in LQTS, so linkage studies are the best approach to detection of the mutation for such genetic heterogeneous diseases.
This study set out to explore the utility of STRs in mutated gene diagnosis for LQTs by presenting the six novel tetra-or pentanucleotide STR markers surrounding the KCNQ1 gene. Heterozygosity and frequency evaluation of these markers has been carried out in the Iranian population.

DNA extraction
Sixty unrelated healthy individuals were selected from the Iranian population. After obtaining informed consent, blood samples were collected in tubes containing ethylenediaminetetraacetic acid (EDTA). The research was approved by the Pasteur Institute of Iran and the local ethics committee. DNA was extracted from blood samples using the KBC Blood-DNA Extraction Kit.

Marker primer design and amplification
Primers were designed using Gene Runner software. We used the multiplex PCR method, which amplifies multiple DNA fragments in one polymerase chain reaction (PCR). Each forward primer was labeled with fluorescent-dye labels (either FAM, VIC, or NED dye). STR markers, primer sequences, and their fluorescent-dye labels are shown in Table 1.
STR loci were reproduced in a single reaction in a volume of 18 µL according to the multiplex PCR method. PCR mixtures and the thermal cycler program are described in Table 2. Fragment amplification was carried out on an ABI 3130XL Genetic Analyzer (Applied Biosystems-Kawsar Biotech Co., Iran). PCR products were examined in Gene-Mapper ID ver. 4.0 (Applied Biosystems).

Statistical significance
Using GenAlEx 6.502 (15), heterozygosity, allelic frequencies, the probability of identity (PI), and the power of exclusion (PE) were estimated. Calculating of the power of discrimination (PD) per locus was performed as a proximate rating using the formula PD = 1 -PI.

Results
STR markers were chosen for the KCNQ1 gene using the TRF and SERV programs. All of the reported STR markers in the map viewer were dinucleotide repeats. To reduce the chance of stutter bands being formed during the PCR, the markers were selected across tetra-or pentanucleotide tandem repeats that connect to the KCNQ1 region of chromosome 11 (19). To reduce the probability of a meiosis recombination incident, the markers were selected using a distance of less than 1.4 Mb upstream (D11SU0.6, **STRs downstream from the genes were given a name beginning with D, those upstream were given a name beginning with U, and those that were intragenic were given a name beginning with I. D11SU2.2, D11SU10.9) and downstream (D11SD13.6, D11SD8.3) of the gene; thus, an intragenic marker (D11SI) was selected. The Iranian population's allele frequencies and genetic analysis data (statistical characteristics) for STR markers are shown in Table 3 and Table 4, respectively.

Discussion
In total, 77 alleles were detected and the average number of alleles per locus was 12.8. For the D11SU10.9, D11SU2.2, D11SU0.6, D11SI, D11SD8.3, and D11SD13.6 STR markers, fragment assay showed 11, 18, 7, 12, 16 and 13 alleles, respectively. The D11SU2.2 locus with a   (20). Our study's conclusion revealed the novel STRs that show a high rate of informativity and high degree of variability that make STR markers very efficient for haplotype analysis and human identity testing. This method is easy to use for detecting the multiplex reproduction pattern, and also a cost-effective method for detection of disease-causative genes and the prenatal diagnosis of heterogeneous cardiovascular diseases compared to direct sequencing, which is usually time consuming and too expensive.