Whole Locus Sequencing Identifies a Prevalent Founder Deep Intronic RPGRIP1 Pathologic Variant in the French Leber Congenital Amaurosis Cohort

Leber congenital amaurosis (LCA) encompasses the earliest and most severe retinal dystrophies and can occur as a non-syndromic or a syndromic disease. Molecular diagnosis in LCA is of particular importance in clinical decision-making and patient care since it can provide ocular and extraocular prognostics and identify patients eligible to develop gene-specific therapies. Routine high-throughput molecular testing in LCA yields 70%–80% of genetic diagnosis. In this study, we aimed to investigate the non-coding regions of one non-syndromic LCA gene, RPGRIP1, in a series of six families displaying one single disease allele after a gene-panel screening of 722 LCA families which identified 26 biallelic RPGRIP1 families. Using trio-based high-throughput whole locus sequencing (WLS) for second disease alleles, we identified a founder deep intronic mutation (NM_020366.3:c.1468-128T>G) in 3/6 families. We employed Sanger sequencing to search for the pathologic variant in unresolved LCA cases (106/722) and identified three additional families (two homozygous and one compound heterozygous with the NM_020366.3:c.930+77A>G deep intronic change). This makes the c.1468-128T>G the most frequent RPGRIP1 disease allele (8/60, 13%) in our cohort. Studying patient lymphoblasts, we show that the pathologic variant creates a donor splice-site and leads to the insertion of the pseudo-exon in the mRNA, which we were able to hamper using splice-switching antisense oligonucleotides (AONs), paving the way to therapies.

Integrative Analysis of the Mutational Landscape of Mouse and Human AML Identifies Functionally Relevant Leukemia Disease Alleles

t(8;21) is the most frequent chromosomal abnormality in acute myeloid leukemia (AML), occurring in 4-12% of adult and 12-30% of pediatric patients. This translocation fuses the N-terminus of AML1 to nearly the entire coding region of ETO, resulting in expression of the fusion protein AML1-ETO. Observations that mice expressing AML1-ETO develop AML only if treated with mutagenic agents have suggested that AML1-ETO requires cooperating disease alleles for leukemogenesis. Consistent with this, t(8;21)+ AML patients harbor multiple genetic abnormalities. Recent exome/genome sequencing studies have expanded the number of known mutations in t(8;21)+ AML patients; however, efforts to distinguish driver from passenger mutations have yielded few cooperative events and the requirements for AML1-ETO leukemogenesis remain largely unknown. To better define the genetic landscape in AML and distinguish driver from passenger mutations, we compared the mutational profiles of two specific AML1-ETO driven mouse models of leukemia to the mutational profiles of human AML patients. We found that the mouse models of AML1-ETO driven AML were phenotypically similar in terms of their extensive latency, myeloid progenitor immunophenotype, and the acquired secondary disease alleles. The first model relies upon the expression of AML1-ETO in transplanted p21 null cells, while the second model relies upon the expression of AML1-ETO9a, a splice variant of AML1-ETO, in transplanted wild type cells. p21 is neither disrupted, nor methylated in t(8;21)+ AML. Because loss of p21 prevents the repair of damaged DNA, leukemogenesis may occur in this model once a cooperating disease allele has been naturally acquired in an AML1-ETO positive hematopoietic progenitor. AML1-ETO9a itself deregulates the expression of several DNA repair genes, suggesting that AML1-ETO9a could similarly facilitate the acquisition of a cooperating disease allele. When we compared the mutational landscape of these murine leukemias to AML patients, we found that the murine leukemias enrich for disease alleles present in human AML (hypergeometric p ≤ 4.26x10-20) and that there is a significant tendency for disease alleles mutated in both species to possess mutations in the same protein domain (hypergeometric p ≤ 4.23x10-3). Furthermore, domains mutated in both species were affected by recurrent mutations (Spearman correlation of domain p-values r = 0.53, p ≤ 2.73x10-8). While the frequency with which various protein classes were affected by mutations was significantly different in MLL-AF9 and AML1-ETO/AML1-ETO9a positive murine AML compared to MLL-fusion and t(8;21)+ positive human AML (p = 0.049), the protein classes targeted in AML1-ETO/AML1-ETO9a murine AML vs. human t(8;21)+ AML were not significantly different (p = 0.327). To identify disease alleles capable of cooperating with AML1-ETO, we determined that of the 424 genes mutated in both species, 38 of those genes were significantly mutated in human AML (Genome MuSiC SMG FDR ≤ 30%). These 38 genes represented 45 mouse orthologues, 38 of which were significantly mutated in AML1-ETO driven murine leukemias (FDR ≤ 10%). These 38 orthologues corresponded to 32 human orthologues, 3 of which were annotated in COSMIC as cancer-related genes: TET2, PTPN11, and THRAP3. Using retroviral transduction and transplantation experiments, we demonstrated that the expression of AML1-ETO in transplanted Tet2 null cells or PTPN11 D61Y cells was sufficient for leukemogenesis. At euthanasia, mice exhibited leukocytosis, anemia, thrombocytopenia, splenomegaly, and an expansion in the myeloid progenitor compartment. Our identification of Tet2 loss as a cooperating allele implicates mutations in epigenetic regulators as potential driving events in t(8;21)+ AML, while the discovery of PTPN11 D61Y solidifies the role of constitutive MAPK signaling in t(8;21)+ AML. This integrative genetic profiling approach allowed us to accurately predict cooperating events in t(8;21)+ AML in a robust and unbiased manner, while also revealing functional convergence in mouse and human AML. Collectively, these findings illustrate the power of integrating murine and human genomic profiling to identify functionally relevant disease alleles in AML. Disclosures Levine: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.