Editors: Donna E. Hansel, MD, PhD, division head of pathology and laboratory medicine, MD Anderson Cancer Center, Houston; James Solomon, MD, PhD, assistant professor, Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York; Erica Reinig, MD, assistant professor and medical director of molecular diagnostics, University of Wisconsin-Madison; Marcela Riveros Angel, MD, molecular genetic pathology fellow, Department of Pathology, Oregon Health and Science University, Portland; Maedeh Mohebnasab, MD, assistant professor of pathology, University of Pittsburgh; Alicia Dillard, MD, associate clinical laboratory director, Omniseq/Labcorp, Buffalo, NY; and Richard Wong, MD, PhD, assistant professor of pathology, University of California San Diego.
Employing long-read sequencing in testing for Prader-Willi and Angelman syndromes
March 2025—Genomic imprinting is an epigenetic process that results in expression of only one copy of a gene—either from a person’s mother or father—while the other parent’s copy of the same gene is silenced. A cluster of genes affected by imprinting on the proximal part of the long arm of chromosome 15 (15q11-q13) are associated with syndromic conditions. Deficient expression of the maternally inherited copy of UBE3A in this region results in Angelman syndrome, which is characterized by severe developmental delay, gait ataxia, and an apparent happy demeanor with profuse smiling, frequent laughing, and excitability. In contrast, deficient expression of paternally inherited genes in this region causes Prader-Willi syndrome. Clinical features of this condition can vary but often include developmental delay, short stature, hyperphagia, and obesity. Four underlying genetic mechanisms primarily cause the deficient expression that results in these disorders. Most commonly, a copy number deletion of four to seven megabases occurs, as this region of chromosome 15 is particularly susceptible to microdeletions. Alternatively, uniparental disomy, in which both copies of chromosomes are inherited from one parent, or imprinting defects that result in aberrant expression can occur. Point mutations or small insertions or deletions too can affect relevant genes. In a minority of cases, the underlying genetic mechanism remains unknown. Because of the diverse underlying genetic causes of Angelman and Prader-Willi syndromes, clinical evaluation often requires multiple sequential assays. Some modern methods, such as methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA), can be used to evaluate methylation status and copy number changes. However, MS-MLPA still requires follow-up testing to distinguish uniparental disomy from imprinting defects. Additionally, if the testing reveals no abnormalities, sequencing is required to look for single-gene pathogenic alterations. Moreover, sequencing studies often require sequencing the parents because comparing the sequencing results with maternal and paternal sequences is necessary to phase any de novo alterations. Given the limitations of current standard-of-care methods, the authors evaluated whether whole genome long-read sequencing on an Oxford Nanopore platform could be used as a single comprehensive test for both syndromes. The platform sequences DNA fragments that are kilobases in length and allows for evaluation of single nucleotide variants, insertions and deletions, copy number alterations, and structural variants, along with DNA modifications, such as methylation. The authors established a pipeline that generated a single-page summary of the data from the chromosome 15q11-q13 region that included information about read depth, zygosity of single nucleotide polymorphisms, methylation status, and sequence alterations. To evaluate the pipeline, 22 patient samples—from 20 patients diagnosed with Angelman or Prader-Willi syndrome and two controls who did not have either syndrome—were sequenced. Three analysts blindly reviewed the data and determined the correct molecular diagnosis in all cases. Overall, the study demonstrates the utility of long-read sequencing as a comprehensive clinical assay for evaluating imprinting disorders.
Paschal CR, Zalusky MPG, Beck AE, et al. Concordance of whole-genome long-read sequencing with standard clinical testing for Prader-Willi and Angelman syndromes. J Mol Diagn. doi.org/10.1016/j.jmoldx.2024.12.003
Correspondence: Dr. Danny E. Miller at dm1@uw.edu
Use of KRAS mutant dosage to prognosticate overall survival in pancreatic cancer
With a 13 percent five-year survival rate for all stages combined, pancreatic cancer is the third leading cause of cancer deaths in the United States after lung and colon cancer. More than 90 percent of pancreatic cancers are associated with an activating mutation in KRAS that results in constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. As RAS-targeted therapies progress through clinical trials, understanding the biology of KRAS-mutated pancreatic cancer is crucial. The authors conducted a study in which they leveraged the germline and somatic profiling of 2,336 patients with pancreatic adenocarcinoma (PDAC) to analyze the genomic and clinical correlates of disease outcomes. They found that 95 percent of the tumors harbored oncogenic alterations in KRAS, while three percent had other MAPK driver alterations, and the remaining two percent were wild type for MAPK pathway genes. Patients with KRAS-mutated pancreatic carcinoma had the worst overall survival. Many of the other MAPK mutations, including in NTRK, ERBB2, and BRAF, are targetable, which contributes to improved survival, and the survival benefit remains for wild-type tumors, even when excluding patients who received targeted therapies. Some KRAS mutant tumors have copy number allelic imbalance, which can occur through such molecular mechanisms as loss of the wild-type allele or copy number gain of the mutant allele. The authors used the bioinformatics tool FACETS to determine how many integer copies of each allele were present. They found that 42 percent of tumors had allelic imbalance, including focal or arm-level amplifications, shallow broad gains, loss of heterozygosity, or losses after whole genome duplication. Ninety-three percent of all imbalance events in this cohort involved preferentially gaining or retaining the KRAS mutant allele. Furthermore, tumors with a copy number gain of the mutant allele were associated with significantly worse overall survival than tumors with only one mutant allele, and mutant allele dosage gains were found to be more common in metastatic tumors. The p.G12D KRAS mutation was the most common alteration, seen in 41 percent of tumors, followed by p.G12V (32 percent) and p.G12R (16 percent). Interestingly, patients with p.G12R-mutated tumors had better survival outcomes than those with p.G12D tumors. The study findings demonstrate that KRAS mutant dosage impacts prognosis, but its effect on antitumor response and therapeutic outcomes is unclear. As RAS-targeted therapies progress through clinical trials, KRAS mutant dosage may be found to affect treatment efficacy, and stratifying patients based on KRAS mutant dosage may need to be considered. Currently, however, many comprehensive genomic profiling assays used clinically are unable to analyze allele-specific copy number. If this metric becomes an important biomarker, molecular pathologists may have to consider incorporating this analysis into assays when evaluating pancreatic cancer.
Varghese AM, Perry MA, Chou JF, et al. Clinicogenomic landscape of pancreatic adenocarcinoma identifies KRAS mutant dosage as prognostic of overall survival. Nat Med. 2025. doi.org/10.1038/S41591-024-03362-3
Correspondence: Dr. Chaitanya Bandlamudi at bandlamc@mskcc.org or Dr. Eileen M. O’Reilly at oreillye@mskcc.org