Summary of the chapter dealing with DNA evidence of Bossetti’s appeal against his first grade conviction.

As is known, since the pre-trial stage, following the outcome of an expert report of

geneticists (Dr. Carlo Previderè, Dr. Pierangela Grignani) appointed by the Public

Prosecutor, this defense has highlighted the inexplicable situation that we can briefly

define as the absence of mitochondrial DNA of the accused in the traces

attributed to him as regards nuclear DNA and collected on the briefs and leggings

worn by the victim.

Such a "fallacy" (as it was called the Court of Review of Brescia) found no solution at

trial, nor the ruling takes care to offer an explanation able to overcome the

reasonable doubt that we are - at best - in front of a glaring error.

This is, as evident, the theme of greater delicacy to be addressed and resolved in

order to correctly attribute the trace itself [to a suspect].

Indeed to a statistically certain attribution from the nuclear [DNA] point of view,

corresponds an exclusion (this one certain in biological terms, because of the

complete lack of DNA) from the mitochondrial point of view.

The defence has always maintained that the absence of the mitochondrial DNA

component of the accused in traces attributed to him was indicative of something

unnatural, especially given that such a situation has never been observed in nature

and for which a scientific explanation has not been found.

The thesis continually reaffirmed, however, by the Prosecution, outright adopted by

the Court in the absence of expert opinion [by Court appointed and hence

independent experts], is that only the nuclear DNA has identification capabilities,

which can not be said for mitochondrial, the absence of which, moreover, could be

justified by various reasons, which ones, however, is never explained with reference

to this specific case.

Now, to say, as stated on page. 86) of the judgment, that the mitochondrial DNA

lacks any identification capabilities, "even for mere exclusion" is a serious

mistake, which shows that the Court has certainly misunderstood the importance of

this scientific datum.

Moreover, the wording is clearly logically inconsistent with what was stated by the

Court a few pages later. On page. 95) of the judgment we read that: "the search for

the mitochondrial DNA in samples from mixed traces (ie with multiple

contributors) is not advisable, because it may lead to false exclusions."

But if the mitochondrial [DNA] lacks even exclusion capabilities, it may not lead to

false exclusions.

So, what was told at the preliminary hearing on the subject of mitochondrial DNA?

The Head of RIS - Col. Lago - questioned by the public prosecutor.

Prosecutor: "Instead, the mitochondrial DNA is just as useful to discriminate, to

identify a person over another?”

Lago: "The mitochondrial DNA of a mother will pass equal to itself, identical to

itself, to sons and daughters, and will in turn pass identical to their brothers, to his

mother, to his grandmother, and so on. ..to find a mitochondrial DNA in a trace

means finding not the DNA of that person, but the DNA of the family, of the

maternal line. Therefore the mitochondrial DNA does not allow identification of a

single individual".

No identification, therefore, of a single individual but of the entire maternal line.

Same and substantial answer has been provided on this point also by Dr. Giardina,

another PM consultant who, questioned by the defense (p. 90 of the transcript of the

hearing of 18/11/2015):

Counselor Camporini:"From the point of view, instead, of the exclusion of a person,

is mitochondrial DNA an important finding?

Consultant Giardina:"Certainly when we are in presence of individual DNA profiles,

mitochondrial DNA analysis is an analysis that is also used to exclude,

sure". The consultant therefore believes the analysis of mitochondrial DNA is also useful to

exclude "individual" profiles.

But, concerning the possibility of obtaining the same results even on mixed traces

have weight the considerations expressed by Dr. Casari, another advisor to the PM,

when (p. 115 of transcript of the hearing of 20/11/2015), when questioned on the

point by the defense lawyer.

Camporini:"However with a the mixed trace the result comes out?"

Consultant Casari "Yes".

Camporini:"And it would be reliable?"

Consultant Casari:"Very reliable, and the number of sequences is proportional to

the the contribution of the initial mitochondrial genome".

So, whether it is a mixed trace or whether it is a trace with only one contributor, the

investigation of the mitochondrial DNA gives a very reliable result

according to the Prosecution consultants themselves. The above mentioned statements are in stark and obvious contrast to what was

stated by the Court, which considers the mitochondrial DNA as not even useful to

achieve a mere exclusion , especially with reference to the investigation of traces

mixed and degraded, which are considered, however, inadvisable.

Inadvisable does not mean they can not be done and especially that they may not

lead to clear and interpretable results!

In this case, the study of mitochondrial DNA was entrusted to the University of

Florence - Department of Molecular Anthropology - specialized precisely in analysing

mtDNA from complex traces (with degradation and mixture).

It must be noted as this study of the mitochondrial DNA has led to the exhaustion "in

their entirety" of the samples relative to the best traces for quality and quantity

attributed to Unknown 1.

Now, that it may be belittled the importance of such an expensive scientific

investigation as one having mere heuristic purposes and also the fact that one may

"consume" entirely for these purposes the only traces from which an interpretable

nuclear profile was obtained, leaves one truly amazed.

According to the scientific community and according to the PM

consultants themselves, as we saw, not finding the mitochondrial DNA

corresponding to the nuclear DNA extracted from the same trace of a

person, leads to EXCLUDE the individual and all his maternal line; but

this does not apply for the Court.

The reason why is, however, missing in the ruling.

How it can be said that mitochondrial DNA is irrelevant even to the purpose of

exclusion (p. 86 of the judgment) remains a mystery as well as a groundless

statement, devoid of scientific evidence, but also a strongly misleading element

in the logical and legal route of the decision being appealed. As regards the "identifying" capability of the mitochondrial DNA (not of the

individual but of the entire maternal line), we will further recall the words of Dr.

Lago as well as expressed in his consultant report to the PM in the present

proceedings, which reads, p. 28:

"In the context of genetic identification, starting from a biological sample, in

addition to the study of the polymorphisms of nuclear DNA, molecular biology

provides the possibility of investigating the sequence of polymorphisms of the

mitochondrial DNA(mtDNA)".

Continuing the reading of the Lago report, p. 30, we read:

"To summarize, the molecule of mtDNA plays a forensic role because:

• it only transmits through the mother;

• it presents a nucleotide variability, especially in the non-coding D-loop region,

sufficiently high to allow a discrimination between unrelated individuals;

• it has a high degree of conservation also in tissues subjected to noteworthy

chemical-physical and biological stresses;

• it also appears in cell derivatives in which nuclear DNA is not found (for example

the stem of a human hair);

• the circularity and, therefore, the structuring in molecules without free ends

protects a priori from the autolytic effects of a class of degradative enzymes

(exonuclease)".

What reported above (report submitted on 28.02.2013) is not, however, fully

confirmed by the testimony given by the same consultant in court (23.10.2015

hearing) where, at p. 17/18 of the transcript, one reads:

"... the procedure itself of the mitochondrial DNA study maintains a much more

experimental character in general ... the mitochondrial DNA is not included among

the procedures validated for forensic use."

What, did we not just read that the mitochondrial DNA, because of its

characteristics, "plays a forensic role" and that in genetic identification

research it allows "discrimination between unrelated individuals"?

Why Colonel Lago expresses himself at the hearing in direct conflict with what

himself reported and signed in previous written reports has only one answer: to

support the Prosecution's case.

What originally reported by Lago in a non suspicious context, scientifically correct

and confirmed by the converging opinion of the whole scientific community, is at

odds with the thinking of the Court, which does not take into account the original

positions of the consultant and with laconic unsubstantiated claims, tries to demote

the scientific significance of mitochondrial DNA. Only through such groundless "demotion" it was possible for the Court to enhance

the data provided by nuclear DNA and consider that one as the only useful data to

the identification of an individual even in the presence of incompatible information

coming from mtDNA.

To reduce the study to a "purely investigative" purpose (p. 81 above) - and here is the

"demotion" - ie for purposes of "finding, also using experimental techniques, markers different from those used for identification and which can provide more

information about the physical characteristics and / or geographical origin of the

subject " appears in an open and irreconcilable conflict with the scientific logic

underlying the studies on mitochondrial DNA conferred by the prosecution.

Experimental techniques?

Information on physical and / or geographical origin of the subject?

Undoubtedly, a survey of mitochondrial DNA led by Dr. Lago had, in fact, this

purpose, namely "the study of some genetic DNA markers of recent discovery able to

provide information about the somatic type". For this activity, this one really

experimental, Dr. Lago took advantage of the George Washington University

Department of Forensic Sciences - USA

Another part of the work was, however, dedicated to the "study of genetic markers of

mitochondrial DNA which may provide detailed direct information about

Unknown 1" (p. 3 of the Lago report) but also identify his mother: so nothing

experimental, but something apt to personal identification!

Precisely for this reason the Lago report consists of two distinct and separate

sections.

See, in this regard, what stated by Dr. Lago in the same report (p. 3).

“The present report, following the logic outlined above, is divided in two distinct

sections, with a clear division of the document following, in turn, an as well clear

separation of the technical activities.”

Clear division of the document that follows an equally clear separation of technical

activities, precisely to separate what is experimental from activity performed for

identification purposes.

With identification as aim, then, Dr. Giardina had to identify, through the

comparison of mitochondrial DNA content in traces identified by RIS with that of

532 individuals which were thought to include the mother of Unknown 1, the woman

who shared the same genetic heritage of Unknown 1 and that, therefore, was his

mother.

Hence not experimental investigative purposes, as suggested by the Court but quite

different identification purposes, concerning both Unknown 1 and his maternal line!

As proposed by this defense, the discrepancy between nuclear DNA and

mitochondrial DNA could be explained by unforgivable errors that occurred in

the technical investigations (to which this defense has never been able to take part),

if one wants to give credit to the not scientifically proven thesis, not scientifically

proven that we are in the presence of a DNA actually deposed as a result of

direct contact between the victim and the accused at the time of the aggression.

Any deposition of biological material of any origin cannot, in fact, do

without the inevitable transfer of both nuclear and mitochondrial genetic

contribution in its entirety.

Without, however, forgetting how the genetic analysis, is not able to determine the

age and the mode of deposition of the individual traces, a fortiori when you do not

have reliable data as to the nature of the trace (epithelial, sperm, saliva, blood, etc.).

And please do not object that the conditions of degradation of the traces, which in

this case are equivalent to the standards of similar cases, were able to create a

situation never seen before in which:

• the nuclear components of the trace remain while the mitochondrial components

vanish;

• simultaneously exactly the opposite happens for the contribution of the victim, the

mitochondrial component remaining in the trace, while the nuclear one disappears.

At the end of these first observations one can certainly say that, despite the claims

made by the Court, the mitochondrial DNA was not used to acquire information on

the physical characteristics and/or geographical origin of a person, but to identify the

maternal line of origin as well as to derive "direct detailed information about

Unknown 1" and therefore with identification purposes.

This is the weak point of the entire ruling that shows how science has been

manipulated to make appear as valid only the results that one likes while those not

compatible with the assumption one wants to demonstrate, are disregarded, ie,

diminished in their scientific relevance.

This approach, in a proceeding based essentially on DNA evidence is inadmissible.

On the issue of alleged "lack of investigative significance" of the mitochondrial DNA

supported by the Court, it is interesting to bring to the attention of this [ie appellate]

Court what was stated by Dr. Giardina (a PM consultant) in the web site of the

University Tor Vergata, on the page of the Master in Forensic Genetics, which you

may find at: http://www.mastergeneticaforense.it/cms/approfondimento/6/

USE OF MITOCHONDRIAL DNA IN FORENSICS

Thanks to its biological properties mitochondrial DNA is an important

tool for forensic applications. Consider first the morphological and structural

characteristics. In particular, the double membrane of the mitochondrion

effectively protects DNA from breakage and damage induced by

environmental stresses. In addition, the circular nature of [mt]DNA

provides less susceptibility to exonucleases (enzymes that cut DNA),

allowing the mitochondrial DNA molecule to be preserved better in the

course of time. To all this must be added the considerable advantage of

having a number of mitochondrial genomes per cell vastly greater than

nuclear DNA, hence increasing the chances of success in obtaining a

profile. Mitochondrial DNA is often used in cases where the biological

material is degraded or available in limited quantity.

However, the use of mtDNA is not limited solely to cases of samples in

bad state of conservation. The matrilineal transmission mode also makes mitochondrial DNA useful for

integration of classical STRs markers in cases in which it is not possible to make a

direct comparison between the subjects. "

In a nutshell, for Dr. Giardina:

• mitochondrial DNA is an important tool for forensic applications; •the characteristics of said component of the cell make it especially resistant to

damage induced by environmental stress;

• its circular nature enables the mtDNA molecule to be preserved better in time;

• being able to have a number of mitochondrial genomes per cell enormously greater

than nuclear DNA increases the chances of success in profiling; • the mitochondrial DNA is often used in cases where the biological material is

degraded.

Needless to emphasize that the outlined scientific principles are perfectly compatible

with the needs of this case, in which you have a degraded trace yes, but with a DNA

of an amazing quality and quantity.

Additional litmus test of intellectual honesty of those who preach one way and then

acts in another is, on the one hand what reported at the hearing (23.10.2015, p. 17-18

of the transcripts) by Col. Lago, Head of the RIS of Parma:"the mitochondrial DNA

is not among the procedures validated for forensic use … hence

mitochondrial DNA has an essentially experimental nature."

On the other hand, the website Carabinieri.it - Lago belongs to the Italian Carabinieri

Corp - to this days advertises with reference to its section of genetics:

"The section also … performs sequencing DNA using technology based on

fluorescent techniques. This technical support allows us to operate in what is

currently the most modern and interesting field of forensic molecular

biology: mitochondrial DNA."

The macroscopic contrast is quite obvious.

In the context of this proceeding, the scientific evidence and in particular that

relating to DNA has undoubtedly the greatest importance.

The Public Prosecutor has in fact benefited from the advice of several specialists who

have studied, at different times, every aspect of the DNA and, above all, as is

evident from the assignment lines, mitochondrial DNA. Likewise, the vast majority of the fact finding at trial found in DNA evidence its most

relevant subject.

The RIS of Parma was responsible for the scientific investigation on nuclear DNA; dr.

Lago, on his own [as PM consultant], in collaboration with the University of Florence

and in collaboration with the George Washington University - Department of

Forensics Science - USA, oversaw the mitochondrial DNA study; doctors Cattaneo

and Piccinini examined the issue of Giuseppe Guerinoni’s paternity, with particular

reference to the study of new regions of nuclear DNA, while doctors Previderè and

Grignani, having to analyze the hair formations, focused on the study of

mitochondrial DNA and Dr. Casari also studied the mitochondrial DNA.

The problems relating to the mitochondrial DNA of Unknown 1 has emerged, as

already said, in the reports signed by consultants Previderè and Grignani, who, in

analyzing some hairy formations sampled on the victim on the one hand, and on the

truck in to Bossetti on the other, in order to answer the question posed by the PM,

chose to use the study of the mitochondrial component attributable to the victim and

to the defendant, despite the hair having, in the majority, the bulb (as is known, in

the absence of bulb, is only the mitochondrial component is analyzable).

The outcome of this examination has shown that, surprisingly, in no

trace, isolated and analyzed first by RIS and then by Lago, was present a

mitochondrial component attributable to Bossetti and that indeed, there

was a minority component certainly belonging to another person, other

than the victim or Bossetti.

The data to be taken into consideration are two. The absence of mitochondrial DNA

of the accused and the simultaneous presence of the mitochondrial DNA of another

person, remained unknown.

As can be seen from the table of the Lago Report, p. 36):

But also from the report of Professor Casari, p. 8): “The analysis of the NGS sequences confirmed consistently the YG [Yara Gambirasio] haplotype as primary, with a secondary mitochondrial DNA component with a CRS reference sequence”.

And finally, as certified by Previderè and Grignani about the non-compatibility of

Bossetti’s mitochondrial DNA with the one detected in the traces attributed to

Unknown 1.

See, in this regard, the table below from p. 110) of the report Previderè-Grignani.

In summary, four different Prosecution consultants in three reports, at

different times, with different objectives and with different instruments,

have reached the same and incontrovertible result, totally incompatible

with the attribution of the said traces to the person of Massimo Giuseppe

Bossetti, a result that the ruling belittle with the sole purpose of saving the results

of a scientific investigation that, at this point, if deemed anyway of some value, would

provide an a-scientific answer, indeed an answer against nature.

Why a-scientific? Why against nature?

Because science, as represented by all the consultants heard at trial, could not

provide real explanations but mere abstract hypotheses, detached from the case

under judgment.

That, in the opinion of this defense, makes the fact finding at trial completely

unreliable, since, as stated by Dr. Capra [defense geneticist] at one hearing, a trace

can not be defined of forensic interest if it does not fulfill the

fundamental requisite of accuracy and completeness.

Only a DNA complete of the two inseparable nuclear and mitochondrial components,

free from defects, inaccuracies, properly interpreted according to verified scientific

canons and repeated as the dictated by the Supreme Court [re the number of

amplifications], may be suitable to identify the owner of the trace that contains that

DNA.

The question that must be asked is: could Unknown 1 have transferred only the

nuclear component of its DNA?

Is it justifiable in nature a DNA missing the necessary mitochondrial component?

Can this happen in nature?

The answer to these questions can only be provided through the proper use of the

scientific method. The validity of any scientific law is not measured in terms of its

verifiability, but in those of his falsifiability, so the parameter of its validity is

permanent resistance to falsification.

Indeed just the success of a single operation designed to falsify refutes the thesis (in

this regard, it is known in the scientific literature that a DNA trace can be falsified,

that is artificially fabricated).

The lack of acceptance by the generality of the scientific community of the validation

of a hypothesis means in fact scientific uncertainty.

Can we safely say that the scientific community accepts that the identification of a

person can be performed through nuclear DNA even when there is an unjustified

absence of the corresponding mitochondrial component?

That the scientific method has been greatly neglected in this proceeding even if it

apparently was the cornerstone of each argument, is proven first by Lago’s words at

trial, then by prosecutor herself, and, finally, by the Court which completely

abstained from debating about mitochondrial DNA, aligning itself in a uncritical and

apodictic way to the hypotheses made by the prosecution’s experts, instead of

appointing a panel of independent experts.

As stated, the words of Dr. Lago answering to the question of the PM (p. 22 of the

transcript of the hearing of 23.10.2015) are enlightening on the (non) scientific

answers that one would have expected:

"Can you say that it is unnatural, against nature, that the forensic analysis of an

exhibit, only detects the nuclear DNA or the mitochondrial DNA?

Consultant Lago: Let's say that even before giving a scientific answer, an answer,

let us say, philosophical, must be given. I go to Mars, I find stones in the shape of a

pyramid, I did not expect it and I say, it is something that is not possible. Simply

because my experience on this planet is that the stones are basically round. In fact it

could also be that I have been particularly unfortunate, I am landed at a point

where there are only stones of that form; or I can do a study on all that planet and

[find that] the stones there are almost all in that way. I do not know."

The prosecutor believes that science should take a step back in courtrooms (p. 184 of

the trans of the hearing of 13/05/2016):

"This is not the place in which to find scientific explanations".

But in the face of such a lack of scientific explanations, the issue is not all that complex. That in a cell, clearly composed of a nucleus and mitochondria, these two elements

are inseparable is not only common knowledge but, also, an elementary knowledge.

The apparent situation found (possible loss of mitochondrial DNA) was never

detected in the literature, except in one experimental study well-known by dr. Lago

enough to be mentioned but not to be deemed relevant to the refutation of

the reliability of the results obtained [on mtDNA].

Moreover, the cited study of Montesino concerns particular situations and

technical procedures definitely not applicable to this case.

Indeed, in the Lago Report on mitochondrial DNA, one reads (p.37):

"The sequences [mtDNA sequences] obtained, evaluated according to

the guidelines of the international forensic science community, meet

qualitative and quantitative parameters, such as to also be useful for

identification purposes". This (Lago’s) study led to the result of identifying in the best trace under

examination, 31G20, in addition to the mitochondrial DNA of the victim, always

present, the mitochondrial DNA remained unknown and NEVER the one belonging

to the defendant. Also Prof. Casari, with specific reference to the absence of the mitochondrial DNA of

Bossetti, confirms that would have expected a different considering the sizable

presence of nuclear DNA, indeed (p. 138 of the transcript of the hearing of

20/11/2015), he so reports to the Court:

"it should also be said - maybe I have not stressed this enough in the report - that on

these samples the amount of DNA of Unknown 1, compared always of course to the

nuclear DNA of Yara, is 40-50%. I should expect a 40 or 50% of CRS sequence, if

this were a minority sequence that comes from Unknown 1. While ... I found

nothing. I do not know if I have make this clear enough."

On the existence of a minority component different from that of the Bossetti

and from the majority component belonging to Yara there is the

convergence of the opinion of all the consultants, except for a few,who, apparently in

an attempt to diminish it, consider the component as uninterpretable without

providing, however, any scientific justification.

The fact remains that none of the consultants considered that the data in

question were wrong. In conclusion, we recall the words of the PM said during her closing arguments,

which essentially summarized the thinking of her consultants:

"The defendant's mitochondrial DNA is certainly different from the mtDNA of the

traces that were studied, the ones I mentioned earlier, including the minority

component. It's certainly a different mitochondrial DNA".

That statement shows that, even the Prosecution is aware of the existence of a

minority component, again with reference to the mitochondria of the cell, that it

could be interpreted [ie a sequence of markers obtained] so as to be compared and

that the comparison has returned an outcome absolutely not compatible with the

mitochondrial component of the defendant's DNA.

This is, in short, the problem of the trial that needs to be solved, because from a

scientific point of view people can not transfer their DNA if not in its

entirety (nuclear and mitochondrial). The lack of an essential part of the cell structure is food for thought and, in a way,

even cause of alarm.

The unproven and non-technical conclusions reached by the Court in terms of

mitochondrial DNA can be summarized as follows:

• The mitochondrial DNA was used with a purely investigative purpose,

namely to detect, even using experimental techniques, markers other than those used

for identification, that can to provide further information on the physical

characteristics and / or geographical origin of the subject;

• international scientific studies on the analysis of the mitochondrial DNA in mixed

traces are very few and they all assert that the variables involved are so many that it

is better not to use them for forensic purposes;

• the mitochondrial DNA does not have identification capabilities even for the mere

purpose of exclusion.

Those listed above are the points extrapolated from the ruling that are thought to be

the basis of the reasoning that led the Court to deny the relevance of the investigation

on mitochondrial DNA and of the indisputable results which it achieved. According

to the Court, therefore, the mitochondrial DNA would not have the value that the

defense attributes to it, basically because the investigation into the mitochondria is a

complex investigation, is not recommended for mixed traces, and is not useful for

identification purposes, even just for exclusion.

This assumption is, however, widely denied, on the one hand by some simple logical

considerations and on the other by the evidence provided by the prosecution experts

themselves, as well as by those of the defense.

This defence points out that, as the mitochondrial study was carried out by a laboratory of absolute excellence, which deals exclusively with mitochondrial DNA in mixed and degraded traces (University of Florence - Dr. Pilli) as highlighted in

the opening words of the report, and led to a sure result, never belied by other consultants and indeed confirmed by the findings of other professionals, like Dr. Carlo Previderè of the Pavia University and Dr. Giorgio Casari of the San Raffaele Hospital in Milan. The report of Previderè - Grignani had for object the comparison of hairy formations found on the victim's body with the genetic profile of the defendant. None of the seven hairy formations which are not attributable to the victim showed a mitochondrial haplotype compatible with that of the accused. In the aforementioned report, in Section 4.4 -analysis of mitochondrial DNA polymorphisms - there is an element considered by this defense of fundamental importance and overwhelming extent. "The analysis of the mitochondrial DNA extracted from traces 31G19 and 31G20 revealed an unusual situation, in the opinion of the writers. In the face of a sizable amount of male DNA in these samples (about 50% in the sample 31G19 and about 70% in the sample 31G20), as documented in the RT-PCR present in the RIS report, amounts such as to produce autosomal profiles with a clear component attributable to a male subject ( "Unknown 1"), the analysis of mitochondrial DNA showed a mixed haplotype with a major component due to the victim and a minor component of difficult interpretation (see electropherograms attached to the Lago Report). The 31G19 sample even produced a haplotype exclusively attributable to the victim. This situation was apparently in contradiction to what is expected by the genetic analysis of mixed biological samples. In fact, in general, the genetic profile of a mixed trace reflects the proportion of the different cellular fractions, the latter originating from different quantities (major, minor or comparable) of biological material of the various contributors. It can, therefore, be that, in a mixed trace, a subject has contributed with a number of more cells than another. This condition will also be represented in the genetic profile where the various contributions will be present proportionally as alleles of the majority component and alleles of the minority [component]." This inversion (between nuclear and mitochondrial) of the quantities of DNA attributable to the victim and to Unknown 1 is the first “unusual situation”. But there is also the second and far more important situation left without an explanation: the non-compatibility, indeed even the absence of the mitochondrial DNA of Bossetti in a trace attributed to him, with at the same time place the presence of a genetic profile of a subject still unknown. The study of nuclear DNA on the samples mentioned above led to a male subject Unknown 1, later associated with Bossetti, and to the victim, while as regards mitochondrial DNA it was possible to profile the victim's component, together with a minority component not attributable to the victim or to Bossetti. One of the hypotheses proposed by the prosecution experts was initially that the victim and the male subject could be related through the mother and, therefore, share the same mitochondrial DNA. The same consultants of the prosecution, however, have ruled out this hypothesis by investigating the maternal line of Yara Gambirasio.

On the basis of the scientific literature on the subject, Dr. Previderè and Dr. Grignani speculate, then, that the two subjects contributing of the mixed trace have brought a different quantity of mitochondrial DNA. According to the observations of Dr. Sarah Gino, defense expert, "it becomes, however, difficult to then give explanation of the fact that at least in the 31G20 sample there is no trace of the nuclear DNA of the victim". To better understand the scope of this statement it must be considered that the prosecution case involves the simultaneous deposition of two biological fluids (of the victim and of the killer) which will be then exposed to the same environmental conditions. It is unique, and as such should be read in the statement of Prof. Gino, that one of biological fluids may have "lost" its nuclear DNA while the nuclear DNA of the other appears to be of extraordinary quality. The last scientific hypothesis proposed by the prosecution experts to justify the absence of Bossetti’s mtDNA is the mixture of biological material coming from two specific biological fluids, ie blood and semen. But Lago itself stated: "according to laboratory analyses carried out by the RIS of Parma to determine the nature of the traces on the briefs, the tests have provided in general and in particular for trace 31G20, negative results for the presence of saliva and sperm and positive results only for human blood." "It was also pointed out by the RIS that given the good quality of the trace in terms of DNA quantification and electropherogram results, we could trust the results of the tests determining the source of the trace, given that to a protein degradation such as to provide a false negative for saliva or semen could not possibly correspond a so good conservation of the genetic material." It is recalled, moreover, that to exclude the spermatic nature of the trace in question the RIS, unique case, has carried out the tests with three different kits validated for use in forensics and able to detect the presence of constituents of the human seminal fluid, also degraded , in infinitesimal quantities. "The SPERM HY LITER test was created for the search and identification of human sperm found in traces on the occasion of sexual violence, the test is able to detect a single sperm present in a mixture of epithelial or vaginal fluids. It is absolutely specific for human sperm and thus does not provide any positive with other body fluids or sperm from other species." On the nature of the traces and, therefore, the possibility that we are talking about sperm, the words of the RIS seem to leave no room for any possibility that what they have analyzed, with specific reference to the male component may result from human sperm. We read:"it seems unreasonable to think that a possible false negative of a diagnostic test may be associated to a genotypic profile of extraordinarily high quality as is, for example, the one based on that sample." It is, therefore, the same RIS to affirm that it would be "absurd" to think that the trace in question may be attributable to sperm.

In summary, and in essence, this defence believes that the identification of a subject occurs, obviously, through the analysis of autosomal markers of nuclear DNA if and only if the mitochondrial DNA is present and is corresponding with that nuclear, given that the lack of the mitochondrial component of the cell would not in any way have a scientific explanation and would make what observed something alien, an artifact, or the product of an error. Every defensive motion aimed at finding a solution to this obvious inconsistency has ALWAYS been frustrated with a blatant violation of the right of defense. In addition, we make another consideration. As stated on p. 65 of the ruling, the Court considered "superfluous the request of the defense of the accused to confer to appoint a special expert panel to re-inspect the clothes of the victim, in order to check on them if it were possible, moreover, at a distance of over five years from their retrieval, to obtain further biological traces attributable to the accused or to any other contributors". So the time elapsed is given as a motive for rejection, for reasons that seem not only quite frankly to be in complete contradiction with current scientific knowledge but also with the judicial practice that we all know. Cases of investigation conducted through the study of the DNA at a distance of many years from the murder are well known. We name only the best known: Alberica Filo della Torre, Elisa Claps, Simonetta Cesaroni and, recently, also the case of Lydia Macchi, who died thirty years ago (in the latter case, consultants for the study of mitochondrial DNA are again Dr. Lago and Dr. Pilli of the University of Florence). For the Court, as it has often been said, all reasoning on mitochondrial DNA turns out to be "indecisive" and is therefore unnecessary, in view of the results obtained on the nuclear component of the DNA, that allows the attribution of the same with such a high statistical probability to be considered equivalent to certainty. Is this certainty the offspring of a survey conducted in compliance with international best practices? As demonstrated by the defence at trial and, here, there is no analysis (with reference to nuclear DNA), which is compliant to the formal requirements of the scientific community, which in short prescribes that for an outcome to be reliable it has to be repeated in the presence of three essential requirements: • the kits used in each phase of the study shall be valid; • the "negative control" which is also to be made with reference to each electrophoretic run, shall have, of course, a negative outcome; • the so-called "positive control" must, similarly, return the DNA of the sample analyzed. So as to exclude any risk of exogenous contamination. Now, these features are not jointly present in any analysis and, therefore, in no repetition. By an examination of the raw data this defense was able to ascertain that the presence of the three requirements stated above is not found in any electrophoretic run.

See, for example, the table refers to trace 31G20.

In this case, the expiration date of the kit used was at a few months old and with regard to some repetitions even more than a year old.

The non-compliance of the negative controls of the amplification of 25.10.2011 on trace 31G20 (the only seemingly perfect repetition even in terms of compliance with the guidelines) denounced by the defense and "long debated by the parties during the discussion" because of the presence in the negative control of a peak that showed a possible contamination, for the Court was justified because "the consultant of Civil Party Dr. Portera explained that the presence in one of the negative controls on the marker FGA of a 88 rfu peak does not affect the result of the clearly interpretable electrophoretic runs "(p.94 of the judgment). Justifying such a serious fault (it is recalled that the peak in question, present in the negative control, which should not present any allele, is a feature present in the nuclear DNA of the accused) with the words of a consultant of one if the parties, who deems the electrophoretic runs, however, clearly interpretable, leaves much amazed. It is indeed well known that a single allelic variation of a single component identifies different individuals! In extreme synthesis, and for greater clarity, a negative control that should, by definition, return a “flat” electropherogram, therefore not contaminated, presents, instead, a peak. And not just any peak, but a peak present in the defendant's DNA. The chain of custody and "multiplication" of the test tubes. The last but not least argument on the subject of DNA, relates to what this defence believes to be the "solid proof" that the chain of custody was managed “sloppily”. During the closing arguments, in fact, the defense emphasized again what their consultant, Dr. Capra, had already expressed during his testimony. There was no question by the prosecution or the counselors of the civil parties on this specific point during cross examination. They opted not to challenge the statements of the defense consultant reported below (hearing of 02/03/2016 from p. 163): "Consultant Capra- I note some inconsistencies that I can see in the technical surveys carried out by Colonel Lago and the RIS. List of exhibits acquired by the RIS from Parma. Volumes and concentrations are listed below. Quantifications have been produced using the Plexor kit, marketed by Promega. It is noted that 31G1Ext, "critical sample", 31G1Int and 31.18 were used for the genetic study of the physical features. While 31G19 and 31G20 samples were used for the profiling of the mitochondrial DNA. Let's see the table. It gives me 31G1Ext 12,700 picograms, 4,880 picograms men. 31G1Int 19 and 20, 18, 236, 136, 301, 179. 31G20, 2,530, 1,680. And then there's the bra sample used as a reference standard. the problem is that these concentrations of DNA, which are critical to the execution of all subsequent investigations, which are the data we have seen that some have tried to use, in my opinion not in the right way, to determine whether a sample was mixed or not and so on. They are completely different from those we found in the RIS table. This is the same test tube, of the same sample always analyzed by the RIS, and here we have 12,700, and originally - that is reported more than once - we had 2500. That is, this tube here suddenly had a concentration five times higher than what was there before. The DNA is leavened, from one became five. 2,500 and 1,000 male DNA, becomes

12,700 and 4,880. And also there are differences, less significant, certainly not in the order of five times, as occurred in this case, also for all the other samples. We can find explanations, because everything can be explained. The problem is that these explanations have not left traces in all the various reports, or a justification that could, in some way, explain this, which is a further very strong anomaly. I repeat, one single specimen, that has a good concentration of DNA, requires no further problems, which suddenly gives a value of one, I have a kilo of DNA, I measure it again and I get five kilos of DNA. It's something that, objectively, puzzles me a lot”. What stated by the defense and its consultant is perceived in its full extent by the simple comparison of the two tables, which are given below for convenience, the first written by RIS, the second by Dr. Lago, on the same subject - DNA concentration - and, above all, on the same test tubes. [In this summary the order of these two tables is reversed]. The significant change in concentration - indeed a multiplication - is evident. This staggering figure is unjustifiable if not with a breach of the chain of custody, meant as a sheer tampering, or outcome of a serious error. Analyzing the quantification results for the G1EXT trace and trace G20, ie the traces most representative of the presence of Unknown 1, you may see, as the first, having initially a total DNA concentration of 2500 pg/ml of DNA, with a male component of 1000 pg/ml, then grown to 12700 pg/ml total DNA and 4880 pg /ml for male DNA, or more than five times as much. Similarly, the increase involved also trace G20.

For this event there are only three possible explanations, all of them shocking: • or some material was added, hence varying the concentration and the quantity; • or there has been a substitution of tubes; • or we are facing yet another mistake of the investigators, detected by this defense, but not recognized by either the Prosecution or the ruling. The reason for this further anomaly remains - to this date - a mystery. Again, the simplest hypothesis, namely a glaring mistake, is, not without reason, not even taken into account by the Court. Otherwise, admitting the mistake, would generate a reasonable doubt about the quality of the results achieved by the RIS, with the logical need of completely redoing the genetic investigation, in addition to admitting the fallibility of consultants that one wants, instead, to look infallible. That the chain of custody has been violated is demonstrated, also, by what is already in the court documents and also stigmatized by this defense in the closing arguments. The following dates, inferable from the official papers of the investigation, patently confirm what was said. On 22.09.2011, Dr. Lago was appointed as technical consultant by the public prosecutor of Bergamo.

On 26.09.2011, Dr. Lago was given a specific task as consultant. On 28.09.2011, with the delivery of the samples [by the RIS], Dr. Lago began his investigation on the extracted DNA, hence, in this very date, he made the first and necessary investigation of the tubes that had been delivered, ie the verification of volumes and concentrations. On 25.10.2011 the RIS performed the three analyses on trace 31G20, whose electropherograms were also the object of analysis by the Civil Party. On this date, however, the tubes of the best trace [31G20] and the others entrusted to dr. Lago, had to be in possession of the Prosecution consultant (Lago) and not of the RIS. The question that this defense arises, and which was also asked in the closing arguments, is how was it possible for the RIS to carry out these analyses when the tube was in fact no longer in their availability having been, indeed, already delivered to Dr. Lago. To assert that Dr. Lago was given just a fraction of the exhibits in question, in the absence of any documentation that certifies it and in the presence of opposite written statements is an unsupported claim, moreover conflicting with court documents. If that did, however, took place, the ruling would consider as absolutely ordinary a fact which would on the contrary be quite serious and which would without a doubt devastate the reliability of the evidence. There would be, in fact, no certainty about the chain of custody of the exhibits. Each passage of hands of the test tubes must be verifiable otherwise the genetic investigations will not be valid, there being absolute uncertainty about who had what and at what time. Thank you for your patience in reading all of this lengthy document. I also hope, however that it aroused both your interest and your perplexities in the strangeness (if not uniqueness) of this case, as far as DNA evidence is concerned. Luca Cheli