Forensic scientist Mitchell Holland of Pennsylvania State University (Penn State) has spent much of his career using DNA to solve identity mysteries, from royal family members to unknown soldiers and victims of mass disasters. However, there is one case that particularly stands out in his memory. In the early 2010s, Holland was called upon to help identify a nun being considered for sainthood by the Pope. Finding and preserving her remains was crucial for her sainthood. To aid in this process, an international team of forensic scientists came together, marking one of the first times DNA evidence played a key role in this sacred process.1
Sister Marija Krucifiksa Kozulić was a Croatian nun who dedicated her life to caring for and educating disadvantaged, abandoned, and orphaned children. She was part of the community of the Society of Sisters of the Sacred Heart of Jesus. In the 1890s, she established the Catholic order of the Daughters of the Sacred Heart of Jesus in Rijeka, Croatia. She remained a tireless advocate for those in need, until her passing in 1922 from a stroke.
Her legacy of compassion and service did not go unnoticed: When Pope John Paul II visited Rijeka in 2003, the process of sainthood was initiated. Sister Dobroslava Mlakić, a member of the Society of Sisters of the Sacred Heart of Jesus, spearheaded efforts to advance this cause. The first step was beatification, a papal declaration that a person is “Blessed,” after a confirmed miracle.
“In order to be considered for sainthood, you have to have two documented miracles that you’re associated with,” said Holland. For example, if someone gravely ill prayed to Sister Marija and experienced instantaneous healing, it must be scientifically verified. Once all the documentation is submitted, the miracles are investigated by a Vatican committee in Rome. The details of the miracles are not shared until the process is officially concluded. “And you also have to identify the remains of the individual,” added Holland.
However, identifying the location of Sister Marija’s grave was no easy task. Sister Dobroslava painstakingly pieced together historical evidence and the necessary permissions to locate the grave. At last, Sister Marija’s purported remains were exhumed in 2011 and DNA testing commenced.
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Holland and his American colleagues joined forces with the Croatian team. Together, this identification effort was led by geneticist and forensic scientist Dragan Primorac and his colleagues from the University of Split, including forensic anthropologist Željana Bašić. According to historical records, Sister Marija and her biological sister Tereza had been buried in a tomb belonging to the Society of Sisters of the Sacred Heart of Jesus—alongside some 35 others in tin and wooden coffins.
The remains in the coffins were identified through archaeological and forensic anthropological analysis by the Croatian team, but Sisters Marija and Tereza were not accounted for in the coffins. However, additional skeletal remains, likely reorganized to make space, had been placed in plastic bags, accelerating their degradation in the tomb’s humid environment. The researchers believed Sister Marija might be among these bags containing multiple remains.
“The remains were co-mingled,” remarked Holland, which made the project much more complicated. “They took cuttings of the bone and sent them to us, here at Penn State.”
Image of Sister Marija Krucifiksa Kozulić.
Sister Marija Krucifiksa Kozulić dedicated her life to serving others, tirelessly advocating for those in need through both her spirituality and actions.
Fraxinus Croat, CC BY-SA 3.0
Scientists at Penn State and the Armed Forces Medical Examiner System performed mitochondrial DNA (mtDNA) analysis on femoral and humeral bone samples to determine which remains belonged to Sister Marija. They searched for matching haplotypes to confirm maternal relatives, as Sisters Marija and Tereza were the only known relatives buried in the tomb.
Among 14 samples, they identified six unique mitochondrial genome haplotypes which are specific regions of mtDNA that are inherited together, suggesting at least six individuals. Then, the team assessed haplogroups, which are clusters of similar haplotypes that share a common ancestor. They found that the six profiles were distributed across five haplogroups (H1a, H1bu, H1e1b, V, and K1a5a).
Notably, two individuals in haplogroup H1bu exhibited similar but not identical profiles, both sharing a point heteroplasmy—where multiple copies of mitochondrial DNA (mtDNA) differ at a specific nucleotide position. This trait is common among close maternal relatives. “This suggests that those could be the bones of the two sisters,” said Holland.
Although the remains were confirmed to be sisters, were they the Kozulić sisters? To confirm, the researchers compared the bone samples to a cheek swab from their only living relative, Fides Kozulić, their paternal niece. They used autosomal short tandem repeat (STR) and single nucleotide polymorphism (SNP) sequencing to analyze the DNA of all three individuals. Due to the degraded nature of the sisters’ samples, they were only able to obtain partial STR and SNP profiles, but fortunately, a kinship analysis was still possible. This step was crucial, as no direct DNA references from Sister Marija such as hair from a brush or personal clothing were available.
For the kinship analysis, the team made two hypothetical family trees using the case findings and sequencing data. Their probability calculations confirmed that the DNA profiles linked the skeletal remains to Kozulić, confirming them as Sisters Marija and Tereza.
This painstaking research took nine years, as the team worked with highly degraded, porous bones. Even though researchers were ultimately unable to determine which sister was Marija and which was Tereza, this breakthrough still allowed the church to proceed with Sister Marija’s beatification. In 2020, both sisters were officially laid to rest together. While the beatification process is still ongoing, these efforts not only honored Sister Marija’s legacy but also highlighted the intersection of science and religion.
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