Historically, carrier screening involved a patchwork of approaches. Testing guidance varied between populations — largely based on self-reported ethnicity — with a confusing array of rules specific to each group, and analysis involved testing different sets of genes with different technologies, creating a substantial burden for clinical and reference laboratories. These inconsistencies created laboratory challenges and inequitable healthcare for prospective or expecting parents.
Clearly, consistency is needed. To unify guidelines in the interest of health equity, the American College of Medical Genetics and Genomics (ACMG) published new carrier screening recommendations in 2021, abandoning ethnicity-specific guidelines for a uniform approach to carrier screening across all populations. The updated guidelines call for screening 113 genes — 97 autosomal recessive genes plus 16 X-linked genes — in all women expecting or considering childbirth. Results from that screening would inform the need for subsequent carrier screening in partners.
However, ACMG’s guidance to screen far more genes than were on previous carrier panels has increased the complexity for laboratories. The greatest difficulty comes from nine of the 113 genes: SMN1, SMN2, FMR1, HBA1, HBA2, F8, GBA, CYP21A2, and TNXB. Though they represent a small fraction of the total screening set, these genes contain a large proportion of pathogenic variants or are so challenging to resolve that they take more time and resources to analyze than all the others combined.
That’s because the genetic variation underlying certain diseases and disorders is impossible to characterize using standard short-read sequencing technology. With reads of only about 300 bases, it is difficult to accurately align and assemble sequence data. Repeat expansions collapse upon themselves, making sizing difficult; a gene and its pseudogene appear to be one element, making copy number resolution difficult; large structural variants extend well beyond the limits of these short reads, making them tough to identify reliably; and certain critical variants exist in GC-rich sequence regions that are particularly difficult to enrich due to challenges with amplification. Six of the 10 most common neonatal genetic conditions are caused by complex variants in these tough-to-resolve genes.
These challenges leave clinical laboratory teams turning to a host of technologies to resolve these intractable genes. Approaches might include qPCR, long-range PCR with Sanger sequencing, multiplex ligation-dependent probe amplification, and PCR with capillary electrophoresis, among others. Having to implement and validate each method, train staff members, and stock appropriate reagents makes carrier screening far more burdensome than most labs can justify to support multiple single gene tests.
Resolving Challenging Genes With Long Reads
Ideally, carrier screening could be carried out in just two streamlined, complementary workflows: a short-read workflow for most genes, and a unified workflow that would handle the most challenging genes with a single technology platform.
Long-read sequencing — specifically, long-read nanopore sequencing — fits the bill. Long-read sequencing provides enough context that even closely related genes and pseudogenes can be distinguished from one another, and variants can be accurately phased into alleles.
While there are various long-read sequencing platforms available today, nanopore sequencing offers the unique benefit of a small lab footprint without requiring a large capital expenditure to implement, as well as accurate sequencing data for carrier screening — significant advantages that make nanopore sequencing a more practical fit for most clinical labs.
Recently, scientists at Asuragen, a Bio-Techne brand, used nanopore sequencing as the foundation for a new kit covering the nine genes that are challenging to resolve with short-read sequencing plus two additional genes that should be included in carrier screening. The research-use-only AmplideX Nanopore Carrier Plus Kit*† was designed to give laboratories a streamlined workflow that can reduce testing complexity and replace the previous patchwork approach that required several different technologies. With this kit, all 11 genes can be analyzed from a single run leveraging short- and long-range PCR enrichment, nanopore sequencing, and automated analysis. These genes represent about 70 percent of all pathogenic variants associated with hereditary diseases that affect neonates. The remaining genes recommended by ACMG can be analyzed in a single assay with short-read sequencing.
Taken together, these two streamlined workflows make it feasible and cost-effective for clinical laboratories to bring carrier screening capabilities in-house rather than continuing with slow, expensive send-out testing.
Evaluation of the AmplideX Nanopore Carrier Plus Kit
Prior to commercial launch, the AmplideX Nanopore Carrier Plus Kit was run on thousands of unique samples to assure robust and reliable performance. In a validation study conducted with Anne-Sophie Lebre at the Centre Hospitalier Universitaire de Reims (CHU Reims) with samples provided by several other clinical sites in France, researchers assessed the performance of the kit.
The study included 155 residual clinical DNA samples, most of which had previously been genotyped. Target regions were enriched with four PCR mixes and then barcoded, pooled, and sequenced on MinION flow cells at CHU Reims. Analysis was performed using custom-built bioinformatics pipelines designed to automate calls for structural variants, gain or loss of exons or whole genes, and gene-pseudogene fusions. Single-nucleotide variants, insertions, and deletions were identified with Clair3 and Sniffles2, respectively.
Results of samples that passed quality control metrics were compared to existing genotype data or were considered wild type if no comparator data was available and no variants were observed. For example, analysis of the FMR1 gene revealed that the kit delivered 100 percent concordance with CGG repeat sizing performed by PCR and capillary electrophoresis.
The study demonstrated that this approach has the potential to greatly reduce variant detection complexity, reflex testing, and turnaround times (two days from sample to answer) compared to conventional workflows. Accompanying software simplifies data navigation, provides QC metrics, provides variant call information, and allows in-depth investigation of sequencing data and analysis results.
Conclusion
The AmplideX Nanopore Carrier Plus Kit paired with nanopore sequencing supports a robust, practical workflow that deals with all of the typically intractable genes recommended for carrier screening research. By implementing this kit alongside a standard short-read sequencing workflow for the remaining genes, clinical labs can achieve rapid and cost-effective carrier screening clinical research that complies with ACMG guidelines. In the future, decentralizing carrier screening across clinical laboratories will make it easier for more people to get tested and should contribute to more equitable screening for all prospective and/or expecting parents.
Intractable Genes
Of the 113 genes recommended for carrier screening by ACMG, the AmplideX Nanopore Carrier Plus Kit covers 11. Nine of these contain variants or sequence characteristics that make them difficult to resolve using short-read sequencing.
Here they are, with descriptions of what makes them so challenging.
CYP21A2: This gene may contain pathogenic variants responsible for congenital adrenal hyperplasia. It is difficult to genotype thanks to a similar pseudogene, chimeras, and copy number variation.
F8: Large inversions in two introns (1 and 22) of F8 are responsible for nearly half of all cases of severe hemophilia A.
FMR1: This gene contains a repeat expansion that causes fragile X syndrome. In addition to accurately quantifying the number of trinucleotide repeats, possibly stretching into the hundreds, laboratories also must detect any AGG interruptions in the region to determine risk of expansion.
GBA: The gene linked to Gaucher disease, GBA is virtually identical to the GBAP1 pseudogene. Complex and recombinant alleles must be characterized accurately to ensure correct answers for carrier screening
HBA1, HBA2: These genes must be scanned to detect risk for alpha thalassemia, but they contain both small variants (single point mutations) and large variants (copy number variation, exon deletions, and structural variants) that must all be identified reliably.
SMN1 and SMN2: Variants in the SMN1 gene can cause spinal muscular atrophy. Resolving copy number of both genes, fully phasing variants to alleles, and differentiating SMN1 gene content from its pseudogene SMN2, which shares more than 99 percent sequence similarity, pose challenges.
TNXB: This gene may contain pathogenic variants responsible for Ehlers-Danlos Syndrome and is difficult to genotype due to a similar pseudogene, chimeras, and copy number variation.
*For Research Use Only. Not for use in diagnostic procedures.
†This product is under development; performance characteristics and final product features to be determined.
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