CpG Island Hypermethylation Mediated by DNMT3A Is a Consequence of AML Progression.

DNMT3A mutations occur in ∼25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3AR882H, has dominant negative activity that reduces DNA methylation activity by ∼80% in vitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patients with or without DNMT3AR882 mutations. Non-leukemic hematopoietic cells with DNMT3AR882H displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing and was essentially absent in AMLs with DNMT3AR882 mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Our findings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3AR882, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression.

The origin and evolution of mutations in acute myeloid leukemia.

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

Role of TP53 mutations in the origin and evolution of therapy-related acute myeloid leukaemia.

Therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. There are several features that distinguish t-AML from de novo AML, including a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, complex cytogenetics and a reduced response to chemotherapy. However, it is not clear how prior exposure to cytotoxic therapy influences leukaemogenesis. In particular, the mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unknown. Here, by sequencing the genomes of 22 patients with t-AML, we show that the total number of somatic single-nucleotide variants and the percentage of chemotherapy-related transversions are similar in t-AML and de novo AML, indicating that previous chemotherapy does not induce genome-wide DNA damage. We identified four cases of t-AML/t-MDS in which the exact TP53 mutation found at diagnosis was also present at low frequencies (0.003-0.7%) in mobilized blood leukocytes or bone marrow 3-6 years before the development of t-AML/t-MDS, including two cases in which the relevant TP53 mutation was detected before any chemotherapy. Moreover, functional TP53 mutations were identified in small populations of peripheral blood cells of healthy chemotherapy-naive elderly individuals. Finally, in mouse bone marrow chimaeras containing both wild-type and Tp53(+/-) haematopoietic stem/progenitor cells (HSPCs), the Tp53(+/-) HSPCs preferentially expanded after exposure to chemotherapy. These data suggest that cytotoxic therapy does not directly induce TP53 mutations. Rather, they support a model in which rare HSPCs carrying age-related TP53 mutations are resistant to chemotherapy and expand preferentially after treatment. The early acquisition of TP53 mutations in the founding HSPC clone probably contributes to the frequent cytogenetic abnormalities and poor responses to chemotherapy that are typical of patients with t-AML/t-MDS.

Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing.

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.

Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia.

BACKGROUND: Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear. METHODS: We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis. RESULTS: AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcription-factor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumor-suppressor genes (16%), DNA-methylation-related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories. CONCLUSIONS: We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.).

Genomic impact of transient low-dose decitabine treatment on primary AML cells.

Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized.

Association Between Mutation Clearance After Induction Therapy and Outcomes in Acute Myeloid Leukemia.

IMPORTANCE: Tests that predict outcomes for patients with acute myeloid leukemia (AML) are imprecise, especially for those with intermediate risk AML. OBJECTIVES: To determine whether genomic approaches can provide novel prognostic information for adult patients with de novo AML. DESIGN, SETTING, AND PARTICIPANTS: Whole-genome or exome sequencing was performed on samples obtained at disease presentation from 71 patients with AML (mean age, 50.8 years) treated with standard induction chemotherapy at a single site starting in March 2002, with follow-up through January 2015. In addition, deep digital sequencing was performed on paired diagnosis and remission samples from 50 patients (including 32 with intermediate-risk AML), approximately 30 days after successful induction therapy. Twenty-five of the 50 were from the cohort of 71 patients, and 25 were new, additional cases. EXPOSURES: Whole-genome or exome sequencing and targeted deep sequencing. Risk of identification based on genetic data. MAIN OUTCOMES AND MEASURES: Mutation patterns (including clearance of leukemia-associated variants after chemotherapy) and their association with event-free survival and overall survival. RESULTS: Analysis of comprehensive genomic data from the 71 patients did not improve outcome assessment over current standard-of-care metrics. In an analysis of 50 patients with both presentation and documented remission samples, 24 (48%) had persistent leukemia-associated mutations in at least 5% of bone marrow cells at remission. The 24 with persistent mutations had significantly reduced event-free and overall survival vs the 26 who cleared all mutations. Patients with intermediate cytogenetic risk profiles had similar findings. [table: see text]. CONCLUSIONS AND RELEVANCE: The detection of persistent leukemia-associated mutations in at least 5% of bone marrow cells in day 30 remission samples was associated with a significantly increased risk of relapse, and reduced overall survival. These data suggest that this genomic approach may improve risk stratification for patients with AML.

Biome representational in silico karyotyping.

Metagenomic characterization of complex biomes remains challenging. Here we describe a modification of digital karyotyping-biome representational in silico karyotyping (BRISK)-as a general technique for analyzing a defined representation of all DNA present in a sample. BRISK utilizes a Type IIB DNA restriction enzyme to create a defined representation of 27-mer DNAs in a sample. Massively parallel sequencing of this representation allows for construction of high-resolution karyotypes and identification of multiple species within a biome. Application to normal human tissue demonstrated linear recovery of tags by chromosome. We apply this technique to the biome of the oral mucosa and find that greater than 25% of recovered DNA is nonhuman. DNA from 41 microbial species could be identified from oral mucosa of two subjects. Of recovered nonhuman sequences, fewer than 30% are currently annotated. We characterized seven prevalent unknown sequences by chromosome walking and find these represent novel microbial sequences including two likely derived from novel phage genomes. Application of BRISK to archival tissue from a nasopharyngeal carcinoma resulted in identification of Epstein-Barr virus infection. These results suggest that BRISK is a powerful technique for the analysis of complex microbiomes and potentially for pathogen discovery.

DNMT3A mutations in acute myeloid leukemia.

BACKGROUND: The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown. METHODS: Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations. RESULTS: A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P<0.001 for both comparisons). The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months vs. 41.1 months, P<0.001). DNMT3A mutations were associated with adverse outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 mutations, regardless of age, and were independently associated with a poor outcome in Cox proportional-hazards analysis. CONCLUSIONS: DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome. (Funded by the National Institutes of Health and others.).

Integrative Genomic Analysis of Pediatric Myeloid-Related Acute Leukemias Identifies Novel Subtypes and Prognostic Indicators.

Genomic characterization of pediatric patients with acute myeloid leukemia (AML) has led to the discovery of somatic mutations with prognostic implications. Although gene-expression profiling can differentiate subsets of pediatric AML, its clinical utility in risk stratification remains limited. Here, we evaluate gene expression, pathogenic somatic mutations, and outcome in a cohort of 435 pediatric patients with a spectrum of pediatric myeloid-related acute leukemias for biological subtype discovery. This analysis revealed 63 patients with varying immunophenotypes that span a T-lineage and myeloid continuum designated as acute myeloid/T-lymphoblastic leukemia (AMTL). Within AMTL, two patient subgroups distinguished by FLT3-ITD and PRC2 mutations have different outcomes, demonstrating the impact of mutational composition on survival. Across the cohort, variability in outcomes of patients within isomutational subsets is influenced by transcriptional identity and the presence of a stem cell-like gene-expression signature. Integration of gene expression and somatic mutations leads to improved risk stratification. SIGNIFICANCE: Immunophenotype and somatic mutations play a significant role in treatment approach and risk stratification of acute leukemia. We conducted an integrated genomic analysis of pediatric myeloid malignancies and found that a combination of genetic and transcriptional readouts was superior to immunophenotype and genomic mutations in identifying biological subtypes and predicting outcomes. This article is highlighted in the In This Issue feature, p. 549.

Clonal dynamics of donor-derived myelodysplastic syndrome after unrelated hematopoietic cell transplantation for high-risk pediatric B-lymphoblastic leukemia.

Donor-derived hematologic malignancies are rare complications of hematopoietic cell transplantation (HCT). Although these are commonly either a myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), in general, they are a heterogeneous group of diseases, and a unified mechanism for their development has remained elusive. Here we report next-generation sequencing, including whole-exome sequencing (WES), whole-genome sequencing (WGS), and targeted sequencing, of a case of donor-derived MDS (dMDS) following HCT for high-risk B-lymphoblastic leukemia (B-ALL) in an adolescent. Through interrogation of single-nucleotide polymorphisms (SNPs) in the WGS data, we unequivocally prove that the MDS is donor-derived. Additionally, we sequenced 15 samples from 12 time points, including the initial B-ALL diagnostic sample through several post-HCT remission samples, the dMDS, and representative germline samples from both patient and donor, to show that the MDS-related pathologic mutations, including a canonical ASXL1 (p.Y700*) mutation, were detectable nearly 3 yr prior to the morphological detection of MDS. Furthermore, these MDS mutations were not detectable immediately following, and for >1 yr post-, HCT. These data support the clinical utility of comprehensive sequencing following HCT to detect donor-derived malignancies, while providing insights into the clonal progression of dMDS over a 4-yr period.

Germline SAMD9 and SAMD9L mutations are associated with extensive genetic evolution and diverse hematologic outcomes.

Germline SAMD9 and SAMD9L mutations cause a spectrum of multisystem disorders that carry a markedly increased risk of developing myeloid malignancies with somatic monosomy 7. Here, we describe 16 siblings, the majority of which were phenotypically normal, from 5 families diagnosed with myelodysplasia and leukemia syndrome with monosomy 7 (MLSM7; OMIM 252270) who primarily had onset of hematologic abnormalities during the first decade of life. Molecular analyses uncovered germline SAMD9L (n = 4) or SAMD9 (n = 1) mutations in these families. Affected individuals had a highly variable clinical course that ranged from mild and transient dyspoietic changes in the bone marrow to a rapid progression of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with monosomy 7. Expression of these gain-of-function SAMD9 and SAMD9L mutations reduces cell cycle progression, and deep sequencing demonstrated selective pressure favoring the outgrowth of clones that have either lost the mutant allele or acquired revertant mutations. The myeloid malignancies of affected siblings acquired cooperating mutations in genes that are also altered in sporadic cases of AML characterized by monosomy 7. These data have implications for understanding how SAMD9 and SAMD9L mutations contribute to myeloid transformation and for recognizing, counseling, and treating affected families.

The genomic landscape of pediatric myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are uncommon in children and have a poor prognosis. In contrast to adult MDS, little is known about the genomic landscape of pediatric MDS. Here, we describe the somatic and germline changes of pediatric MDS using whole exome sequencing, targeted amplicon sequencing, and/or RNA-sequencing of 46 pediatric primary MDS patients. Our data show that, in contrast to adult MDS, Ras/MAPK pathway mutations are common in pediatric MDS (45% of primary cohort), while mutations in RNA splicing genes are rare (2% of primary cohort). Surprisingly, germline variants in SAMD9 or SAMD9L were present in 17% of primary MDS patients, and these variants were routinely lost in the tumor cells by chromosomal deletions (e.g., monosomy 7) or copy number neutral loss of heterozygosity (CN-LOH). Our data confirm that adult and pediatric MDS are separate diseases with disparate mechanisms, and that SAMD9/SAMD9L mutations represent a new class of MDS predisposition.

PML-RARA requires DNA methyltransferase 3A to initiate acute promyelocytic leukemia.

The DNA methyltransferases DNMT3A and DNMT3B are primarily responsible for de novo methylation of specific cytosine residues in CpG dinucleotides during mammalian development. While loss-of-function mutations in DNMT3A are highly recurrent in acute myeloid leukemia (AML), DNMT3A mutations are almost never found in AML patients with translocations that create oncogenic fusion genes such as PML-RARA, RUNX1-RUNX1T1, and MLL-AF9. Here, we explored how DNMT3A is involved in the function of these fusion genes. We used retroviral vectors to express PML-RARA, RUNX1-RUNX1T1, or MLL-AF9 in bone marrow cells derived from WT or DNMT3A-deficient mice. Additionally, we examined the phenotypes of hematopoietic cells from Ctsg-PML-RARA mice, which express PML-RARA in early hematopoietic progenitors and myeloid precursors, with or without DNMT3A. We determined that the methyltransferase activity of DNMT3A, but not DNMT3B, is required for aberrant PML-RARA-driven self-renewal ex vivo and that DNMT3A is dispensable for RUNX1-RUNX1T1- and MLL-AF9-driven self-renewal. Furthermore, both the PML-RARA-driven competitive transplantation advantage and development of acute promyelocytic leukemia (APL) required DNMT3A. Together, these findings suggest that PML-RARA requires DNMT3A to initiate APL in mice.

Comprehensive genomic analysis reveals FLT3 activation and a therapeutic strategy for a patient with relapsed adult B-lymphoblastic leukemia.

The genomic events responsible for the pathogenesis of relapsed adult B-lymphoblastic leukemia (B-ALL) are not yet clear. We performed integrative analysis of whole-genome, whole-exome, custom capture, whole-transcriptome (RNA-seq), and locus-specific genomic assays across nine time points from a patient with primary de novo B-ALL. Comprehensive genome and transcriptome characterization revealed a dramatic tumor evolution during progression, yielding a tumor with complex clonal architecture at second relapse. We observed and validated point mutations in EP300 and NF1, a highly expressed EP300-ZNF384 gene fusion, a microdeletion in IKZF1, a focal deletion affecting SETD2, and large deletions affecting RB1, PAX5, NF1, and ETV6. Although the genome analysis revealed events of potential biological relevance, no clinically actionable treatment options were evident at the time of the second relapse. However, transcriptome analysis identified aberrant overexpression of the targetable protein kinase encoded by the FLT3 gene. Although the patient had refractory disease after salvage therapy for the second relapse, treatment with the FLT3 inhibitor sunitinib rapidly induced a near complete molecular response, permitting the patient to proceed to a matched-unrelated donor stem cell transplantation. The patient remains in complete remission more than 4 years later. Analysis of this patient's relapse genome revealed an unexpected, actionable therapeutic target that led to a specific therapy associated with a rapid clinical response. For some patients with relapsed or refractory cancers, this approach may indicate a novel therapeutic intervention that could alter outcome.

The R882H DNMT3A mutation associated with AML dominantly inhibits wild-type DNMT3A by blocking its ability to form active tetramers.

Somatic mutations in DNMT3A, which encodes a de novo DNA methyltransferase, are found in ∼30% of normal karyotype acute myeloid leukemia (AML) cases. Most mutations are heterozygous and alter R882 within the catalytic domain (most commonly R882H), suggesting the possibility of dominant-negative consequences. The methyltransferase activity of R882H DNMT3A is reduced by ∼80% compared with the WT enzyme. In vitro mixing of WT and R882H DNMT3A does not affect the WT activity, but coexpression of the two proteins in cells profoundly inhibits the WT enzyme by disrupting its ability to homotetramerize. AML cells with the R882H mutation have severely reduced de novo methyltransferase activity and focal hypomethylation at specific CpGs throughout AML cell genomes.

Functional heterogeneity of genetically defined subclones in acute myeloid leukemia.

The relationships between clonal architecture and functional heterogeneity in acute myeloid leukemia (AML) samples are not yet clear. We used targeted sequencing to track AML subclones identified by whole-genome sequencing using a variety of experimental approaches. We found that virtually all AML subclones trafficked from the marrow to the peripheral blood, but some were enriched in specific cell populations. Subclones showed variable engraftment potential in immunodeficient mice. Xenografts were predominantly comprised of a single genetically defined subclone, but there was no predictable relationship between the engrafting subclone and the evolutionary hierarchy of the leukemia. These data demonstrate the importance of integrating genetic and functional data in studies of primary cancer samples, both in xenograft models and in patients.

Notch signaling in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is initiated by the PML-RARA (PR) fusion oncogene and has a characteristic expression profile that includes high levels of the Notch ligand Jagged-1 (JAG1). In this study, we used a series of bioinformatic, in vitro, and in vivo assays to assess the role of Notch signaling in human APL samples, and in a PML-RARA knock-in mouse model of APL (Ctsg-PML-RARA). We identified a Notch expression signature in both human primary APL cells and in Kit+Lin-Sca1+ cells from pre-leukemic Ctsg-PML-RARA mice. Both genetic and pharmacologic inhibition of Notch signaling abrogated the enhanced self-renewal seen in hematopoietic stem/progenitor cells from pre-leukemic Ctsg-PML-RARA mice, but had no influence on cells from age-matched wild-type mice. In addition, six of nine murine APL tumors tested displayed diminished growth in vitro when Notch signaling was inhibited pharmacologically. Finally, we found that genetic inhibition of Notch signaling with a dominant-negative Mastermind-like protein reduced APL growth in vivo in a subset of tumors. These findings expand the role of Notch signaling in hematopoietic diseases, and further define the mechanistic events important for PML-RARA-mediated leukemogenesis.

Expression and function of PML-RARA in the hematopoietic progenitor cells of Ctsg-PML-RARA mice.

Because PML-RARA-induced acute promyelocytic leukemia (APL) is a morphologically differentiated leukemia, many groups have speculated about whether its leukemic cell of origin is a committed myeloid precursor (e.g. a promyelocyte) versus an hematopoietic stem/progenitor cell (HSPC). We originally targeted PML-RARA expression with CTSG regulatory elements, based on the early observation that this gene was maximally expressed in cells with promyelocyte morphology. Here, we show that both Ctsg, and PML-RARA targeted to the Ctsg locus (in Ctsg-PML-RARA mice), are expressed in the purified KLS cells of these mice (KLS = Kit(+)Lin(-)Sca(+), which are highly enriched for HSPCs), and this expression results in biological effects in multi-lineage competitive repopulation assays. Further, we demonstrate the transcriptional consequences of PML-RARA expression in Ctsg-PML-RARA mice in early myeloid development in other myeloid progenitor compartments [common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs)], which have a distinct gene expression signature compared to wild-type (WT) mice. Although PML-RARA is indeed expressed at high levels in the promyelocytes of Ctsg-PML-RARA mice and alters the transcriptional signature of these cells, it does not induce their self-renewal. In sum, these results demonstrate that in the Ctsg-PML-RARA mouse model of APL, PML-RARA is expressed in and affects the function of multipotent progenitor cells. Finally, since PML/Pml is normally expressed in the HSPCs of both humans and mice, and since some human APL samples contain TCR rearrangements and express T lineage genes, we suggest that the very early hematopoietic expression of PML-RARA in this mouse model may closely mimic the physiologic expression pattern of PML-RARA in human APL patients.

Effect of circadian clock gene mutations on nonvisual photoreception in the mouse.

PURPOSE: Mice lacking rods and cones retain pupillary light reflexes that are mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs). Melanopsin is necessary and sufficient for this nonvisual photoreception. The mammalian inner retina also expresses the potential blue light photopigments cryptochromes 1 and 2. Previous studies have shown that outer retinal degenerate mice lacking cryptochromes have lower nonvisual photic sensitivity than retinal degenerate mice, suggesting a role for cryptochrome in inner retinal photoreception. METHODS: Nonvisual photoreception (pupillary light responses, circadian entrainment, and in vitro sensitivity of intrinsically photosensitive retinal ganglion cells) were studied in wild-type, rd/rd, and circadian clock-mutant mice with and without rd/rd mutation. RESULTS: Loss of cryptochrome in retinal degenerate mice reduces the sensitivity of the pupillary light response at all wavelengths but does not alter the form of the action spectrum, suggesting that cryptochrome does not function as a photopigment in the inner retina. The authors compounded the rd/rd retinal degeneration mutation with mutations in other essential circadian clock genes, mPeriod and Bmal1. Both mPeriod1⁻/⁻; mPeriod2⁻/⁻;rd/rd and Bmal1⁻/⁻;rd/rd mice showed significantly lower pupillary light sensitivity than rd/rd mice alone. A moderate amplitude (0.5 log) circadian rhythm of pupillary light responsiveness was observed in rd/rd mice. Multielectrode array recordings of ipRGC responses of mCryptochrome1⁻/⁻;mCryptochrome2⁻/⁻ and mPeriod1⁻/⁻;mPeriod2⁻/⁻ mice showed minimal sensitivity decrement compared with wild-type animals. mCryptochrome1⁻/⁻;mCryptochrome2⁻/⁻;rd/rd, mPeriod1⁻/⁻;mPeriod2⁻/⁻;rd/rd and Bmal1⁻/⁻;rd/rd mice all showed comparable weak behavioral synchronization to a 12-hour light/12-hour dark cycle. CONCLUSIONS: The effect of cryptochrome loss on nonvisual photoreception is due to loss of the circadian clock nonspecifically. The circadian clock modulates the sensitivity of nonvisual photoreception.