Dissertation committee:
Ειρήνη Λαμπρινουδάκη, Καθηγήτρια, Ιατρική, ΕΚΠΑ
Κωνσταντίνος Πανουλής, Αν. Καθηγητής, Ιατρική, ΕΚΠΑ
Βιολέττα Καψιμάλη-Βαϊοπούλου, Αν. Καθηγήτρια, Ιατρική, ΕΚΠΑ
Χρυσούλα-Ηλιάνα Νικολάου, Αν. Καθηγήτρια, Ιατρική, ΕΚΠΑ
Μακάριος Ελευθεριάδης, Επ. Καθηγητής, Ιατρική, ΕΚΠΑ
Παναγιώτα Περβανίδου, Επ. Καθηγήτρια, Ιατρική, ΕΚΠΑ
Αικατερίνη Ντόμαλη, Επ. Καθηγήτρια, Ιατρική, ΕΚΠΑ
Summary:
Paternity testing requires the use of polymorphic genetic loci, inherited according to Mendel's laws and determined by accurate and reproducible methods. Αutosomal Short Tandem Repeats (aSTRs) and Human Leukocyte Antigens (HLA) constitute genetic systems widely used in disputed paternity/maternity assessment. Both systems have disadvantages: linkage disequilibrium, predominance of certain alleles in particular ethnic groups for HLA, and high mutation rate affected by parental age and sex for STRs.
The aim of the present study was to evaluate the use of polymorphic microsatellite marker DNA analysis and to establish this analysis as the method of choice for parentage investigations.
Material and methods: Among 792 civil disputed parentage tests addressed to Immunology and Histocompatibily lab of Evangelismos General Hospital previously examined for HLA class I (-A*, -B*, -Cw*), and class II (-DRB1*, -DQB1*, -DPB1*) alleles using PCR-SSOP and/or PCR-SSP methodologies (low/high resolution), a cohort of 147 cases (405 individuals, 110trios/37duos) was selected, (including one DNA grandparent testing). DNA-typing was generated from co-amplification of 15-16 autosomal STR DNA markers (aSTRs) and the sex determining Amelogenin marker, using fragment analysis methodology by DNA sequencing on 3130 Genetic Analyzer. Genomic DNA was isolated from either whole blood or buccal swabs. Power of Exclusion (PE), Random Man Not Excluded (RMNE), Combined Parentage Index (CPI), and Probability of Parentage (W) values were calculated by Buckleton (for aSTRs) and Essen-Möller (for HLA) values, based on allele frequency of Caucasoid and Greek population database, respectively.
Results: DNA polymorphism analysis was informative for both exclusion and confirmation of the disputed paternity. 38 out of 147 (28trios/10duos) cases were sufficient for exclusion of fatherhood (at least 3 genetic STRs loci) and 108 (82trios/26duos) were not excluded. The results of both approaches (HLA and STRs) were in agreement and the combined discriminating power of the two methods resulted in assigning paternity for all cases, except in one, where the two alleged fathers were relatives. In this case, there was exclusion by aSTRs, but not by HLA alleles. The low W rates (0.9870-0.9999) using HLA typing were increased up to 0.9999999 using STRs. The CPI by STR genotyping was ranged from 333 (duo) to 6,76x1013 (trio) with statistically significant difference between trios and duos (p˂0.0001), RMNE was ranged from 1.14x10-5 to 9.64x10-13 p˂0.0001, while PE was estimated up to 0.999999 using aSTRs. Additionally, seven aSTRs mutations (2 on D12S391 and 1 for each SE33, D10S1248, vWA, D2S1338, D21S11 locus) and 1 null allele (SE33) were observed in 8 (8/188, 4.26%) different parent/child allele transfers (2 of them was motherless case), without ruling out fatherhood. All mutations were single or double-step (repeat loss or gain). The ratio of paternal versus maternal mutations/null allele was 7:1 (p=0.036). In the above cases, HLA approach was used for paternity confirmation (WHLA value ranged from 0.9972 to 0.9999). No parental exclusion was confirmed by both approaches. However, despite the presence of mutations in those cases, the low W, as determined by aSTRs typing (0.9970-0.9999), was increased up to 0.99999999 using both systems.
Conclusions: The above results underline the usefulness and power of the DNA-based methods and approaches in the field of disputed paternity. The use of DNA-typing with 15-16 aSTRs loci provides an accurate and high-sensitivity method which is easier to perform and more rapid than an accepted standard technology, such as HLA genotyping. It offers a highly discriminating test suitable for trio paternity testing, increasing the W rate in comparison to HLA genotyping, and is useful in cases whereas the maternal DNA is not available or tested individuals originate from the same lineage. Nevertheless, when a mutation/null event occurs in motherless cases, combination of HLA and aSTRs polymorphisms offers high level of information, and also diminishes the possibility of false exclusion due to aSTRs mutations/nulls, or minimizes the risk of wrong inclusion due to the absence of mother’s genotype.
