Monozygotic twins: Identical or distinguishable for science and law?

Introduction

In forensic genetics, individual identification based on the use of autosomal and gonosomal short tandem repeats (STRs) and their genotyping by capillary electrophoresis (CE) is established as the analytical gold standard. The high number of STRs available ensures high discrimination power and therefore unique genetic profiles, giving the ‘DNA testing’ an undisputed probative value in courtrooms as well as in civil (determination of paternity and kinship) and penal situations (criminal investigations).

However, there are circumstances in which analysis by STRs/CE fails to provide unequivocal identification of an individual leaving biological traces or to identify the presumed biological father. One such situation concerns monozygotic twins, which have always been considered genetically identical because they originated from the same zygote (i.e. a single ovum fertilised by a single sperm). Situations involving monozygotic twins are not common. However, the use of assisted reproductive technologies and the use of fertility drugs, partly motivated by advanced maternal age at the time of childbirth, have contributed to increasing twin births over the years.^1–3

Epidemiological studies have reported that the birth rate of dizygotic twins is constantly growing, while the birth rate of monozygotic twins has a relatively constant trend, but has nevertheless doubled in the world population (approximately 4 per 1000 live births).^4–6

In the case of monozygotic twins, genotyping of their genomic DNA by STR/CE is unable to differentiate them because they share the same genetic heritage and have the same STR profile. STR/CE analysis is based mainly on detection of DNA fragments of different lengths (short tandem repeats) that depend on individual variation(s). Overall, in relation to the number of genetic markers analysed, it provides a unique unequivocal genetic profile for each individual, except monozygotic twins. In the case of twins, the markers and analytical technology show their limits, being unable to detect genetic variations that could discriminate monozygotic twins.

The possibility of differentiating monozygotic twins genetically is important not only for genetics itself but also in legal practice. There have been criminal cases (e.g. thefts and sexual assaults) and determinations of paternity involving monozygotic twins, in which DNA testing has been unable to solve the question scientifically (inadequate ‘DNA evidence’), meaning that the solution then had to rely on circumstantial and other evidence.

It has been reported that exposure to various environmental factors during the lifetime can cause genetic modifications mainly occurring on the DNA chemical component, resulting in the alteration of its structure (epigenetics). In particular, changes due to DNA methylation and histone acetylation processes have been observed. The focus has mainly been on the DNA methylation process that consists in the addition of a methyl group to carbon in position 5′ of the cytosine when this is followed by guanine in DNA sites called cytosine-phosphate-guanine (CpG) dinucleotides. Various methodologies, mainly based on the detection of bisulphite conversion of CpG sites, such as high-resolution melting (NRM), pyrosequencing and next generation sequencing (NGS) technology, have been proposed to reveal DNA methylation.

In some studies conducted on monozygotic twins, it has been possible to ascertain how the quantity and pattern of DNA methylation have a different evolution during the growth of individuals, becoming, in turn, a peculiar characteristic of each individual. ⁷

Therefore, these non-genetic age-dependent differences in DNA methylation have been introduced into forensic genetics to evaluate their potential applications (such as age prediction and body fluid identification), and among these, the ability to differentiate monozygotic twins seems to be one of the most promising applications.^8,9

However, it seems that the new gold standard in forensic genetics is the genome sequencing carried out by NGS technology, which has recently allowed genetic differences due to ex novo somatic mutations in monozygotic twins to be detected, which already seem to occur in the embryonic stage. While minimal, these differences make it possible to distinguish monozygotic twins unequivocally.^10,11

NGS, also known as massively parallel sequencing (MPS), analysis of DNA fragments (e.g. STRs) and genetic markers in general, is conducted in sequence, making it possible to detect variations in single bases of the DNA sequence (e.g. single nucleotide polymorphisms (SNPs)). This new analytical method could therefore solve the problem of monozygotic twins, were it not for a series of factors. The initially high costs of NGS have limited its use in forensic genetic laboratories, while the subsequent the need for in-depth validation of NGS performance and interpretation of the results have precluded its routine use in forensic genetic investigations and its consequent introduction as evidence in the courtroom.

Stimulated by the verdict of a case of determination of biological paternity that came to our attention, in which the presumed father had a monozygotic twin brother, we decided to research the orientation of international jurisprudence in similar cases when the only biological proof comes from classical forensic genetic analysis by autosomal and/or gonosomal STRs/CE, and the legal probative value, if recognised, of NGS technology.

Case

An adult woman living in Verona (Italy) renounced the paternity of her legal father and applied for determination of biological paternity of a presumed father, indicated on circumstantial grounds as the monozygotic twin of the legal father. The Court of Verona accepted the application on the basis of testimonial evidence supporting the circumstance of a sentimental relationship at the time of conception more than 60 years earlier between the applicant’s mother and the presumed father, who, due to different life circumstances, was phenotypically distinguishable from his twin.

Forensic genetic testing was conducted on the applicant and the presumed father by STRs/CE with 22 autosomal STRs (PowerPlex® Fusion System; Promega, Madison, WI) and 12 gonosomal STRs of chromosome X (Investigator Argus X-12 Kit; Qiagen, Valencia, CA).^12,13 The daughter and presumed father shared at least one allelic variation for each autosomal STR marker investigated and the X-STRs haplotype, supporting the hypothesis of biological paternity. The biostatistical analysis confirmed the hypothesis with a probability of paternity (W) of 0.999993. On the basis of these results that corroborated the testimonial evidence, the Court of Verona (First Level of Justice in Italy) declared the status of daughter.

However, the legitimate children of the twin who had been declared the applicant’s biological father insisted on the theoretical possibility that the applicant’s biological father was not their father but his monozygotic twin, his uncle. This cast uncertainty on the validity of the scientific proof.

The Appeal Court of Venice (Second Level of Justice in Italy) accepted the arguments of the appellants. Since from a strictly genetic viewpoint it was impossible to distinguish which of the monozygotic twins conceived the appellee, and since the Court did not consider that the testimonial evidence presented constituted proof, the following judgment was handed down:

… the arguments considered by the Court do not appear determinant, in the sense that they do not achieve the necessary sufficiency of proof … the scientific data, which cannot be ignored, is that the applicant has the genetic heritage of the twins … it is not possible to state that she has that of the twin indicated … it is therefore impossible to use genetic markers to define with sufficient margin of certainty the relationship of paternity.

Discussion

At the current state of the art, the present case is paradigmatic. Faced with scientific proof, DNA analysis, sufficient to determine repudiation of biological paternity of the applicant with respect to her legal father, Italian judges only partly accept its probative value, pointing out its scientific limit as regards ascertaining the biological paternity of the presumed father who has a monozygotic twin, while not excluding the possibility that the applicant belonged to the genealogy of the monozygotic twin brother. Therefore, in this case, two fathers were identified, but paternity was not legally attributed to either of them. In Italy, proof of filiation can be provided, in a court of law, by any means established by art. 241 of the Italian Civil Code. It is the task of the judge, observing art. 30, par. 4 of the Italian Constitution, to evaluate the nature and admissibility of the evidence. The only limitation is indicated by art. 269, par. 4 of the Civil Code, and it concerns not so much the ‘hierarchical/chronological’ order of the evidence, but the fact that a legal determination of paternity cannot be based ‘solely on the declaration of the mother and on the sole existence of relations between the mother and the alleged father at the time of conception’.

In light of the above decision, we decided to study the orientation of international jurisprudence in similar cases. We found that while international jurisprudence evidences a ‘scientific shortcoming’ in solving cases involving fathers with a monozygotic twin, it nevertheless avails itself of the genetic test classically performed by STRs/CE to link the offspring to the genealogical context of the monozygotic presumed fathers, and at the same time considers certain testimonial evidence to be probatory, including the declaration of the woman who has had a sentimental relationship with the presumed father.

In this sense, the sentence of the Court of Appeal of Missouri in 2007 regarding determination of paternity in the Miller case is exemplary.

The judge found that the genetic investigations were unable to determine unequivocally which of the monozygotic twins, Raymon or Richard Miller, was the father, both having a probability of paternity (W) of 0.9999. The testimonial evidence of the woman, who identified Raymon as the one with whom she had a relationship at the time of conception, was the only evidence that was useful for the attribution of paternity. ¹⁴ In a similar recent case in which a mother requested that the father of her daughter contribute financially to the child’s maintenance, a Brazilian judge in Goiàs State ruled that because it was impossible to establish which of the monozygotic twins was the biological father on the basis of DNA testing, both were ordered to contribute financially to the daughter’s maintenance. The judge also ordered both twins to be included as the father on the girl’s birth certificate. ¹⁵

Although from the legal point of view it is de facto impossible on the sole basis of classical STRs/CE markers to attribute paternity in cases where the father has a monozygotic twin, NGS/MPS has the power to detect genetic variations (e.g. SNPs) distinctive of apparently identical genetic profiles, such as those of monozygotic twins. This technology could therefore compensate for the inability of the CE and solve the problem. The potential applications of NGS technology in forensic genetics have been known since 2011 and have been confirmed over the years.

In 2014, Eurofins Genomics and Forensics Campus (Ebersberg, Germany) demonstrated that using ultra-deep NGS, it is possible to search in the whole genome the potential somatic mutations and therefore distinguish monozygotic twins.^10,11

Forensic geneticists have shown cautious optimism in forcing the introduction of this technology as evidence in court, and this has given rise to evident resistance by judges to accept NGS as legal proof due to a lack of ‘scientific validation’ by the international scientific community. It therefore cannot be taken as ‘scientific evidence’.

In criminal cases and cases of sexual assault perpetrated by subjects having a monozygotic twin, NGS technology has been proposed as a solution but rejected by the court. Two past criminal cases are of particular interest. Jewellery was stolen by a monozygotic twin in 2009 from Berlin’s DaDeWe department store. His DNA was found on the gloves used to carry out the theft. In the McNair rape and robbery case involving two female victims in Boston in 2004, the suspect had a monozygotic twin. In both cases, DNA testing by the methods that were current in forensic genetics at the time failed to establish which twin the genetic profile belonged to. The court was unable to determine the guilty party: ‘I can’t be sending the wrong person to prison’. In 2014, both cases were reopened on the basis of the results of genetic testing by NGS, but the evidence was not admitted because it had not yet been accepted as scientific proof.

In the McNair case, the evidence based on NGS technology provided by the Suffolk County District Attorney indicated that Dwayne McNair was two billion times more likely to be the source of the DNA at the scene of the crime than his monozygotic twin brother Dedham. However, the Suffolk Superior Court judge accepted the motion of Dwayne’s defense lawyer regarding the inadmissibility of the NGS evidence presented by the District Prosecutor, albeit admitting that ‘testing and analysis were based on generally accepted, scientific and statistical principles’. In January 2018, based on evidence provided by conventional DNA testing, which narrowed the suspects to the McNair twins, but principally on the evidence of an eyewitness, the Suffolk jury found Dwayne McNair guilty of all the accusations, and he was sentenced to imprisonment for 16 years.

It is therefore clearly necessary to attribute legal probative value to the NGS technique. This can only happen by a process of validation by the international scientific community. Recently, Peter de Knijff pointed out that since 2010, genetic analysis of target sequences or whole genomes are conducted by NGS/MPS in genetic diagnostic disciplines, such as oncogenetics and clinical genetics. This is in contrast with the practice in forensic genetics where NGS is not yet in routine use. ¹⁶

In NGS, the main interpretative problems concerning STR markers arise principally in the phase of alignment of the marker sequence, which may have mutations in one or more nitrogen bases, with the reference sequence. However, the STRs are not what make it possible to distinguish monozygotic twins by NGS, but rather any SNPs due to germinal and/or somatic mutations of a nitrogen base in a DNA filament, the same markers used in genetic diagnostics.

If NGS is considered valid for the diagnosis of tumours and/or genetic diseases, why is it not considered valid for the identification of individuals? Knijff also underlines the need to formulate recommendations and guidelines suggesting what technological and interpretative criteria to adopt, as well as the need to create a database of polymorphisms analysed by NGS. ¹⁶ The lack of technical and interpretative data and dedicated databases is certainly due to the moderately high cost of NGS technology, owned by few laboratories, and precludes its routine use in forensic genetics. It is therefore essential that this technique be used in forensic research to acquire technical profiles and case series that can fully meet the criteria and standards required by the international scientific community for its validation as legal evidence.

Conclusion

This case of the determination of paternity of a presumed father having a monozygotic twin is currently unique in Italian jurisprudence. However, it is unlikely to remain an exception, considering the increasing incidence of multiple births, including births of monozygotic twins partly as a result of increasing average maternal age leading to medically assisted reproductive technology. Our review of the orientation of international jurisprudence for cases involving monozygotic twins indicates that there is a situation of delay in establishing the probative value of evidence provided by NGS technology. Although we are convinced that NGS technology can solve legal cases involving monozygotic twins, in our opinion, it is evident that NGS does not yet fully meet the strict validation criteria required by the international scientific community for its introduction as evidence of legal value. This conclusion again confirms the hiatus between ‘scientific evidence’ for clinical diagnostic and for forensic purposes.