Mismatch repair is a highly conserved cellular pathway responsible for repairing mismatched dsDNA. Errors are detected by the MutS enzyme, which most likely senses altered mechanical property of damaged dsDNA rather than a specific molecular pattern. While the curved shape of dsDNA in crystallographic MutS/DNA structures suggests the role of DNA bending, the theoretical support is not fully convincing. Here, we present a computational study focused on a base-pair opening into the minor groove, a specific base-pair motion observed upon interaction with MutS. Propensities for the opening were evaluated in terms of two base-pair parameters: Opening and Shear. We tested all possible base pairs in anti/anti, anti/syn and syn/anti orientations and found clear discrimination between mismatches and canonical base-pairs only for the opening into the minor groove. Besides, the discrimination gap was also confirmed in hotspot and coldspot sequences, indicating that the opening could play a more significant role in the mismatch recognition than previously recognized. Our findings can be helpful for a better understanding of sequence-dependent mutability. Further, detailed structural characterization of mismatches can serve for designing anti-cancer drugs targeting mismatched base pairs.

The B-family multi-subunit DNA polymerase ζ (Polζ) is important for translesion DNA synthesis (TLS) during replication, due to its ability to extend synthesis past nucleotides opposite DNA lesions and mismatched base pairs. We present a cryo-EM structure of Saccharomyces cerevisiae Polζ with an A:C mismatch at the primer terminus. The structure shows how the Polζ active site responds to the mismatched duplex DNA distortion, including the loosening of key protein-DNA interactions and a fingers domain in an "open" conformation, while the incoming dCTP is still able to bind for the extension reaction. The structure of the mismatched DNA-Polζ ternary complex reveals insights into mechanisms that either stall or favor continued DNA synthesis in eukaryotes.

Single-stranded oligodeoxyribonucleotide (ssODN)-mediated repair of CRISPR/Cas9-induced DNA double-strand breaks (DSB) can effectively be used to introduce small genomic alterations in a defined locus. Here, we reveal DNA mismatch repair (MMR) activity is crucial for efficient nucleotide substitution distal from the Cas9-induced DNA break when the substitution is instructed by the 3' half of the ssODN. Furthermore, protecting the ssODN 3' end with phosphorothioate linkages enhances MMR-dependent gene editing events. Our findings can be exploited to optimize efficiencies of nucleotide substitutions distal from the DSB and imply that oligonucleotide-mediated gene editing is effectuated by templated break repair.

We have developed an enhanced form of reduced representation bisulfite sequencing with extended genomic coverage, which resulted in greater capture of DNA methylation information of regions lying outside of traditional CpG islands. Applying this method to primary human bone marrow specimens from patients with Acute Myelogeneous Leukemia (AML), we demonstrated that genetically distinct AML subtypes display diametrically opposed DNA methylation patterns. As compared to normal controls, we observed widespread hypermethylation in IDH mutant AMLs, preferentially targeting promoter regions and CpG islands neighboring the transcription start sites of genes. In contrast, AMLs harboring translocations affecting the MLL gene displayed extensive loss of methylation of an almost mutually exclusive set of CpGs, which instead affected introns and distal intergenic CpG islands and shores. When analyzed in conjunction with gene expression profiles, it became apparent that these specific patterns of DNA methylation result in differing roles in gene expression regulation. However, despite this subtype-specific DNA methylation patterning, a much smaller set of CpG sites are consistently affected in both AML subtypes. Most CpG sites in this common core of aberrantly methylated CpGs were hypermethylated in both AML subtypes. Therefore, aberrant DNA methylation patterns in AML do not occur in a stereotypical manner but rather are highly specific and associated with specific driving genetic lesions.

Dicer partner proteins Drosophila Loquacious-PB (Loqs-PB) and human TRBP tune the length of miRNAs produced by Dicer from a subset of pre-miRNAs and thereby alter their target repertoire, by an unknown mechanism. Here, we developed a novel high-throughput method that we named Dram-seq (Dice randomized pre-miRNA pool and seq) to study length distributions of miRNAs produced from thousands of different pre-miRNA variants. Using Dram-seq, we found that a base-mismatch in the pre-miRNA stem can alter the length of miRNAs compared with a base-pair at the same position in both Drosophila and human, and is important for the miRNA length tuning by Loqs-PB. Loqs-PB directly bound base-mismatched nucleotides in the pre-miRNA stem. We speculate that Loqs-PB tunes miRNA length by changing the conformation of base-mismatched nucleotides in the pre-miRNA stem to that of base-paired ones and thereby altering the distance of the pre-miRNA stem.

Mutation accumulation experiments followed by whole-genome sequencing have revealed that, for several bacterial species, the rate of base-pair substitutions (BPSs) is not constant across the chromosome but varies in a wave-like pattern that is symmetrical about the origin of replication. The experiments reported here demonstrated that, in Escherichia coli, several interacting factors determine the wave. The origin is a major driver of BPS rates. When it is relocated, the BPS rates in a 1,000-kb region surrounding the new origin reproduce the pattern that surrounds the normal origin. However, the pattern across distant regions of the chromosome is unaltered and thus must be determined by other factors. Increasing the deoxynucleoside triphosphate (dNTP) concentration shifts the wave pattern away from the origin, supporting the hypothesis that fluctuations in dNTP pools coincident with replication firing contribute to the variations in the mutation rate. The nucleoid binding proteins (HU and Fis) and the terminus organizing protein (MatP) are also major factors. These proteins alter the three-dimensional structure of the DNA, and results suggest that mutation rates increase when highly structured DNA is replicated. Biases in error correction by proofreading and mismatch repair, both of which may be responsive to dNTP concentrations and DNA structure, also are major determinants of the wave pattern. These factors should apply to most bacterial and, possibly, eukaryotic genomes and suggest that different areas of the genome evolve at different rates.IMPORTANCE It has been found in several species of bacteria that the rate at which single base pairs are mutated is not constant across the genome but varies in a wave-like pattern that is symmetrical about the origin of replication. Using Escherichia coli as our model system, we show that this pattern is the result of several interconnected factors. First, the timing and progression of replication are important in determining the wave pattern. Second, the three-dimensional structure of the DNA is also a factor, and the results suggest that mutation rates increase when highly structured DNA is replicated. Finally, biases in error correction, which may be responsive both to the progression of DNA synthesis and to DNA structure, are major determinants of the wave pattern. These factors should apply to most bacterial and, possibly, eukaryotic genomes and suggest that different areas of the genome evolve at different rates.

In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5'-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine-Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14-C25 base pair. The mismatch base pair in the wt RNA thermometer (A8-G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA.

T:G base pair arising from spontaneous deamination of 5mC or polymerase errors is a great challenge for DNA repair of hyperthermophilic archaea, especially Crenarchaea. Most strains in this phylum lack the protein homologues responsible for the recognition of the mismatch in the DNA repair pathways. To investigate whether Cren7, a highly conserved chromatin protein in Crenarchaea, serves a role in the repair of T:G mispairs, the crystal structures of Cren7-GTAATTGC and Cren7-GTGATCGC complexes were solved at 2.0 Å and 2.1 Å. In our structures, binding of Cren7 to the AT-rich DNA duplex (GTAATTGC) induces opening of T2:G15 but not T10:G7 base pair. By contrast, both T:G mispairs in the GC-rich DNA duplex (GTGATCGC) retain the classic wobble type. Structural analysis also showed DNA helical changes of GTAATTGC, especially in the steps around the open T:G base pair, as compared to GTGATCGC or the matched DNAs. Surface plasmon resonance assays revealed a 4-fold lower binding affinity of Cren7 for GTAATTGC than that for GTGATCGC, which was dominantly contributed by the decrease of association rate. These results suggested that binding of Cren7 to DNA leads to T:G mispair opening in a sequence dependent manner, and therefore propose the potential roles of Cren7 in DNA repair.

We show that in silico design of DNA secondary structures is improved by extending the base pairing alphabet beyond A-T and G-C to include the pair between 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one and 6-amino-3-(1'-β-d-2'-deoxyribofuranosyl)-5-nitro-(1H)-pyridin-2-one, abbreviated as P and Z. To obtain the thermodynamic parameters needed to include P-Z pairs in the designs, we performed 47 optical melting experiments and combined the results with previous work to fit free energy and enthalpy nearest neighbor folding parameters for P-Z pairs and G-Z wobble pairs. We find G-Z pairs have stability comparable to that of A-T pairs and should therefore be included as base pairs in structure prediction and design algorithms. Additionally, we extrapolated the set of loop, terminal mismatch, and dangling end parameters to include the P and Z nucleotides. These parameters were incorporated into the RNAstructure software package for secondary structure prediction and analysis. Using the RNAstructure Design program, we solved 99 of the 100 design problems posed by Eterna using the ACGT alphabet or supplementing it with P-Z pairs. Extending the alphabet reduced the propensity of sequences to fold into off-target structures, as evaluated by the normalized ensemble defect (NED). The NED values were improved relative to those from the Eterna example solutions in 91 of 99 cases in which Eterna-player solutions were provided. P-Z-containing designs had average NED values of 0.040, significantly below the 0.074 of standard-DNA-only designs, and inclusion of the P-Z pairs decreased the time needed to converge on a design. This work provides a sample pipeline for inclusion of any expanded alphabet nucleotides into prediction and design workflows.

We show that in silico design of DNA secondary structures is improved by extending the base pairing alphabet beyond A-T and G-C to include the pair between 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo-[1,2- a ]-1,3,5-triazin-(8 H )-4-one and 6-amino-3-(1'-β-D-2'-deoxyribofuranosyl)-5-nitro-(1 H )-pyridin-2-one, simply P and Z. To obtain the thermodynamic parameters needed to include P-Z pairs in the designs, we performed 47 optical melting experiments and combined the results with previous work to fit a new set of free energy and enthalpy nearest neighbor folding parameters for P-Z pairs and G-Z wobble pairs. We find that G-Z pairs have stability comparable to A-T pairs and therefore should be considered quantitatively by structure prediction and design algorithms. Additionally, we extrapolated the set of loop, terminal mismatch, and dangling end parameters to include P and Z nucleotides. These parameters were incorporated into the RNAstructure software package for secondary structure prediction and analysis. Using the RNAstructure Design program, we solved 99 of the 100 design problems posed by Eterna using the ACGT alphabet or supplementing with P-Z pairs. Extending the alphabet reduced the propensity of sequences to fold into off-target structures, as evaluated by the normalized ensemble defect (NED). The NED values were improved relative to those from the Eterna example solutions in 91 of 99 cases where Eterna-player solutions were provided. P-Z-containing designs had average NED values of 0.040, significantly below the 0.074 of standard-DNA-only designs, and inclusion of the P-Z pairs decreased the time needed to converge on a design. This work provides a sample pipeline for inclusion of any expanded alphabet nucleotides into prediction and design workflows.

The use of multiple drugs simultaneously targeting DNA is a promising strategy in cancer therapy for potentially overcoming single drug resistance. In support of this concept, we report that a combination of actinomycin D (ActD) and echinomycin (Echi), can interact in novel ways with native and mismatched DNA sequences, distinct from the structural effects produced by either drug alone. Changes in the former with GpC and CpG steps separated by a A:G or G:A mismatch or in a native DNA with canonical G:C and C:G base pairs, result in significant asymmetric backbone twists through staggered intercalation and base pair modulations. A wobble or Watson-Crick base pair at the two drug-binding interfaces can result in a single-stranded 'chair-shaped' DNA duplex with a straight helical axis. However, a novel sugar-edged hydrogen bonding geometry in the G:A mismatch leads to a 'curved-shaped' duplex. Two non-canonical G:C Hoogsteen base pairings produce a sharply kinked duplex in different forms and a four-way junction-like superstructure, respectively. Therefore, single base pair modulations on the two drug-binding interfaces could significantly affect global DNA structure. These structures thus provide a rationale for atypical DNA recognition via multiple DNA intercalators and a structural basis for the drugs' potential synergetic use.

Translation initiation in eukaryotes and archaea involves a methionylated initiator tRNA delivered to the ribosome in a ternary complex with e/aIF2 and GTP. Eukaryotic and archaeal initiator tRNAs contain a highly conserved A1-U72 base pair at the top of the acceptor stem. The importance of this base pair to discriminate initiator tRNAs from elongator tRNAs has been established previously using genetics and biochemistry. However, no structural data illustrating how the A1-U72 base pair participates in the accurate selection of the initiator tRNAs by the translation initiation systems are available. Here, we describe the crystal structure of a mutant E. coli initiator tRNAfMetA1-U72, aminoacylated with methionine, in which the C1:A72 mismatch at the end of the tRNA acceptor stem has been changed to an A1-U72 base pair. Sequence alignments show that the mutant E. coli tRNA is a good mimic of archaeal initiator tRNAs. The crystal structure, determined at 2.8 Å resolution, shows that the A1-U72 pair adopts an unusual arrangement. A1 is in a syn conformation and forms a single H-bond interaction with U72 This interaction requires protonation of the N1 atom of A1 Moreover, the 5' phosphoryl group folds back into the major groove of the acceptor stem and interacts with the N7 atom of G2 A possible role of this unusual geometry of the A1-U72 pair in the recognition of the initiator tRNA by its partners during eukaryotic and archaeal translation initiation is discussed.

Uracil (U) can be found in DNA as a mismatch paired either to adenine (A) or to guanine (G). Removal of U from DNA is performed by a class of enzymes known as uracil-DNA-glycosylases (UDG). Recent studies suggest that recognition of U-A and U-G mismatches by UDG takes place via an extra-helical mechanism. In this work, we use molecular dynamics simulations to analyze the structure, dynamics and open base pair kinetics of U-A base pairs relative to their natural T-A counterpart in 12 dodecamers. Our results show that the presence of U does not alter the local conformation of B-DNA. Breathing dynamics and base pair closing kinetics are only weakly dependent on the presence of U versus T, with open T-A and U-A pairs lifetimes in the nanosecond timescale. Additionally, we observed spontaneous base flipping in U-A pairs. We analyze the structure and dynamics for this event and compare the results to available crystallographic data of open base pair conformations. Our results are in agreement with both structural and kinetic data derived from NMR imino proton exchange measurements, providing the first detailed description at the molecular level of elusive events such as spontaneous base pair opening and flipping in mismatched U-A sequences in DNA. Based on these results, we propose that base pair flipping can occur spontaneously at room temperature via a 3-step mechanism with an open base pair intermediate. Implications for the molecular basis of U recognition by UDG are discussed.

DNA base editors use deaminases fused to a programmable DNA-binding protein for targeted nucleotide conversion. However, the most widely used TadA deaminases lack post-translational control in living cells. Here, we present a split adenine base editor (sABE) that utilizes chemically induced dimerization (CID) to control the catalytic activity of the deoxyadenosine deaminase TadA-8e. sABE shows high on-target editing activity comparable to the original ABE with TadA-8e (ABE8e) upon rapamycin induction while maintaining low background activity without induction. Importantly, sABE exhibits a narrower activity window on DNA and higher precision than ABE8e, with an improved single-to-double ratio of adenine editing and reduced genomic and transcriptomic off-target effects. sABE can achieve gene knockout through multiplex splice donor disruption in human cells. Furthermore, when delivered via dual adeno-associated virus vectors, sABE can efficiently convert a single A•T base pair to a G•C base pair on the PCSK9 gene in mouse liver, demonstrating in vivo CID-controlled DNA base editing. Thus, sABE enables precise control of base editing, which will have broad implications for basic research and in vivo therapeutic applications.

Genetic stability depends in part on an efficient DNA lesion recognition and correction by the DNA mismatch repair (MMR) system. In eukaryotes, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes, MSH2-MSH6 and MSH2-MSH3, which recognize and bind mismatches and unpaired nucleotides. Plants encode another mismatch recognition protein, named MSH7. MSH7 forms a heterodimer with MSH2 and the protein complex is designated MutSγ. We here report the effect the expression of Arabidopsis MSH2 and MSH7 alone or in combination exert on the genomic stability of Saccharomyces cerevisiae. AtMSH2 and AtMutSγ proteins failed to complement the hypermutator phenotype of an msh2 deficient strain. However, overexpressing AtMutSγ in MMR proficient strains generated a 4-fold increase in CAN1 forward mutation rate, when compared to wild-type strains. Can(r) mutation spectrum analysis of AtMutSγ overproducing strains revealed a substantial increase in the frequency of base substitution mutations, including an increased accumulation of base pair changes from G:C to A:T and T:A to C:G, G:C or A:T. Taken together, these results suggest that AtMutSγ affects yeast genomic stability by recognizing specific mismatches and preventing correction by yeast MutSα and MutSβ, with subsequent inability to interact with yeast downstream proteins needed to complete MMR.

Microarrays provide a means of studying expression level of tens of thousands of genes by providing one or more oligonucleotide probe(s) for each transcript studied. Affymetrix(R) GeneChiptrade mark platforms historically pair each 25-base perfect match (PM) probe with a mismatch probe (MM) differing by a complementary base located in the 13(th) position to quantify and deflate effects of cross-hybridization. Analytical routines for analyzing these arrays take into account difference in expression levels of MM and PM probes to determine which ones are useful for further study. If a single nucleotide polymorphism (SNP) occurs at the 13(th) base, a probe with a higher MM expression level may be incorrectly omitted. In order to examine SNP affects on PM and MM expression levels, known human SNPs from dbSNP were mapped to probe sets within the Affymetrix(R) HG-U133A platform. Probe sets containing one or more probe pairs with a single SNP at the 13(th) position were extracted. A set of twelve microarray experiments were analyzed for the PM and MM expression levels for these probe sets. Over 6,000,000 human SNPs and their flanking regions were extracted from dbSNP. These sequences were aligned against each of the 247,965 probe pair sequences from the Affymetrix(R) HG-U133A platform. A total of 915 probe sets containing a single probe sequence with a SNP mapped to the 13(th) base were extracted. A subset containing 166 probe sets result in complementary base SNPs. Comparison of gene expression levels for the SNP to non-SNP PM and MM probes does not yield a significant difference using chi2 analysis. Thus, omission of probes with MM expression levels higher than PM expression levels does not appear to result in a loss of information concerning SNPs for these regions.

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for T(m) and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct T(m)'s when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher T(m) closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.

Hypermutagenesis refers to marked increase in the number of mutations due to continuous mutagenic process. Hypermutated tumors, have being found in several tumor types, are associated with inherited or acquired alterations in the DNA repair pathways. Hypermutation has been observed in a subset of adult glioma patients as a direct result of temozolomide(TMZ)-induced mutagenesis. In our study, we have identified a rare subset of treatment-naïve adult gliomas with de novo hypermutator phenotype and explored the evolution of spontaneous and treatment-induced hypermutagenesis. We conducted Whole-Exome Sequencing (WES), Whole-Transcriptome Sequencing (WTS), and Single-Cell Sequencing (SCS) of TMZ-naïve and post-TMZ-treated hypermutated tumors to identify distinct clinical or genomic manifestations that contribute to the development of hypermutation in untreated adult gliomas. TMZ-naïve hypermutated tumors were marked by absence of IDH1 somatic mutation and MGMT promoter (pMGMT) methylation, two genomic traits that were significantly associated with the TMZ-induced hypermutagenic event in glioblastoma, and harbored inherited alterations in the mismatch repair (MMR) machinery. The immediate family members of the TMZ-naive hypermutated glioma patients were also previous diagnosed with cancer development history, suggesting that germline dysfunction of the MMR pathway could potentially pose hereditary risk to genetic predisposition of carcinogenesis in gliomas. Lastly, both TMZ-naïve and post-TMZ-treated hypermutated tumors exhibited a significant accumulation of neoantigen loads, suggesting immunotherapeutic alternatives. Our results present new and unique understanding of hypermutagenic process in adult gliomas and an important step towards clinical implication of immunotherapy in glioma treatment.

Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as "pks" encoding the biosynthesis of a secondary metabolite, colibactin. Colibactin-producing E. coli promote colorectal cancer (CRC) in preclinical models, and in vitro induce a specific mutational signature that is also detected in human CRC genomes. Yet, how colibactin exposure affects the mutational landscape of CRC in vivo remains unclear. Here we show that colibactin-producing E. coli-driven colonic tumors in mice have a significantly higher SBS burden and a larger percentage of these mutations can be attributed to a signature associated with mismatch repair deficiency (MMRd; SBS15), compared to tumors developed in the presence of colibactin-deficient E. coli. We found that the synthetic colibactin 742 but not an inactive analog 746 causes DNA damage and induces transcriptional activation of p53 and senescence signaling pathways in non-transformed human colonic epithelial cells. In MMRd colon cancer cells (HCT 116), chronic exposure to 742 resulted in the upregulation of BRCA1, Fanconi anemia, and MMR signaling pathways as revealed by global transcriptomic analysis. This was accompanied by increased T>N single-base substitutions (SBS) attributed to the proposed pks+E. coli signature (SBS88), reactive oxygen species (SBS17), and mismatch-repair deficiency (SBS44). A significant co-occurrence between MMRd SBS44 and pks-associated SBS88 signature was observed in a large cohort of human CRC patients (n=2,945), and significantly more SBS44 mutations were found when SBS88 was also detected. Collectively, these findings reveal the host response mechanisms underlying colibactin genotoxic activity and suggest that colibactin may exacerbate MMRd-associated mutations.

Here, we present the case of a 47-year-old woman diagnosed with luminal B breast cancer subtype and provide an in-depth analysis of her gene mutations, chromosomal alterations, mRNA and protein expression changes. We found a point mutation in the FGFR2 gene, which is potentially hyper-activating the receptor function, along with over-expression of its ligand FGF20 due to genomic amplification. The patient also harbors somatic and germline mutations in some mismatch repair (MMR) genes, with a strong MMR mutational signature. The patient displays high microsatellite instability (MSI) and tumor mutational burden (TMB) status and increased levels of CTLA-4 and PD-1 expression. Altogether, these data strongly implicate that aberrant FGFR signaling, and defective MMR system might be involved in the development of this breast tumor. In addition, high MSI and TMB in the context of CTLA-4 and PD-L1 positivity, suggest the potential benefit of immune checkpoint inhibitors. Accurate characterization of molecular subtypes, based on gene mutational and expression profiling analyses, will be certainly helpful for individualized treatment and targeted therapy of breast cancer patients, especially for those subtypes with adverse outcome.