Distinction of twins and clones on DNA level (ISFG 2001)

Source: Progress in Forensic Genetics (click for .pdf) (B. Brinkmann & Angel Carracedo (eds.)), International Congress Series #1239 (2003):857-859

Discrimination of monocygotic twins (and clones) on the DNA level

Von Daniel Schlieper, Mark Benecke, Andreas Ehlich

Recent DNA typing methods (RFLP, STR, RAPD; e.g. [3]) do not allow discrimination of monocygotic twins. To overcome this restriction, we suggest the use of variable DNA sequences of bone-marrow derived memory B lymphocytes that are likely to be different even in monocygotic twins. Since memory B cells are transported in the blood stream, they can be found in blood stains on crime scenes and checked for a match to the cells of a living pair of twins. The size of the antibody repertoire has been estimated to comprise theoretically up to 1010 specificities [2].

Since each B lymphocyte is endowed with a single antibody specificity, this estimate corresponds to the number of different B cells that can be generated. A major source of antibody diversity in the preimmune repertoire is the stochastic recombination of V, D, and J elements of the immunoglobulin heavy (IgH) chain locus. It takes place in B cell precursors in the bone marrow, and it results in the generation of genes encoding immunoglobulin heavy chains [1,4]. Apart from random selection of V, D, and J elements, diversity is increased by the random addition of non-germline encoded nucleotides (N sequences) and the addition of nucleotides palindromic to the termini of rearranging gene segments (P nucleotides) resulting in extremely diverse stretches of approximately 45 bp.

These stretches should be specific markers to aid forensic differentiation between monocygotic twins because, due to their extreme diversity, they are highly unlikely to be shared by two individuals in the subset of B cells that forms the population of memory cells. Upon stimulation by an antigen, specific B cells are activated. In the germinal centers (in secondary lymphoid organs like spleen, lymph nodes, Peyer's Patches) they proliferate and differentiate into antibody secreting plasma cells and memory cells [8]. At the same time, further diversity is generated by the introduction of point mutations into Ig genes (somatic hypermutation; [10]).

In contrast to naïve, antigen inexperienced B cells, memory cells are clonal. They are long-lived cells [9] and can provide immunity to the specific antigen for decades. Even if two individuals will have undergone an immune response to the same antigen, the pools of memory cells generated are likely to differ due to the variability in naïve B cells that are recruited into the response. This means that from a statistical standpoint, it is unlikely that monocygotic twins will share a majority of identical VDJ stretches in memory B cells. VDJ genes from B lymphocytes in a blood stain can be isolated by PCR and sequenced. Sequences that are derived from memory B cells can be identified by the presence of somatic hypermutations. The PCR primers used are specific for sequences downstream of J and inside of V elements, respectively. This will result in amplification of VDJ regions of all B cells in a given sample. 25% of all amplificates are expected to represent VDJ sequences from memory cells (e.g., [6,7]).

The forensic question to be asked in a case involving monocygotic twins would be: In whom of both twins are memory B cells (i.e. specific VDJ combinations) present that are identical to the ones found in a given stain? Technically, native blood samples of both twins would be taken, and memory B cells would be isolated by fluorescence-activated cell sorting [5]. Then, possible VDJ sequences matching the stain-derived ones would be detected by PCR using primers specific for the sequences in question. If a specific VDJ sequence of one of the twins’ blood cells matches a VDJ sequence in a stain, a possible match is established. Due to the high variance in specific VDJ sequences, false positives are unlikely.

On the other hand, false negatives are possible, as the lack of a specific VDJ sequence in one individual might not exclude this individual: Depending on the size of the memory B cell clone no cell of a given type might be found in an actual sample of native blood. The data needed to calculate the probabilities for the exclusion of the matched twin and for the inclusion of the other twin are not yet fully available. In particular, comprehensive statistical data concerning the diversity of the available B cell repertoire and on the size of memory clones in humans have still to be established. In any case, an important piece of circumstantial evidence might be obtained by our investigation method.

NOTE: Apart from the potential use for forensic purposes, the method described here can also be applied to distinguish individuals within a population of animals, e.g. sheep or cattle. This might be inbred lines, twins, or clones.

Podcast: »DNA 🧬 War Kolumbus Spanier?«

Die ARD Madrid berichtet (oder hat zumindest im März 2025 einen Beitrag bereit gestellt): War Christoph Kolumbus Spanier oder nicht? Das Labor des Kollegen Lorente hat dazu im TV eine neue Untersuchung vorgestellt .

Hier geht es zum Podcast ↓

Meldung des MDR (ARD):

»Christoph Kolumbus stammte einer neuen Theorie zufolge nicht aus dem norditalienischen Genua, sondern aus dem spanischen Mittelmeerraum. Das wollen Forscher der Universität Granada anhand von DNA-Proben herausgefunden haben.

Über die Herkunft des Entdeckers Christoph Kolumbus gibt es eine neue Theorie. Spanische Wissenschaftler der Universität Granada wollen anhand von DNA-Proben des Seefahrers und seines Sohnes herausgefunden haben, dass der Entdecker Amerikas aus dem spanischen Mittelmeerraum stammte. Sie erklärten zudem, das Erbgut seines Sohnes Hernando enthalte auch Merkmale, die mit einer jüdischen Herkunft vereinbar seien. Lange Zeit war angenommen und gelehrt worden, dass Kolumbus aus der italienischen Hafenstadt Genua stammte.

Die spanischen Forscher um José Antonio Lorente erläuterten ihre Theorie von der spanisch-jüdischen Herkunft von Kolumbus in der Dokumentation Colón ADN, su verdadero origen (Kolumbus DNA, seine wahre Herkunft) des spanischen öffentlich-rechtlichen Fernsehsenders RTVE. Das Team hatte zahlreiche Theorien zu Kolumbus' Herkunft überprüft. Unter anderem nahmen die Forscher DNA-Proben von Männern mit dem Nachnamen Colombo in Norditalien, bei denen es jedoch keinerlei genetische Ähnlichkeiten zu Kolumbus gab.

Letztlich kamen Lorente und sein Team zu dem Schluss, dass eine spanische Herkunft des Amerika-Entdeckers am wahrscheinlichsten sei.«

Fingerspuren & DNA (genetische Fingerabdrücke) Training Dez. 2024 🧬

Book Review: 'Who They Were' by Bob Shaler (NYC OCME / WTC DNA) 🌇

This is very valuable book because it tells the inside view straight from the point of laboratory organization and management as well as techniques used after 9/11 in Manhattan. It will disappoint readers who want to know about life stories of single victims of the WTC attack; that is not the topic of the book.

Bob Shaler was very much driven to identify the victims as far as possible in-house and to coordinate as much as possible himself. The Office of Chief Medical Examiner was (and is) under direct command of the Mayor's Office, so it was bound to City of New York and during the WTC times many other policies. (Often, Institutes for Legal aka Forensic Medicine belong to universities, are academic institutions and are therefore run a bit differently.) Our* lab was massively expanding under Bob Shaler, at the end of the 1990s, we were around 50 scientists there. When 9/11 happened, the number of scientists had doubled. Sexual crime had become a focus of attention under then mayor Rudy Giuliani, so many "sexual assault kits" came in, containing underwear, swabs, hair and more, mostly taken from the victims. Shootings and knife wounds were less common compared what some people think because guns and knifes were already strictly banned at that point in the city. (For example, I was scolded twice by Bob Shaler for having a small swiss army knife in my pocket and had to remove it.)

In the middle of this lab expansion, the sexual crime stains pouring in and the urge to speed up case turnover time in the lab, as well as the police bringing in lots and lots of material which they thought might be useful for stain analysis — anything containing a stain which was a lot in blood stain cases, e.g., phone books, sneakers etc. —, the first World Trade Center Tower fell. 

The head of OCME, forensic pathologist Charles Hirsch as well as colleague Ristenbatt from the DNA department immediatly went downtown (which is not too far from the office) and got cought in the fall of the second tower: No more connection to them. This is one of the moments in the book where Shaler mentions that everyone was shocked but does not mention that both survived (until much later in the book). 

Shaler also mentions that he became "less happy" as a person over the course of the events, and that much later, bone fragments were found that came from the persons in the airplanes that crashed into the towers. I mention this as a warning that even though everyone was deeply affected by the events, the book is not emotional. If you wish to read an emotional account, this is not your book.

Instead, Shaler gives a very precise timeline of which laboratories he asked for help and who in the laboratory became responsible for which part of the DNA work. He mentions all the names of persons involved, and it sounds a bit like a historical writing to me — so that the facts will not disappear in time and space.

The determination he put into the WTC work is most obvious to me when the number of identifications went down and — in my opinion — very minor misidentifications took place. Shaler then set up a grid and tried the impossible: To match the three-dimensional structure including every single person who had still been in the building to the comparably two-dimensional, collapsed rubble into which the mostly very small body parts were mixed after the towers fell. I would have thought such calculations to be unfeasible — but he did it. I find the results amazing; they are in the book.

If you come from the field of forensic biology, you will not mind the numerous abbreviations that Shaler uses and that are common use in our field like SNP, STR, KADAP, OCME, DM, MDKAP, WTC CODIS, MFISys. All is explained, of course, but for a reader from a different field, you may wish to consult the glossray and the index at the end of the book. What I find very cool is the 'Cast of Characters', also at the end of the book. It is a honorable move to include many of the scientists and organizational staff that took part in the identification process.

After reading Bob's book, I understand the many decisions that had to be made in-house concerning the involvement of others. One of the software programmers for example, a mathematician, was quite a character and Shaler had to decide which parts of the statistical work (to connect stain to stain to anything the relatives of missing persons delivered, e.g. toothbrushes) to give him. 

Same for Craig Venter, then an absolutlely famous person for decoding the human genome after speeding the 'race for the human genome' to the max. He offered Shaler close to unlimited help which sounded possible and nice on the one hand. On the other hand, Venters experience was not forensics, and his company later decided (Shaler ponders) that unpaid or hardly paid work for a good cause might damage their revenue. 

Also, I now realize how difficult it is to communicate all to everone who needs information in a mass disaster situation (and probably also during day-to-day work in a large lab). 

Some stories are not told, of course, and since there may be a reason for this, I will leave it like that. 

Shaler's book is unusual since it is a popular science book that avoids tearful comments wherever possible and focuses on procedures, processes, genetic fingerprinting, agencies and persons involved. There are brief exceptions, though, mostly relating to the contact with the victim's families. The relatives had high hopes to science and identification even though they were often facing total destruction or decomposition of the body parts: "Their grief was exhausting, and while I tried to remain detached and not watch, it was impossible." 

What Shaler does not write is that the whole area around the office was literally plastered with self-made posters of missing persons, and that many people where standing in front of the Bellevue hospital entrance next to the OCME — quietly, looking for a glimpse of information, not walking away.  

More than once, Shaler mentions that probably god pushed him into the direction of his — this — job. For Bob, it was the most important task of his life. He also mentions that for younger scientists in the lab, it might be difficult to work on the most important case of their lives at the beginning of their careers because afterwards, work might be less intense. From my experience, this matters not because at the end of the day, a case is a case, and all cases should be treated equally. Probably his statements are proof to the shell shock that WTC caused to so many (including me). 

After the World Trade Center DNA investigation was closed, Bob Shaler resigned at the OCME. He then set up a forensic program at Penn State University. In 2010, he retired from forensic work.   

* I worked in the forensic biology (i.e., DNA) department of the NCY OCME  from 1997 to 1999. Bob Shaler was my direct boss; Mecki Prinz from the OCME DNA lab was my first forensic boss in Germany. Briefly after the WTC fell, I went to Manhattan, talked to my colleagues and wrote a popular science article about their DNA efforts → https://home.benecke.com/publications/nicht-sachen-sondern-menschen


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A routine rape case that became tricky (and educational)

After attending this presentation, attendees will understand why it is important to listen to the victim and relatives, who do not fully understand the objective DNA evidence, but who point in a relevant direction relating to procedural failures of police and crime labs.

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Y-chromosomal short tandem repeat haplotypes at the loci DYS393, DYS19, DYS392, and DYS385-I/II, DYS390, DYS389-I/II, and DYS391 in a Filipino population sample

Source: J Forensic Sci (2001) 46(5): 1250–3.

Miranda JJ, Benecke M, Hidding M, Schmitt C

The article as .pdf

Population: Male population sample (n = 106) from the Metro Manila area (largest urban center in the Philippines).

Keywords: forensic science, forensic DNA typing, short tandem repeat (STR), Y chromosome, genetic fingerprint, Philippines

Whole blood samples were obtained from 106 unrelated male individuals living in Metro Manila, Philippines, through the Department of Health, Manila. DNA was extracted by isopropanol fractionation-sodium iodide precipitation (4) and quantified by spectrophotometry. Nine Y-chromosomal short tandem repeats (STR’s) were analyzed from a population sample of 106 unrelated males by means of a quadruplex PCR (DYS393, DYS19, DYS392, DYS385-I/II) and a triplex PCR (DYS390, DYS389-I/II, and DYS391).

Primers were Cy5-labeled, and based on sequences described by Kayser et al. (1). PCR products were separated on ReproGel™ High Resolution polyacrylamide gels, and laser-detected by an ALFexpress sequencer (Amersham Pharmacia Biotech). Allelic ladders, and nomenclature were standardized against allelic ladders from P. de Knijff (Leiden), L. Roewer (Berlin), J. Edelmann (Leipzig), and P. Schneider (Mainz).

Discrimination capacity for the nine-loci system was 83%. Gene diversity was calculated following Kayser et al. (3). Frequencies of the individual alleles are shown in Table 2. Haplotype data (88 distinct haplotypes, 75 of which were unique) are given in Table 1. Gene diversity values ranged between 0.37 for DYS91 and 0.94 for DYS385, which is similar to frequences reported elsewhere (2,3).

Acknowledgments

The authors wish to acknowledge the assistance of Rubigilda Paraguison and Edith Tria in the collection and preparation of the samples. Dr. Miranda was supported by a fellowship of the Deutscher Akademischer Austauschdienst (DAAD, German Academic Exchange Service), Federal Republic of Germany.

References

1. Kayser M, Caglia A, Corach D, Fretwell N, Gehrig C, Graziosi G, et al. Evaluation of the Y-chromosomal STRs: a multicenter study. Int J Legal Med 1997;110:125–33.

2. Pestoni C, Cal ML, Lareu MV, Rodriguez-Calvo, Carracedo A. Y chromosome STR haplotypes: genetic and sequencing data of the Galician population (NW Spain). Int J Legal Med 1998;112:15–21.

3. Rossi E, Rolf B, Schürenkamp M, Brinkmann B. Y-chromosome STR haplotypes in an Italian population sample. Int J Legal Med 1998;112: 78–81.

4. Wang L, Hirayasu K, Ishizawa M, Kobayashi Y. Purification of genomic DNA from human whole blood by isopropanol fractionation with concentrated NaI and SDS. Nucleic Acids Res 1994;22:1774 –5.


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Asian online Y-STR Haplotype Reference Database

For several years Y-chromosomal microsatellites (short tandem repeats, STRs) have been well established in forensic practice. In this context, the genetic characteristics of the Y chromosome (i.e. its paternal inheritance and lack of recombination) render STRs particularly powerful.

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