Twins from different fathers: A heteropaternal superfecundation case report in Colombia

Abstract Heteropaternal superfecundation is an extremely rare phenomenon that occurs when a second ova released during the same menstrual cycle is additionally fertilized by the sperm cells of a different man in separate sexual intercourse. In August, 2018, the Grupo de Genética de Poblaciones e Identificación at Universidad Nacional de Colombia received a request to establish the paternity of a pair of male twins with genetic markers. The following analyses were performed: amelogenin gene, autosomal short tandem repeat (STR), and Y-STR analyses by means of human identification commercial kits, paternity index, and the probability of paternity calculation and interpretation. A paternity index of 2.5134E+7 and a probability of paternity of 99.9999% for twin 2 were obtained while 14 out of 17 Y-chromosome markers and 14 out of 21 autosomal short tandem repeats were excluded for twin 1. The results indicated that the twins have different biological fathers. Although heteropaternal superfecundation is rarely observed among humans given its low frequency, in paternity disputes for dizygotic twins it is mandatory to demand the presence of the two twins in the testing to avoid wrong conclusions.


Twins from different fathers: A heteropaternal superfecundation case report in Colombia
Fernanda Mogollón, Andrea Casas-Vargas, Fredy Rodríguez, William Usaquén Grupo de Genética de Poblaciones e Identificación, Instituto de Genética, Universidad Nacional de Colombia, Bogotá, D.C., Colombia Heteropaternal superfecundation is an extremely rare phenomenon that occurs when a second ova released during the same menstrual cycle is additionally fertilized by the sperm cells of a different man in separate sexual intercourse. In August, 2018, the Grupo de Genética de Poblaciones e Identificación at Universidad Nacional de Colombia received a request to establish the paternity of a pair of male twins with genetic markers. The following analyses were performed: amelogenin gene, autosomal short tandem repeat (STR), and Y-STR analyses by means of human identification commercial kits, paternity index, and the probability of paternity calculation and interpretation. A paternity index of 2.5134E+7 and a probability of paternity of 99.9999% for twin 2 were obtained while 14 out of 17 Y-chromosome markers and 14 out of 21 autosomal short tandem repeats were excluded for twin 1. The results indicated that the twins have different biological fathers. Although heteropaternal superfecundation is rarely observed among humans given its low frequency, in paternity disputes for dizygotic twins it is mandatory to demand the presence of the two twins in the testing to avoid wrong conclusions.
Heteropaternal superfecundation is an extremely rare phenomenon that occurs when a second ova released during the same menstrual cycle is additionally fertilized by the sperm cells of a different man in separate sexual intercourse taking place within a short period of time from the first one (1-4). Wenk, et al., found three cases in 39,000 records of a paternity-test database and showed a frequency of 2.4% heteropaternal superfecundation among dizygotic twins whose parents were involved in paternity disputes (5,6). Nevertheless, the frequency of these cases may vary depending on the population's coital rates and double ovulation rates (3).
This phenomenon was initially presented by Archer in 1810 (7); he showed the phenotypic differences between a white female twin and the other mulatto female twin and his study was later followed by Terasaki, et al., in 1978

Financiación:
This research did not receive any specific grants from funding agencies in the public, commercial, or not-forprofit sectors.
In Colombia, Bravo-Aguilar presented a case of heteropaternal superfecundation as an example in the book "The genetic truth of the paternity" where two cases detected in the laboratory are mentioned, but no further information is given (24).
Here we present a case of dizygotic twins from different fathers detected in Colombia as evidenced by autosomal short tandem repeat (STR) DNA markers.

Case presentation
In August, 2018, the Grupo de Genética de Poblaciones e Identificación at Universidad Nacional de Colombia received a request to establish the paternity of a pair of male twins with genetic markers from the alleged father, who suspected the children's kinship and requested paternity testing. Both twins were males, born after 35 weeks of gestation; the first male was 1,700 g and the second one, 2,380 g. The mother denied receiving blood transfusions during pregnancy, but she mentioned oral contraceptive intake before conception and antecedents of twins on her mother's side. The study was conducted with written consent from both the twins' mother and the alleged father. They also gave their consent for the publication of this work.

Genetic profiles
Blood samples were collected from the twins, their mother, and the alleged father on FTA cards. We obtained two independent samples for each person taken at different times and each sample was investigated in duplicate. These amplifications were done in an Applied Biosystems 2720 Thermal Cycler™. Capillary electrophoresis running and detection of amplified products were conducted with the ABI PRISM 310™ genetic analyzer. Samples were analyzed with GeneMapper ID™ software, version 3.2 (Applied Biosystem).

Alleged father -twin 1
Discrepancies in 14 autosomal STR markers and in 14 STR loci for Y chromosome (table 1) between the alleged father and twin 1 were observed.

Alleged father -twin 2
Results favored the paternity of the alleged father for only one of the twins whom we labeled as 'twin 2' . All 21 autosomal and 17 Y-chromosomal markers were in concordance with paternity (table 1). The paternity index was 2.5134E+7.
As the genetic profile of the undoubted biological mother was available, the autosomal genotyping results indicated that the two twins had different fathers, which was confirmed with Y STR markers.

Discussion
Few cases of heteropaternal superfecundation have been reported worldwide possibly because not all the cases have been involved in paternity disputes, which decreases the probability of their reporting (3,5,22). However, the frequency of cases will probably rise with time due to the availability of molecular techniques that serve as a tool for the correct resolution of such cases, as well as the recent increase in twin births (2).
In this case, the two brothers participated in the paternity study in response to a preview communication from the laboratory support service explaining that the test required the presence of both twins to study each of them separately, as well as the importance of including the mother. We observed 14 alleged father-twin 1 incompatibilities for Y-STRs loci (table 1) while no discrepancies between the alleged father and twin 2 were found allowing us to link twin 2 to the same paternal linage of the alleged father and to exclude twin 1 relationship to him. On the other hand, the analysis of the autosomal markers enabled us to establish the biological paternity of the alleged father in regard to twin 2 while excluding him as the father of twin 1. We also confirmed the biological maternity thus excluding any possibility of an interchange of the twins.
The difference between the birth weight of twin 1 and twin 2 was remarkable (680 g). This has also been mentioned by other authors (4,10,12,22) and is explained by the different gestational age of the children as a consequence of the time elapsed between one fertilization and the second one (21). Some authors assume that the time between the first and the second fertilization is about three or four days, although this time can be even longer (up to 14 days) (1,5,6).
Finally, this outcome allowed us to detect a case of dizygotic twins with different fathers, which is a rare and infrequent event reported in the literature. This case reiterates the need for the participation of both twins in paternity testing as these events happen although their frequency is not high.

Conclusions
The results of the present study confirmed that these twins have different biological fathers.