Plasmodium falciparum is evolving to escape malaria rapid diagnostic tests in Ethiopia

In Africa, most rapid diagnostic tests (RDTs) for falciparum malaria recognize histidine-rich protein 2 antigen. Plasmodium falciparum parasites lacking histidine-rich protein 2 (pfhrp2) and 3 (pfhrp3) genes escape detection by these RDTs, but it is not known whether these deletions confer sufficient selective advantage to drive rapid population expansion. By studying blood samples from a cohort of 12,572 participants enroled in a prospective, cross-sectional survey along Ethiopia’s borders with Eritrea, Sudan and South Sudan using RDTs, PCR, an ultrasensitive bead-based immunoassay for antigen detection and next-generation sequencing, we estimate that histidine-rich protein 2-based RDTs would miss 9.7% (95% confidence interval 8.5–11.1) of P. falciparum malaria cases owing to pfhrp2 deletion. We applied a molecular inversion probe-targeted deep sequencing approach to identify distinct subtelomeric deletion patterns and well-established pfhrp3 deletions and to uncover recent expansion of a singular pfhrp2 deletion in all regions sampled. We propose a model in which pfhrp3 deletions have arisen independently multiple times, followed by strong positive selection for pfhrp2 deletion owing to RDT-based test-and-treatment. Existing diagnostic strategies need to be urgently reconsidered in Ethiopia, and improved surveillance for pfhrp2 deletion is needed throughout the Horn of Africa.

Molecular Inversion Probe-Based Sequencing of USH2A Exons and Splice Sites as a Cost-Effective Screening Tool in USH2 and arRP Cases

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.

Clone expansion of mutation-driven clonal hematopoiesis is associated with aging and metabolic dysfunction in individuals with obesity

Aging is associated with clonal hematopoiesis (CH) caused by somatic mutations in blood cell progenitors. Mutations with ≥2% variant allele frequency (VAF), known as clonal hematopoiesis of indeterminate potential (CHIP) mutations, have been linked to risk of hematological malignancies and cardiovascular disease. Using an ultrasensitive single-molecule molecular inversion probe (smMIP) sequencing assay, we identified clonal hematopoiesis driver mutations (CHDMs) in a set of established CH driver genes in individuals with obesity from the Swedish Obese Subjects study. In cross-sectional setting, with samples from 1050 individuals, we identified 273 candidate CHDMs in 216 individuals, with a VAF ranging from 0.01% to 31.15% and with clone sizes and prevalence increasing with age. Longitudinal analysis over 20 years in 40 individuals showed that small clones may grow over time and become CHIP. Greater speed of clone growth was associated with insulin resistance (R=0.04, P=0.025) and low circulating levels of high-density lipoprotein-cholesterol (R=-0.68, P=1.74E-05), suggesting that dysfunctional metabolism may accelerate expansion of CH.

Medulloblastoma in Adults: Cytogenetic Phenotypes Identify Prognostic Subgroups

Adult medulloblastomas (MB) are rare. We investigated the genetic landscape and prognostic impact of genetic aberrations in a cohort of 117 adult medulloblastomas. Histological features and pathway activation were evaluated at the protein level; 14.5% showed wingless-type activation, 63.3% SHH activation, and 22.2% were classified as non-WNT/non-SHH-MB. Genome-wide copy number analysis was performed by molecular inversion probe array technology. MB-related genes were sequenced in WNT- and SHH-activated MBs. 79.7% of SHH-MBs showed desmoplastic/nodular histology; all other MBs had classic histology. WNT-MBs carried oncogenic CTNNB1 mutations in 88.2% and had monosomy 6 in 52.9%. In SHH-MBs, TERT promoter mutations occurred in 97%, mutations in PTCH1 in 38.2%, SMO in 15.5%, SUFU in 7.4%, and TP53-mutations in 4.1%. In all, 84.6% of non-WNT/non-SHH-MBs had an isochromosome 17q. A whole chromosomal aberration (WCA) signature was present in 45.1% of SHH-TP53-wild type (wt)-MBs and 65.4% of non-WNT/non-SHH-MBs. In 98 cases with survival data, WNT-MBs had a 5-year overall survival (OS) of 68.6%. SHH-MBs TP53wt type and non-WNT/non-SHH-MBs showed 5-year OS of 80.4% and 70.8%, respectively. TP53-mutant SHH-MBs represented a prognostically unfavorable entity; all patients died within 5 years. Patients with a WCA signature showed significantly increased OS (p = 0.011 for SHH-TP53wt-MBs and p = 0.048 for non-WNT/non-SHH-MBs).

Multiclonal complexity of pediatric acute lymphoblastic leukemia and the prognostic relevance of subclonal mutations

Genomic studies of pediatric acute lymphoblastic leukemia (ALL) have shown remarkable heterogeneity in initial diagnosis, with multiple (sub)clones harboring lesions in relapse-associated genes. However, the clinical relevance of these subclonal alterations remains unclear. We assessed the clinical relevance and prognostic value of subclonal alterations in the relapse-associated genes IKZF1, CREBBP, KRAS, NRAS, PTPN11, TP53, NT5C2, and WHSC1 in 503 ALL cases. Using Molecular Inversion Probe sequencing and breakpoint-spanning PCR we reliably detected alterations below 1% allele frequency. We identified 660 genomic alterations in 285 diagnosis samples of which 495 (75%) were subclonal. RAS pathway mutations were common, particularly in minor subclones, and comparisons between RAS hotspot mutations revealed differences in their capacity to drive clonal expansion in ALL. We did not find an association of subclonal alterations with unfavorable outcome. Particularly for IKZF1, an established prognostic marker in ALL, all clonal but none of the subclonal alterations were preserved at relapse. We conclude that, for the genes tested, there is no basis to consider subclonal alterations detected at diagnosis for risk group stratification of ALL treatment.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda

Background In Uganda artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine (SP) for chemoprevention during pregnancy, but drug resistance may limit efficacies. Methods Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and qPCR assays. Results Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of PfCRT 76T decreased, but varied markedly between sites (0-48% in 2018; 0-22% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For PfMDR1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14/16 sites, and gene amplification was not seen. Considering mutations associated with high level SP resistance, prevalences of PfDHFR 164L (up to 80%) and PfDHPS 581G (up to 67%) were high at multiple sites. Considering PfK13 propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 40%, respectively). Conclusions We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.