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Putting Expanded Carrier Screening Into Practice Requires Considerations to Avoid Pitfalls

NEW YORK – Driven by recent advances in genome technologies, expanded carrier screening, defined as testing for more than 20 hereditary disorders across ancestral groups, has become increasingly popular.

But laboratories offering such testing must be aware of still-controversial areas and potential pitfalls, according to Linda Hasadsri, co-director of the genomics laboratory and an assistant professor in the Department of Laboratory Medicine and Pathology at the Mayo Clinic.

During a workshop sponsored by Thermo Fisher Scientific at the Association of Molecular Pathology 2020 virtual annual meeting this week, Hasadsri said that professional organizations such as the American College of Obstetricians and Gynecologists and the American College of Medical Genetics and Genomics have for decades recommended carrier screening in certain populations, such as Ashkenazi Jews, focusing on genetic disorders they are at high risk for.

More recently, though, societies have acknowledged the benefits of expanded carrier screening across populations and ethnicities, she said, and new technologies have made it both convenient and economical to test for carriers of large numbers of mostly autosomal recessive disorders. ECS is especially beneficial to those who are unsure what population they belong to because they were adopted or are of mixed ancestry, she said. At the Mayo Clinic, interest among Ob/Gyns in ordering ECS has been growing, she said, and patients who in the past were not especially interested in cystic fibrosis carrier screening, for example, are much more keen on ECS.

The Mayo Clinic, where carrier screening makes up the bulk of germline genetic testing performed by the genomics laboratory, currently provides ECS for more than 200 genes that were carefully vetted for meeting guidelines and recommendation, she said.

While the inclusion of severe childhood disorders that were previously tested for was not an issue, "there is still quite a bit of controversy and debate over whether one should include disorders with variable expressivity or incomplete penetrance, as well as adult-onset disorders," Hasadsri said. An example is hereditary hemochromatosis, she said, which can lead to iron overload and severe liver disease. However, only about 40 percent of people homozygous for the most common mutation in Northern Europeans actually develop serious iron overload, and it "can be really tricky to counsel expecting parents on diseases such as this," she said.

Similarly, there is debate over whether to include adult-onset diseases or cancer predisposition syndromes, which the American Academy of Pediatrics advises against testing for in asymptomatic children, she said. As a result, Mayo Clinic, for the time being, has decided not to include such disorders in its ECS.

On the other hand, childhood disorders that are treatable, whether or not they are severe, might be considered for inclusion in ECS, "especially those where early diagnosis is going to make a difference or the condition is not one that's detected by newborn screening," Hasadsri said. As an example, she cited biotin thiamine responsive basal ganglia disease, a rare disorder that occurs almost exclusively in individuals of Middle Eastern background and can be treated with two vitamins. It is currently not included in newborn screening in the US and "early intervention is going to make all the difference in the world for that patient," Hasadsri said, noting that Mayo Clinic has seen a number of patients with this disorder.

What is important to include in the ECS report is the patient's residual post-test risk of still being a carrier for a disorder even if the result was negative. This can be calculated based on the carrier frequency in the population the patient belongs to and the specific disease-causing mutations considered by the test, she explained, but the calculation can be tricky because the carrier frequency in a certain location is not always known or it is unclear what population a patients belongs to.

Another controversial issue around the ECS report is whether variants of unknown significance should be included, or whether labs should update reports if a VUS gets reclassified as a likely pathogenic variant. This is only an issue with sequencing-based tests, Hasadsri noted, not with assays that only test for known pathogenic variants. Generally speaking, she said, most VUSs later get classified as benign, which is why many labs do not include them in their reports. However, they should clearly state this, and should also note whether they will inform patients if a classification of an initially unreported VUS changes to likely pathogenic.

Hasadsri mentioned that one feature Mayo Clinic doctors have liked about external labs providing ECS is that they issue combined reports that contain results for both the pregnant woman and her partner, which allows them to determine whether their unborn child is at risk of developing one of the diseases tested for. However, such combined reports are problematic, she said, because in the future, the woman may want to have children with a different partner, yet the genetic information of her previous partner will remain on her carrier screening report, which might violate regulations about protected health information.

During the first year that Mayo Clinic providers have ordered ECS — from multiple labs — they found that a lot more carriers than expected were detected. Among the 106 patients tested, Hasadsri said, almost 30 percent were carriers of at least one disorder, and about 10 percent of more than one condition.

In fact, one patient, who carried two mutations in the ALDOB gene, turned out to be affected by one of the disorders — hereditary fructose intolerance, which can be lethal in children — that had previously been undetected. Since childhood, she had avoided food containing fructose because it had always made her sick.

Detecting patients who are not only carriers but actually have a disorder is not that uncommon, Hasadsri noted. Among the 20,000 or so cystic fibrosis carrier screening tests Mayo performed last year, for example, there were three patients who turned out to have atypical cystic fibrosis themselves.

Another example is alpha thalassemia. Mayo Clinic runs more than 4,000 carrier tests each year for copy number variants associated with that disorder. That test uses a control probe for copy number of the X-chromosome and incidentally discovers approximately five patients each year that have a sex chromosomal abnormality. "These types of incidental findings have ramifications for the parents and not just the pregnancy and are something to bear in mind when offering ECS," Hasadsri said.

In addition, ECS performed on a couple may reveal that their unborn child has a different father. Among the three CF-affected patients, for example, was a man who had bilateral absence of the vas deferens, which is caused by the mutations in the CFTR gene, so he was infertile and could not be the father in his partner's pregnancy.

Finally, labs offering ECS need to bear in mind the limitations of the test technologies they use. As an example, Hasadsri mentioned the case of a pregnant patient who had tested positive for Gaucher disease on a carrier screening test performed by an outside lab that suggested she has the disease because she carried two mutations.

When the Mayo lab conducted its own test to see whether the two mutations were affecting only one or both copy of the gene, called GBA, they found neither of them. "The provider was threatening litigation over whichever lab got their result wrong," Hasadsri recalled. As it turned out, neither lab had gotten it completely right or wrong. The patient turned out to have one normal copy of GBA and one mutant copy that had undergone recombination with a nearby pseudogene. The Mayo test had not found that mutant allele because the long-range PCR it performed prior to sequencing the gene did not amplify it, leading to allelic dropout. The patient, in fact, was a heterozygous carrier of the disease. The example illustrates that "pseudogene interference and complex gene-pseudogene rearrangements are important to consider in ECS design" alongside the choice of technologies used, Hasadsri said.