SIBLING PAIR ANALYSIS IN THE IDENTIFICATION PROCESS OF THE MADURESE POPULATION USING STR CODIS LOCI

Background: The most common problem in forensic personal identification, particularly in paternity tests, is the availability of information originating from a mother or a father that can be used as a comparison in the forensic DNA examination process. The use of sibling analysis in paternity tests is still not widely known. Materials and methods: The respondents of this study were 25 families of the Madurese population with 6, 9, and 10 sibling pairs categorized as female-female, male-male, and male-female siblings. The kinship relationship was analyzed by using 13 STR CODIS loci technique (TPOX, D3S1358, FGA, D5S818, CSFIPO, D7S820, D8S1179, THOI, vWA, D13S317, D16S539, D18S51, D21S11), and Amelogenin (Amel) x: 106bp,y: 112bp). Results: The results of DNA contents and purity examination of the extracted DNA sample showed that the average value of DNA contents was 675±5.35 ng/μL with a purity range of 1.05-1.86. The findings proved that the male-male siblings of the Madurese population had the highest allele sharing percentage at the loci of D13S317, D16S539, and D21S11. The highest allele sharing percentage for female-female siblings was at the loci of TPOX and D21S11. Meanwhile, the male-female siblings had the highest allele sharing percentage at the loci of TPOX, D5S818, vWA, D7S820, THO1, vWA, and D13S317. Conclusion: The main STR loci recommended in the male-female siblings of Madurese population identification are TPOX, D13S317, and D21S11.


Introduction
A paternity test is a means to determine whether a man is a biological father. It is a valid legal procedure for determining paternity. Paternity determination is very complicated because many aspects need to be examined. Until now, the solution for paternity problems is assessed by examining the similar or dissimilar features of a child and the alleged father. The similar features here refer to characteristics of irises' color of the eyes, hair, unique manners or way of speaking, and stature (Yudianto, 2015: Yudianto et al., 2021).
The most common challenge faced during the paternity test process is the unavailability of genetic information from the parents. The unavailability of genetic information from both parents as a comparison in an identification process is one of the problems in forensic DNA analysis, particularly in paternity tests (Sykuriani, 2012: Yudianto, 2013. This condition requires a comparison that has a close family line as a solution in the process of forensic DNA analysis such as biological siblings (Gaytmenn et al., 2002).
The forensic identification process of siblings is also conducted through the examination of alleles at the STR loci of nuclear DNA. Based on this reason, the use of siblings as the comparison through STR remains unknown in Indonesia as well as other countries regardless of the demand for ethnic and population diversity, natural disasters, and other events that require forensic readiness. The application usually targets the use of human profiling and typing, forensic application, sibship relationship, craniology tracing or individual identification, and incestuous identification (Consetino et al., : Karbeyas K et al., 2016Yudianto et al., 2021).
The Madurese are one of the ethnic groups with the highest population in Indonesia. Based on National Census 2010, the Madurese population was 7,179,356. They are originating from the island of Madura and its nearby islets (Puteran, Gili Iyang, Sapudi, Gili Raja, Gili Genting, Raas, etc). Additionaly, they are a wandering tribe that can be found in many places across Indonesia. The population can also be found in neighboring countries, such as Malaysia and Singapore.
In general, the Madurese population was chosen in this study because of their unique characteristics in terms of their temperament and unique accents. Most of the Madurese population have strong work ethics and are adventurous. These characteristics make Madurese people tend to migrate from their homeland (Prastowo W

Population and Research Sample
The population of this research was all subjects undergoing paternity examination at Human Genetic Study Group, The Institute of Tropical Disease, Universitas Airlangga. The research sample was originating from the peripheral blood of paternity examination respondents, consisting of a father, a mother, and two children of the Madurese population. The father and the mother serve as the control/reference for allele sharing between the siblings. The inclusion criterion for this research was a family consisting of at least three generations of pure Madurese population with two biological children. This research has obtained ethical clearance from the Faculty of Dentistry Universitas Airlangga Number 275/HRECC.FODM/VI/2020. The number of respondents in this study was 25 with 100 samples. This research was conducted at the Human Genetic Study Group, the Institute of Tropical Disease Universitas Airlangga.

Sample Preparation
One hundred peripheral blood samples of the respondents were stored inside tubes and labeled as A [father], B [mother], and C [children]. The labels were used to indicate that the samples were collected from fathers, mothers, and biological children.

DNA Extraction
The DNA extraction process of 100 peripheral blood samples was carried out using the DNAzol method (McClintock 2014). 50 μL of distilled water was added to the isolated DNA pellet.

DNA Amplification
The DNA amplification process was conducted through Polymerase Chain Reaction (PCR) process (PowerPlex® 21Systems, Promega, USA) targeting specific DNA sequences to replicate the isolated DNA. The amplification of 80 samples was conducted using 13 Short Tandem

Electrophorese Gel
The visualization of PCR results was conducted through vertical electrophoresis using 6% polyacrylamide agarose gel [PAGE] [Bio-Rad Mini-PROTEAN®] with silver nitrate staining ( Figure 1).

Sample Alleles
The reading of PCR visualization on the electrophoresis gel resulted in alleles at each locus with K562 as the control (Table I). The alleles were matched with family members (the father, the mother, and the children) and the value of allele frequency (Table II). The analysis was based on the frequency of allele sharing of kinship analysis between biological siblings at every STR CODIS locus by examining the allele sharing ( Figure 2, 3, 4, 5).

Results
The measurement of DNA contents and DNA purity examination of the extracted DNA samples results in the average value of DNA contents of 675 ± 5.35 ng/μl with a purity range of 1.05-1.86.
The calculation of the Sibship Index (SI) of this study is presented in Table III below:

Discussion
This study indicates the basic principles applied in assessing the probability of general alleles in the examined sample and inherited reference of Identical by Descent (IBD) alleles. There is a 25% probability that two siblings inherit two IBD alleles from their parents, a 50% probability that the siblings inherit one IBD allele and a 25% probability that the siblings do not inherit any IBD allele from the parents. There is no shared allele on the diploid loci. The probability that the two siblings do not share any alleles (25%) at one of the two loci might cause significant issues in identifying individuals who die in mass disasters if there is only one biological sibling alive. The probability that one sibling inherits zero IBD allele from their parents is constant at 25% for every locus. The probability that two biological siblings do not share alleles/partial loci depends on polymorphism information contents (PIC) and heterozygosity (Lee et al., 2012).
In genetics, alleles are the alternative forms of genes on specific loci that are related to the expression of certain characteristics (phenotype). Alleles are formed as the result of variations in nitrogen base sequences caused by mutation. In an individual, allele pairs determine the genotype of the individual. The term 'allele' exists as the result of the use of the term allelomorph in Mendel's Principles of Heredity (Butler 2006: Elvita A et al., 2008: Butler et al., 2012.
Individuals with the same alleles at a locus are called to have a homozygote genotype while individuals with different alleles are heterozygote genotypes. Since genotypes are expressed into certain phenotypes, alleles may cause different appearances among individuals within a population (Butler 2006: Butler 2015. In kinship examination using biological siblings, the sensitivity of variations depends on certainty thresholds. The higher the certainty threshold, the lower the sensitivity. High sensitivity is when the number of false-negative is low while high specificity is when the number of false-positive is low. Although biological siblings do not necessarily have the same alleles, allele sharing in biological siblings can be used to examine whether the siblings have kinship relationships as indicated in the study (Tzeng et al., 2000).
Small variations in allelic distribution between siblings are also affected by endogamous marriage where the authors recommend the use of 25 loci for the Lebanese population (Setyowati et al., 2020). In forensic cases such as the identification of fire disaster victims, siblings are compared with the victims by utilizing the Y-chromosome to identify whether the siblings and the victims come from the same father (Maeda et al., 2015). Many previous studies have been conducted to assess the relationship between biological siblings mainly based on heterozygosity and combined sibship indices (CSIs) (Reid et al., 2004). A study utilizing STR to analyze genetic diversity in Indonesia has been conducted to examine the extent of genetic mixing between Javanese and Arab ethnic groups (Sari, 2017).
A paternity test is a DNA test aimed to determine whether a man is the biological father of a child. Family dispute cases, in the form of doubting parents, are increasingly popular in Indonesian society (Syukriani Y, 2012).
Therefore, a paternity test uses parents as the comparison where the results are statistically close to 100% or about 99.99% (Untoro et al., 2009). The unavailability of information originating from a father, a mother, or a child that can be used as a comparison in forensic DNA examination becomes a problem in forensic DNA examination (Jacewicz et al, 2003). Unlike DNA testing with parental DNA as the comparison, the accuracy rate of personal identifications using siblings' information is not close to 100% (O Connor, 2011).
The principle of identification examination through DNA is based on the process of allelic comparison between the victim or the perpetrator and the alleles from the family line, especially parents, following the Mendelian laws (Leclair B et al., 2004: Reid TM et al 2008: Hares DR, 2015. In case the parental or descendant line is unavailable, a comparison with a close family line is needed as a method taken in an identification examination through DNA, namely biological siblings.
The use of siblings as the comparison is one of the identification methods known as kinship analysis. Similar to a paternity test, the kinship analysis in an identification process also has a possibility of mismatched profiles in the One of the aspects that need to be considered in assessing sibling relationships is minimizing the number of false-positive and increasing the number of the examined loci. The findings of this study show that male-male siblings of the Madurese population have a high percentage of allele sharing at D13S317, D16S539, and D21S11 loci while the female-female siblings at TPOX and D21S11 loci, and male-female siblings at TPOX, D5S818, vWA, D7S820, THO1, vWA, and D13S317 loci. These findings are beneficial in enriching the literature and the analysis of sibling relationships in Indonesian ethnic groups related to the paternity examination process or the identification of mass disaster victims.

Conclusion
This study signifies that two alleles sharing is stronger compared to one-allele sharing and zero-allele sharing. The 50% of two alleles sharing indicates that the interpretation of full siblings is accepted so that the relationship claim is true. When assessing full sibling relationships, careful consideration should be given to STR loci with high frequencies of two alleles sharing. The findings of this study show that male-male siblings of the Madurese population have a high percentage of allele sharing at D13S317, D16S539, and D21S11 loci while the female-female siblings at TPOX and D21S11 loci, and male-female siblings at TPOX, D5S818, vWA, D7S820, THO1, vWA, and D13S317 loci. The main STR loci recommended for male-female siblings of the Madurese population are TPOX, D13S317, and D21S11.

Conflict of Interest:
The authors declare that there is no conflict of interest in this study.