Post mortem genetic determination of age and sex of unidentifiable remains admitted to the Pretoria Medico-Legal Laboratory

Abstract

Unidentified human remains in the South African (SA) medico-legal population is well recognised by the forensic pathologists working in the field and reported on in the media, however a paucity of scientific publications reporting on the magnitude of the problem, proves a challenge. A study conducted at the Pretoria Medico-Legal Laboratory (PMLL) indicated that, despite the current standard operating procedures pertaining to the identification of human remains, unidentified remains continue to be problematic for South Africa (SA). Cases involving unidentified bodies comprise of a wide range of population groups, including homeless individuals, missing persons (both reported and unreported), international (often illegal) migrants, abandoned infants found in rubbish dumps, victims of crime that have been displaced from their hometown, as well as wanted criminals (and even individuals mistaken to be suspects of community crime) that may have been killed and severely mutilated by means of “mob justice”. The problem of human remains being buried or cremated whilst unidentified has far reaching effects, ranging from the psychological trauma and financial burden of next-of-kin to the administrative and financial burdens imposed on the state and indirectly, the imposition on the taxpayers and insurance companies of SA. A solution may be possible with the assistance of genetic analysis and phenotypic depiction of otherwise unidentifiable human remains. The aim of this prospective study was to genetically determine the age and sex of visually unidentifiable human remains admitted to the PMLL from deoxyribonucleic acid (DNA) extracts of bone and teeth samples. Buccal swabs from 70 volunteers (10 individuals per age group, ranging from 1 – 60 years) were analysed, to investigate the practical application of Amelogenin (AMEL) versus Sex-determining Region Y (SRY) genes in establishing sex in a local population, and to investigate the potential inference of biological age based on methylation levels of Fatty Acid Elongase 2 (ELOVL2), Four and a Half LIM domain protein 2 (FHL2), and Proenkephalin (PENK) genes in this population. The DNA was extracted using one of two DNA extraction kits available in the laboratory, according to the two different manufacturer’s protocols, with various adjustments. In the powdered hard tissue samples (pars petrosal- and molar teeth, respectively, collected post mortem from cases admitted to the PMLL), DNA was extracted using according to the kit manufacturer’s protocol, after adjustments were made. Twenty eight of the 70 swabs were selected (representing at least one male- and -female swab sample from each of the six defined age groups) for sex determination. The Amelogenin (AMEL-) and Sex determining Region-Y (SRY) markers were equally successful in determining sex using both real time polymerase chain reaction (PCR) melt curve analysis and agarose gel electrophoresis (AGE) in the buccal swab samples (100%, n = 28 and n = 13, respectively). The AMEL marker was, however, not as efficient to use for sex determination as the SRY marker; in three (11%, n = 28) buccal samples, AGE was required to clarify otherwise ambiguous AMEL-based sex results, based on sex determination by PCR melt curve analysis alone. The SRY marker provided sufficiently clear melt curve data to determine male sex without the need to analyse the SRY marker’s AGE samples’ data. In assessing the possibility of using DNA methylation patterns of the ELOVL2, FHL2 and PENK markers to establish an age-predictive algorithm, universal control DNA dilutions and all 70 swab samples’ DNA isolates were treated with a bisulphite modification kit, according to the manufacturer’s instructions. Primer optimisation attempts for the ELOVL2- and PENK markers were unsuccessful, in contrast to the successful optimisation of the FHL2 marker. Methylation proportion patterns were subsequently analysed using the optimised high-resolution melt (HRM) PCR conditions for the FHL2 marker on the Rotorgene Q (Qiagen, Germany). The obtained data were modelled; the coefficients from which were then utilised for the design of an age-predictive algorithm. The two sex-determining markers and the FHL2 marker were then similarly tested in a decedent population admitted to the Pretoria Medico-Legal Laboratory (PMLL), where 31 case decedents were admitted with a presumed identity, despite being visually unidentifiable due to the conditions that the bodies were subjected to, peri- or post mortem. These conditions included: charring, mutilation, and decomposition, sometimes to the extent of skeletisation. Due to the compromised condition of the remains, a portion of the petrous bone and a molar tooth sample were collected from each case (where possible) for comparative success of molecular determination of sex (using AMEL and SRY markers, respectively) and age (using the FHL2 marker). Uncompromised pars petrosal samples were available more frequently (25; 81%) than uncompromised molar tooth samples (23; 74%) in the 31 cases included. Sex determination by amplification of the AMEL marker was successful in 24 (96%) of the 25 pars petrosal DNA isolates, and in 14 (61%) of the 23 molar tooth DNA samples. Amplification of the SRY marker successfully detected male sex in 16 (94%) of the 17 male pars petrosal specimens, and in 15 (79%) of the 19 male molar teeth samples. Overall, the SRY marker was determined have a marginally higher sensitivity (86%) in determining male sex in the 48 tissue specimens (collected post mortem), than the AMEL marker (85%). Tissue-specific methylation patterns observed with age progression in the DNA isolates originating from the buccal swabs, powdered pars petrosa, and powdered molar teeth specimens, respectively, precluded the envisaged proposal of using the buccal swab age-predictive algorithm developed, for methylation pattern-based age prediction in the post mortem specimens. Consequently, an age-predictive algorithm was developed for all three tissue groups, with varying age-predictive accuracies – based on the tissue donors’ recorded ages, respectively. Specifically, age predicted using DNA isolates from the buccal swabs was most accurate, with a maximum absolute deviation (MAD) value of 6.67 years. In contrast, both post mortem tissue specimen types performed poorly with respect to age predictive information: the molar teeth were accurate to within 27.89 years, while the pars petrosal specimens were least accurate, with a MAD value of 30.42 years. Overall, the results confirm that buccal swabs are an effective method of collecting DNA samples from willing participants – particularly children; as it is a less invasive method (thus improving participation rates) than collection of blood, which also requires a trained phlebotomist for sample collection. Buccal swab isolated DNA yielded between 0.06 µg to 3.99 µg (median = 0.83 µg), which far exceeded the DNA yields obtained in the post mortem specimens. Despite teeth seemingly being considered the ‘gold standard’ in extracted DNA quality and quantity in the forensic field of bodies without soft tissues available, extractions from pars petrosa of the temporal bone yielded a competitively useable sample from most cases: the DNA yields from the 25 petrous bone specimens ranged from 0.12 µg to 0.96 µg (median = 0.26 µg) per 100 mg powdered bone sample. From the 23 molar teeth, extracted DNA yields ranged from < 0.01 µg to 0.71 µg (median = 0.27 µg) per 100 mg powdered tooth sample. The DNA extracted from the petrous bone specimens successfully amplified the AMEL and SRY markers (24 of 25 petrous bone specimens) more frequently than the DNA samples collected from the molar teeth (19 of 23 molar tooth samples). Using only molar teeth-derived DNA for sex determination (instead of petrous bone-derived DNA) demonstrated a significantly higher margin of error (i.e., higher probability of failing to determine correct sex), compared to DNA extracted from the petrous bone counterparts (p-value: 0.03; Perror(MOLAR) = 0.19 vs. Perror(PETROUS) = 0.07). Based on these results, it is recommended that pars petrosal samples be collected for downstream molecular analyses in visually-compromised human remains in the SA forensic field, where buccal swabs are not a viable sampling option. This alternative specimen-type [to tooth specimens] proved to be easier to process than molar teeth, whilst providing DNA samples that successfully amplified various molecular markers (AMEL, SRY, FHL2) more frequently than DNA extracted from molar teeth. The age-predictive model performed well with the buccal DNA samples, based on the accuracy of 6.67 years (MAD-value). Nonetheless, the substantial difference in accuracy of the age-predictive model between the buccal swabs and the post mortem specimens indicates that the age predictive component of this study requires further review (to improve the age-predictive accuracy), before the algorithm would effectively add value to the local forensic environment.