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Determined science

In 2003, Kathleen Folbigg was convicted of killing her four young children, who died suddenly and unexpectedly in their sleep. But several forensic pathologists raised concerns over medical evidence provided at the trial, and in March 2019, a judicial inquiry into Folbigg’s convictions commenced.

According to the medical records, the four children suffered from a series of conditions before they died. The first boy (Caleb) had difficulties breathing since birth. The second boy (Patrick) suffered epileptic seizures. The two girls (Sarah and Laura) had respiratory infections only a few days before their deaths.

These conditions suggested that if there was a single underlying natural cause it was likely to be genetic, with a broad spectrum of manifestations and lethal triggers.

So a multidisciplinary, international team of scientists embarked on a study to find the culprit. The team hypothesised that rare inherited genetic variants could be responsible for the cardiac or respiratory disorders behind the children’s sudden deaths.

The team began by sequencing Kathleen Folbigg’s whole genome.

“Given that it was much more complicated to extract the genome of the children, there was a chance that Kathleen herself might be carrying one of these variants, because they tend to be inherited,” says Professor Carola Vinuesa, from the Australian National University.

It’s not uncommon for some variants that cause sudden death in children to remain silent in some individuals – some can carry the mutation but grow up as a healthy adult. So Folbigg could have been carrying these mutations and passed them on her children. “It was a bit of a long shot,” says Vinuesa – but the team decided to sequence Folbigg’s genome.

They used programs to identify every single base of the genome and pick up every single variant. They discovered that Folbigg carries genetic mutations on CALM2, one of the three CALM genes (CALM1CALM2CALM3).

These three genes all make an identical protein, called calmodulin, responsible for sensing changes in calcium ion concentration and regulating the flow of calcium ions in and out of heart cells. This is essential to maintain a stable and healthy heart rhythm. Research has shown that mutations in any one of the three CALM genes might lead to the production of malfunctioning calmodulin proteins, causing a rare, life-threatening heart disorder called calmodulinopathy, which predominantly affects young children.

The scientists then extracted the genomes of three of the children and the exome – the genetic information that makes proteins – of one of the girls from either frozen samples, immortalised cells or neonatal heel-prick screening cards of the deceased children. They again sequenced every gene in the genome to try and identify any ultra-rare mutations that could cause lethal heart conditions, epilepsy, or sudden unexplained death.

They found a series of variants, “but the ones that looked more interesting to us was the initial calmodulin variant,” says Vinuesa – the same as Folbigg carries.

Like their mother, the two girls carried a mutation in CALM2 called G114R, never seen before. This mutation was very similar to a mutation that only in June last year was linked to lethal cardiac arrhythmias in children. CALM3 G114W had been found in two American siblings, a girl who died suddenly at age 5 and her brother, who suffered a cardiac arrest at age 4.

To prove that CALM2 G114R could also cause heart disorders, a team of calmodulin experts led by Professor Michael Toft Overgaard, from Denmark, measured the affinity between calcium ions and the calmodulin protein encoded in the mutant gene.

They expressed the protein by delivering the genetic code into Escherichia coli cells. Once the cells had produced the calmodulin, they extracted and purified the protein. Then, they measured the affinity and the kinetics of calmodulin binding to calcium ions. They discovered that, just like G114W, the G114R mutation found in Folbigg and the girls impairs calmodulin’s ability to bind to calcium ions.

The deleterious effect of a malfunctioning calmodulin is an improper heartbeat – irregular, too fast or too slow – that can cause sudden cardiac death in children.

What about the two boys?

“I kept re-analysing the genomes after the inquiry,” says Vinuesa, “and at some point I found that the two boys shared two variants in the same gene that we and the other genetic experts had missed.”

The two boys had two rare mutations in a gene called BSN. Vinuesa and her colleagues struggled to find it because it had never been described before as a cause of human disease. But a study in mice showed that when BSN is deleted, the animals suffer fatal seizures at a very young age.

“These mutations are exceptionally interesting because one of them is novel and it’s a case of compound heterozygosity,” says Vinuesa, which means that the mother only had one copy of this rare mutation and the two boys must have also inherited one copy from the father in order to have two mutant variants.

“We still do not know if these particular BSN variants can cause disease, but we are looking into it right now,” says Vinuesa.

How likely is all this to happen in one family?

Each boy had a 1 in 4 chance to get two mutant copies of BSN from their parents, which means that there’s a 1 in 16 chance that two children inherited the mutation.

The girls each had a 1 in 2 chance to inherit the CALM2 mutation from their mother, so there’s a 1 in 4 chance that both girls inherited the mutation.

The possibility that all children inherited a mutation that eventually caused their death is 1 chance in 64.

“If you do not take genetics into account, it would seem an exceptionally rare scenario to have four natural deaths in a family,” says Vinuesa. “Actually it isn’t.”

The are multiple reports in the literature where members of the same family have rare diseases that present with similar clinical manifestations, but are caused by different genetic mutations. An example is breast cancer: “There can be two sisters with breast cancer, you might think they have the same type of cancer, but one has a mutation in BRCA1 and the other has a mutation in BRCA2, two completely different genes, in two different parts of the genome, in two different chromosomes,” Vinuesa says.

“This is just a really terrible set of circumstances,” says Melanie Bahlo, a statistical geneticist and bioinformatician at The Walter and Eliza Hall Institute, in Parkville, Victoria. “But these happen from time to time.”

Kathleen Folbigg was convicted in 2003 on the basis of notes she had written in her diary and the so-called Meadow’s law, named for British paediatrician Roy Meadow.

According to Meadow’s now discredited precept, one sudden infant death is a tragedy, two is suspicious and three must be treated as murder until proved otherwise.

“Well, genetically, we know now that it can happen,” says Bahlo.