Modern carrier screening requires a streamlined approach

One of the biggest changes in women’s healthcare recently has come from the field of carrier screening. Once a niche offering focused on just a few genes for women of specific ancestries, carrier screening has become far more common — and more complex — thanks to updated clinical guidelines that broadened recommendations for who should be tested. The goal of these new recommendations, improving healthcare equity across all women, is admirable. But on the technical front, the result for clinical labs can be quite a burden.

The updated carrier screening guidelines did not just add to the pool of women who should receive testing; these guidelines also added substantially to the list of genes that should be included in the screening process. There are now more than 100 genes recommended for carrier screening. That list includes a number of genes known to be difficult or even impossible to resolve with the short-read sequencing technologies that have become nearly ubiquitous in clinical laboratories.

Clinical lab teams have scrambled to answer the call for expanded carrier screening, often by implementing a variety of technologies to resolve the difficult genes. Carrier screening for a single person might involve four, five, six, or more workflows to generate reliable results about all of the recommended genes. This kind of complexity is simply not tenable for a screening process that might be appropriate for every pregnant woman and all women considering becoming pregnant. Long-read sequencing technologies offer a more practical approach, allowing lab staff to implement two straightforward workflows — one for the difficult genes, and one for everything else. This technique can simplify carrier screening and lower the barrier to adding carrier screening as a routine capability in clinical labs.

The evolution of carrier screening

The early days of carrier screening took place 50 years ago, beginning with ancestry-based guidelines, such as testing for Tay-Sachs disease in people of Ashkenazi Jewish descent. Over time, these recommendations turned into a patchwork that has been difficult for physicians to keep up with. The approach also suffered from limitations in how much patients know about their own genetic ancestry; people who should have gotten screening based on guidelines could easily be missed with the reliance on self-reporting. For a long time, the only genetic conditions for which carrier screening was advised regardless of ancestry were spinal muscular atrophy and cystic fibrosis.

In 2021, the American College of Medical Genetics and Genomics (ACMG) set out to improve equity with a new, universal set of recommendations for carrier screening. The updated ACMG guidelines standardized the list of genes that should be screened — a total of 113 genes, including 16 X-linked genes and 97 autosomal recessive genes — and recommended the same level of screening for almost all women.¹ The guidelines also allow for screening additional genes based on the needs of specific populations or families.

For most clinical labs, the leap from the handful of genes previously included in standard carrier screens to a panel of 113 genes is enormous. But it’s also important. The previous approach to carrier screening allowed too many women to fall through the cracks and failed to test for genetic conditions relevant to each patient. The expanded ACMG gene list represents an effort to bring standardization to carrier screening and to ensure a base level of care for all women who need this valuable test.

Intractable genes

Unfortunately, being important does not translate to being easy. In its report presenting the new guidelines, the ACMG acknowledged that many of the genes included in the recommendations would be burdensome for clinical labs. “Another challenge is for the molecular testing laboratories to adapt new testing strategies since some of the ACMG Tier 3 genes may harbor variants that are not routinely detected by NGS only,” the authors wrote.¹

While most of the 113 genes can be resolved easily enough with short-read tools, about a dozen genes defy characterization. Some examples include the CGG repeat expansion in FMR1 that’s responsible for fragile X syndrome; F8, which can have large inversions in an intron that lead to severe hemophilia A; the GBA gene associated with Gaucher disease, which has a nearly identical pseudogene in GBAP1; and CFTR for cystic fibrosis, a gene that harbors large exon deletions or duplications.

From structural variants that are too large and complex for standard NGS platforms to pseudogenes with high homology to genes targeted by carrier screening, the group of challenging genes requires lab staff to get creative. They usually end up with a number of different technologies: qPCR, long-range PCR, PCR/CE, and Sanger sequencing are all common. Even multiplex ligation-dependent probe amplification, a tedious and cumbersome technique, is often implemented to help with some of these troublesome genes.

Streamline with long-read sequencing

With so many niche technologies deployed to address each gene’s specific challenges, carrier screening can be far too complex for most clinical labs to offer. But long-read sequencing offers a solution to this problem.

While the dozen or so intractable genes are intractable for different reasons, all of the technical issues they present can be overcome with long-read sequencing data. Highly homologous sequence, GC-rich repeat regions, large structural elements — all of these suddenly become tractable simply by having reads long enough to fully span the difficult region with enough flanking sequence to allow for proper alignment. There are a few long-read sequencing technologies available, with read lengths ranging from kilobases to hundreds of kilobases. By pairing these long reads with sufficient depth of coverage, it is now feasible to resolve even the most difficult genes in the human genome.

In addition, long-read sequencing data allows users to phase variants. As a result, clinical lab teams can read out maternal and paternal alleles, with the ability to assign mutations to the correct allele for situations where genome interpretation may differ when two mutations in a gene are on the same allele or on different alleles.

While long-read sequencing can be used for all of the ACMG-recommended genes, it is likely more cost-effective to continue sequencing the easy genes on short-read platforms. Consolidating all of the difficult genes onto a single long-read platform streamlines the carrier screening process, allowing labs to implement just two reliable workflows for the full set of genes.

Incorporating a new long-read sequencing platform may seem impossible for clinical labs with scarce resources. While some platforms require high capital expenditures up front, there are options that don’t. Nanopore sequencing, for example, does not have the same cost structure as other sequencing platforms, so it may be a more affordable option.

Moving forward

ACMG’s updated carrier screening guidelines represent much-needed progress in improving health equity and standardizing an important healthcare tool — but they also dramatically increased the complexity of this screening process for clinical lab teams. Being able to offer carrier screening for all pregnant women, plus all women who plan to become pregnant, means that labs must have robust and streamlined workflows capable of resolving all 113 recommended genes. Current approaches using many different technologies to process the most challenging genes require too much hands-on time and involve more complexity than is desirable for such a frequently ordered test.

A better approach reduces that complexity to a simple dual workflow: standard short-read sequencing for the hundred or so easy genes, plus a single long-read sequencing assay covering all of the tough genes. This model minimizes manual intervention and delivers results more reliably than the patchwork method used by many labs today. For a healthcare need as important as carrier screening, that’s a truly meaningful improvement.

Reference

1. Gregg AR, Aarabi M, Klugman S, Leach NT, et al. Screening for autosomal recessive and X-linked conditions during pregnancy and preconception: a practice resource of the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2021 Oct;23(10):1793-1806. doi: 10.1038/s41436-021-01203-z.