Study of the five AMPFLPs in paternity investigation.

In this study a disputable paternity testing of 252 trios "mother, child and putative father" carried out at the DNA laboratory of the Lithuanians Institut of Forensic Medicine during 1999-2000 is reviewed. Paternity testing was performed at first using preliminary evaluation of the ABO system and leter - VNTR polymorphism of five loci - apoB, Col2A1, Ig-JH, D1S80 and D17S30.

It should be noted that paternity exclusion in 10 cases was obtained using ABO polymorphism. Paternity exclusions according to VNTR loci performed using two loci in 22 cases, according to three loci in 17 cases, our loci in 4 cases and five loci only in 1 case. More informative systems in the paternity exclusion were apoB - 36 cases and D1S80 - 29 cases. Three other loci were less informative: D17S30 - 22, Ig-JH - 22 and Col2A1 - 15 cases.

In all cases alleged paternity was verified, as rool, when coincidens of polymorphism were established not less than in two systems. If an alleged father cannot be excluded, several values, based upon the frequency of occurrence of the obligate paternal allele at each locus, are then calculated.

Introduction

As far back as 1902 when Karl Landsteiner and Max Richter established ABO blood system, it was affirmed that different individuals could be individualized by heritable features. Later some other blood features, according to which people could have different indices were discovered and applied successfully. Child's features are inheritable from his biological parents according to the classical Mendelian law so it could be used in positive paternity testing. On the other hand, paternity denial could be established when an alleged father have no any feature, established at investigated child.

For many years, the resolution of disputed paternity cases by genetic means was performed at laboratory testing red cell antigens and serum proteins. But these systems generally are characterised as lacking high polymorphism and having low powers of persons exclusion.

Only after 1985, when Alec Jeffreys invented DNA polymorphism analysis, genetics and forensic medicine experts had got a very powerful tool for persons individualization and identification. Genetic variability could be detected by analysing highly polymorphic DNA loci, specifically stretches of DNA containing variable number of short repetitive sequences known as VNTRs (variable number tandem repeats). Analysing the allelic pattern of several polymorphic VNTR loci of the individuals one could achieve probabilities for investigating biological relationships.

Material and methods

In this study paternity testing of 252 trios consisting from mother, child and alleged father at DNA laboratory of the State Forensic Medicine Service of Lithuania during 1999-2000 is reviewed. Paternity testing was performed by two steps: at first we used preliminary evaluation of blood ABO system. It should be noted that paternity exclusion in 10 cases was obtained using ABO polymorphism only. On the second step - polymorphism of five VNTR loci - apolipoprotein B, D1S80, D17S30, Colagen2A1 and Imunoglobulin JH. Characteristics of the five VNTR loci used in our study is presented in Table 1.

Table 1. Characteristics of the vntr loci used in this study

Locus	Chromosome localisation	Number of alleles	Repeat unit bp	Alleles size bp
ApoB	2	25	13-15	510-1100
D1S80	1	23	16	398-775
D17S30	17	14	70	170-1080
Col2A1	12	12	31-34	500-920
Ig-JH14	14	13	50	420-1020

For examination the blood was taken from mother, child and alleged father. DNA samples for the amplification were prepared by Chelex extraction. The utility of VNTR probes were evaluated by examining the homozygosity/ heterozygosity levels and the power of exclusion. These parameters were calculated for all five of the VNTR probes routinely used in our laboratory with data obtained from over 500 paternity cases. The homozygosity and heterozygosity levels were determined using data from the mother and alleged father. The children were excluded from this measurement because a noticeable portion of the paternity cases we conduct involve incestuos relations.

At the First International Symposium for Human Identification in 1989, Brenner and Morris presented several mathematical formulae including one which demonstrated the relationship between the percent heterozygosity and the mean exclusion probability. The average Power of Exclusion of a probe could be calculated using the equation:

Average PE = h² (1 - hH²),

where H = the percent homozygosity at a specific VNTR locus and

h = the percent heterozygosity.

Brenner and Morris pointed out that it is essential that the criteria for determining the percent heterozygosity versus homozygosity must be identical to those used for determining exclusion versus nonexclusion.

The criteria chosen were: one must be able to distinguish two discrete bands in the phenotype of an individual, and that both alleles of the alleged father must be visually distinguishable from the obligate paternal allele. Based upon these values the corresponding Average Power of Exclusion was calculated. The calculated values for the Mean Exclusionary Probability were consistent with, although slighly lower than those determined from our limited amount of case material. The Power of Exclusion (PE) for each of the five probes was determined from our initial case material and is presented in Table 2.

Table 2. Polymorphism and average power of exclusion

Locus	Homo	Hetero	PE %
ApoB	0,176	0,824	66,13
D1S80	0,165	0,835	68,10
D17S30	0,270	0,730	50,47
Col2A1	0,217	0,783	59,03
Ig-JH	0,325	0,675	42,36

Results

Study of 5 VNTR loci of 252 trios in 44 cases proved exclusion of alleged father. Paternity exclusions according to VNTR loci performed using two loci in 22 cases, according to three loci in 17 cases, four loci in 4 cases and five loci only in 1 case. More informative systems in the paternity exclusion were apoB?- 36 cases and D1S80 - 29 cases. Three other loci were less informative: D17S30 - 22, Ig-JH - 22 and Col2A1 - 15 cases.

Conclusions

1. Alleged fathers were excluded when they had not obligate paternal allele at a minimum of 2 independent loci.

2. Each VNTR locus was treated as an independent system.

3. The analysis of VNTRs by PCR could provide a very rapid and extremely informative approach to investigating parentage.The judicious selection of VNTRs allows a resolution of discrete alleles and, subsequently, a straight forward assignment of population frequencies.

4. If an alleged father cannot be excluded, several values, based upon the frequency of occurrence of the obligate paternal allele at each locus, are then calculated. These values are the Paternity Index (PI) and the Probability of Paternity (W).