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IDna Genetics Limited, Scottow Enterprise Park, Norfolk, United KingdomSchool of Chemistry, College of Science, University of Lincoln, Lincolnshire, United Kingdom
In this study, the impact of consanguineous marriage and inbreeding on heterozygosity was evaluated by comparing levels of heterozygosity in children of closely related parents with those of unrelated parents. Compared to the average expected heterozygosity, the average observed heterozygosity was lower in the children with first-cousin parents. This was not the case in the children with unrelated parents, where an increase in the average observed heterozygosity was noted. Differences in the average heterozygosity between parents-children also observed between related and unrelated parents. First-cousin parents had higher average heterozygosity than the children compared to the unrelated parents where the average heterozygosity was lower than the children. Further investigation with bigger sample size and different populations will provide better understanding, however this study showed that a careful approach should be taken when dealing with parentage testing involving closely related parents.
]. This is evident when using a limited number of Short Tandem Repeat (STR) markers in kinship testing, when trying to differentiate related and unrelated individuals [
]. Thus, the heterozygosity in children with related or unrelated parents in South Asian families was studied using autosomal STR markers to identify any changes in heterozygosity ratios.
2. Materials and methods
2.1 Sample collection
Buccal swab samples from twenty South Asian trios with self-proclaimed ethnicity for three generations were collected with informed consent. Ten of the trios were with first-cousin parents and the other ten with unrelated parents.
2.2 DNA profiling
Samples were extracted using Prep-n-Go™ Buffer (Applied Biosystems™) and amplified using VeriFiler™ Express PCR Amplification Kit (Applied Biosystems™), following an optimized reduced volume method [
]. Capillary electrophoresis was performed on the ABI Prism® 3500xL Genetic Analyzer (Applied Biosystems™) and alleles were determined using the GeneMapper® ID-X v1.6 software (Applied Biosystems™).
2.3 Statistical analysis
Statistical calculations for the relationships between tested trios were carried out using Converge™ software (Applied Biosystems™) [
]. Expected and observed heterozygosities in children’s profiles were generated using FORSTAT, a web-based forensic and population genetics analysis tool [
First-cousin relationships between the parents were verbally verified during sample collection. They were also statistically confirmed using a likelihood ratio (LR) threshold of 10 (probability >90 %). The same LR threshold was also used for confirmation of unrelated parents. Parentage was confirmed with combined paternity/maternity index greater than 10,000 (probability >99.99 %). All these statistical analyses were carried out using the developed DNA profiles with 23 autosomal STR markers.
The heterozygosity results showed that the average observed heterozygosity among children with first-cousin parents was 69.57 % (σ: 19.65 %) but the average expected heterozygosity was higher at 74.83 % (σ: 8.06 %). This was the opposite in children with unrelated parents, where the average observed heterozygosity was 82.17 % (σ: 13.47 %) compared to the average expected heterozygosity of 74.83 % (σ: 7.77 %) (Fig. 1).
Fig. 1Heterozygosity in children with related and unrelated parents.
These results were also supported by the average heterozygosity observed among the parents. The average heterozygosity among unrelated parents was 77.72 % (σ: 12.76 %), increased to 82.17 % (σ: 13.47 %) among the children, while the average heterozygosity among first-cousin parents was 76.63 % (σ: 10.01 %), reduced to 69.57 % (σ: 19.65 %) among the children (Fig. 2). The highest reduction was in a child with only 13 heterozygotes (56.52 %) while the parents had 17 (73.91 %) and 19 (82.61 %) heterozygotes. Conversely, no reduction in heterozygosity was observed in children with unrelated parents.
Fig. 2Comparison of heterozygosity between parents and children.
This study demonstrates that there is a reduction in heterozygosity in children whose parents are closely related. A further study with larger data set and different populations will provide a better understanding of this phenomenon. However, this study indicates that additional familial information should be gathered when test samples are collected, especially for duo parentage testing with closely related parents. Any potential mutation in this situation should be treated carefully and supplementary markers should be added to produce a conclusive result.
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