Distinct Patterns of Clonal Evolution Drive Myelodysplastic Syndrome Progression to Secondary Acute Myeloid Leukemia.

Clonal evolution in myelodysplastic syndrome (MDS) can result in clinical progression and secondary acute myeloid leukemia (sAML). To dissect changes in clonal architecture associated with this progression, we performed single-cell genotyping of paired MDS and sAML samples from 18 patients. Analysis of single-cell genotypes revealed patient-specific clonal evolution and enabled the assessment of single-cell mutational cooccurrence. We discovered that changes in clonal architecture proceed via distinct patterns, classified as static or dynamic, with dynamic clonal architectures having a more proliferative phenotype by blast count fold change. Proteogenomic analysis of a subset of patients confirmed that pathogenic mutations were primarily confined to primitive and mature myeloid cells, though we also identify rare but present mutations in lymphocyte subsets. Single-cell transcriptomic analysis of paired sample sets further identified gene sets and signaling pathways involved in two cases of progression. Together, these data define serial changes in the MDS clonal landscape with clinical and therapeutic implications. SIGNIFICANCE: Precise clonal trajectories in MDS progression are made possible by single-cell genomic sequencing. Here we use this technology to uncover the patterns of clonal architecture and clonal evolution that drive the transformation to secondary AML. We further define the phenotypic and transcriptional changes of disease progression at the single-cell level. See related article by Menssen et al., p. 330 (31). See related commentary by Romine and van Galen, p. 270. This article is highlighted in the In This Issue feature, p. 265.

Acquisition of aneuploidy drives mutant p53-associated gain-of-function phenotypes.

p53 is mutated in over half of human cancers. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. To study mutant p53 GOF mechanisms and phenotypes, we genetically engineered non-transformed and tumor-derived WT p53 cell line models to express endogenous missense mutant p53 (R175H and R273H) or to be deficient for p53 protein (null). Characterization of the models, which initially differed only by TP53 genotype, revealed that aneuploidy frequently occurred in mutant p53-expressing cells. GOF phenotypes occurred clonally in vitro and in vivo, were independent of p53 alteration and correlated with increased aneuploidy. Further, analysis of outcome data revealed that individuals with aneuploid-high tumors displayed unfavorable prognoses, regardless of the TP53 genotype. Our results indicate that genetic variation resulting from aneuploidy accounts for the diversity of previously reported mutant p53 GOF phenotypes.

Clinical diagnosis of neurofibromatosis type I in multiple family members due to cosegregation of a unique balanced translocation with disruption of the NF1 locus: Testing considerations for accurate diagnosis.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder that causes a predisposition to develop tumors along the peripheral nervous system. The NF1 gene, located at 17q11.2, has the highest mutation rate among known human genes and about half of NF1 patients have de novo pathogenic variants. We present a case of clinical NF1 diagnoses in multiple family members with phenotypes ranging from mild to severe. Chromosome analysis of the 3-year-old female proband with NF1 resulted in an abnormal karyotype that was inherited from her mother: 46,XX,t(4;17)(q21.3;q11.2) mat. However, no NF1 genetic variants were identified by either NGS analysis of NF1 DNA coding regions, deletion-duplication studies, or by cytogenomic microarray copy number analysis. Follow-up chromosome studies of the proband's two male siblings demonstrated cosegregation of the same balanced translocation and a clinical diagnosis of NF1. Based on the cosegregation of the translocation with the NF1 clinical presentation in this family, we hypothesized that the NF1 gene may have been disrupted by this unique rearrangement. Subsequent fluorescence in situ hybridization (FISH) analysis of the metaphase cells of an affected sibling revealed a disruption of the NF1 gene confirming the underlying basis of the clinical NF1 presentation in this family. The utilization of traditional cytogenetic as well as evolving molecular methods was not only pivotal in the diagnosis of NF1 and management for this family, but is also pertinent to other patients with a family history of NF1.

Institutional Training Opportunities for PhD Students in Laboratory Medicine: An Unmet Career Development Need?

In the United States, the credentialing of PhD-scientists as medical directors of clinical laboratories is driven by formal postdoctoral training programs. Prior to acceptance in one these accredited fellowships, however, a trainee's exposure to the field can be far less standardized, with significant ramifications for their awareness and competitiveness. In the current article, we describe our recent experiences in developing local, institution-based immersion opportunities for PhD experiences in the subdisciplines of laboratory medicine (clinical microbiology, clinical chemistry, and molecular genetics/genomics). It is our hope that this article-and a corresponding online survey-can prompt reflection and discussion on the status of early career training opportunities in these key clinical areas.

A unique evolution of the kidney phenotype in a patient with autosomal recessive Alport syndrome.

Alport syndrome is due to mutations in one of the genes encoding (α3,4,5) type IV collagen resulting in defective type IV collagen, a key component of the glomerular basement membrane (GBM). The GBM is initially thin and, with ongoing remodeling, develops a thickened basket-woven appearance. We report a unique case of a 9-year-old boy who underwent biopsy for hematuria and proteinuria, diagnosed as IgA nephropathy, with normal GBM appearance and thickness. Because of a family history of hematuria and chronic kidney disease, he subsequently underwent genetic evaluation, and a mutation of α3 type IV collagen (COL4A3) was detected. Additional studies of the initial biopsy demonstrated abnormal type IV collagen immunostaining. A repeat biopsy 4 years later showed characteristic glomerular basement membrane morphology of Alport syndrome and scarring consistent with sequelae of IgA nephropathy. This is the first description of this unusual transition from an initial normal appearance of the glomerular basement membrane to the classic Alport phenotype.

Limited Utility of Fluorescence In Situ Hybridization for Recurrent Abnormalities in Acute Myeloid Leukemia at Diagnosis and Follow-up.

OBJECTIVES: Acute myeloid leukemia (AML) is classified in part by recurrent cytogenetic abnormalities, often detected by both fluorescent in situ hybridization (FISH) and karyotype. The goal of this study was to assess the utility of FISH and karyotyping at diagnosis and follow-up. METHODS: Adult AML samples at diagnosis or follow-up with karyotype and FISH were identified. Concordance was determined, and clinical characteristics and outcomes for discordant results were evaluated. RESULTS: Karyotype and FISH results were concordant in 193 (95.0%) of 203 diagnostic samples. In 10 cases, FISH detected an abnormality, but karyotype was normal. Of these, one had a FISH result with clinical significance. In follow-up cases, 17 (8.1%) of 211 showed FISH-positive discordant results; most were consistent with low-level residual disease. CONCLUSIONS: Clinically significant discordance between karyotype and AML FISH is uncommon. Consequently, FISH testing can safely be omitted from most of these samples. Focused FISH testing is more useful at follow-up, for minimal residual disease detection.

The duplication 17p13.3 phenotype: analysis of 21 families delineates developmental, behavioral and brain abnormalities, and rare variant phenotypes.

Chromosome 17p13.3 is a gene rich region that when deleted is associated with the well-known Miller-Dieker syndrome. A recently described duplication syndrome involving this region has been associated with intellectual impairment, autism and occasional brain MRI abnormalities. We report 34 additional patients from 21 families to further delineate the clinical, neurological, behavioral, and brain imaging findings. We found a highly diverse phenotype with inter- and intrafamilial variability, especially in cognitive development. The most specific phenotype occurred in individuals with large duplications that include both the YWHAE and LIS1 genes. These patients had a relatively distinct facial phenotype and frequent structural brain abnormalities involving the corpus callosum, cerebellar vermis, and cranial base. Autism spectrum disorders were seen in a third of duplication probands, most commonly in those with duplications of YWHAE and flanking genes such as CRK. The typical neurobehavioral phenotype was usually seen in those with the larger duplications. We did not confirm the association of early overgrowth with involvement of YWHAE and CRK, or growth failure with duplications of LIS1. Older patients were often overweight. Three variant phenotypes included cleft lip/palate (CLP), split hand/foot with long bone deficiency (SHFLD), and a connective tissue phenotype resembling Marfan syndrome. The duplications in patients with clefts appear to disrupt ABR, while the SHFLD phenotype was associated with duplication of BHLHA9 as noted in two recent reports. The connective tissue phenotype did not have a convincing critical region. Our experience with this large cohort expands knowledge of this diverse duplication syndrome.

Tnni3k modifies disease progression in murine models of cardiomyopathy.

The Calsequestrin (Csq) transgenic mouse model of cardiomyopathy exhibits wide variation in phenotypic progression dependent on genetic background. Seven heart failure modifier (Hrtfm) loci modify disease progression and outcome. Here we report Tnni3k (cardiac Troponin I-interacting kinase) as the gene underlying Hrtfm2. Strains with the more susceptible phenotype exhibit high transcript levels while less susceptible strains show dramatically reduced transcript levels. This decrease is caused by an intronic SNP in low-transcript strains that activates a cryptic splice site leading to a frameshifted transcript, followed by nonsense-mediated decay of message and an absence of detectable protein. A transgenic animal overexpressing human TNNI3K alone exhibits no cardiac phenotype. However, TNNI3K/Csq double transgenics display severely impaired systolic function and reduced survival, indicating that TNNI3K expression modifies disease progression. TNNI3K expression also accelerates disease progression in a pressure-overload model of heart failure. These combined data demonstrate that Tnni3k plays a critical role in the modulation of different forms of heart disease, and this protein may provide a novel target for therapeutic intervention.

Association of hereditary prostate cancer gene polymorphic variants with sporadic aggressive prostate carcinoma.

BACKGROUND: ELAC2, MSR1, and RNASEL are candidate genes for hereditary prostate carcinoma (HPC). While, studies have demonstrated that single nucleotide polymorphisms (SNPs) in these genes are associated with sporadic disease as well as HPC, these results are often not replicated in follow-up studies. Given that the majority of patients studied had localized disease and up to 50% of localized prostate cancer is clinically insignificant, the inability to replicate the initial findings may reflect that some subjects had indolent tumors. Herein, we examine patients with metastatic disease to determine if an association exists between HPC SNPs and unambiguously significant prostate cancer. METHODS: We examined polymorphisms within ELAC2 (S217L, A541T, E622V), MSR1 (P275A, R293X, aIVS5-59c), and RNASEL (E265X, R462Q, D541E) in 150 European-Americans with metastatic prostate cancer and 170 prostate cancer-free controls using pyrosequencing assays. RESULTS: Only ELAC2 217L (37% cases vs. 29% controls (P=0.034)) and RNASEL 541E (61% cases vs. 53% controls (P=0.045)) were over-represented. Analysis of genotypes revealed that presence of the leucine ELAC2 allele (OR 1.54: 95% CI=0.99-2.41, SS vs. SL, LL) and homozygosity for the glutamic acid RNASEL allele (OR 1.68: 95% CI=1.04-2.70, EE vs. DE, DD) were associated with increased risk. Patients with both genotypes were of particularly high-risk (OR 2.66: 95% CI=1.36-5.19). CONCLUSIONS: These results suggest that, in a European-American population, ELAC2 217L and RNASEL 541E are associated with metastatic sporadic disease. ELAC2 and RNASEL SNP analysis may prove useful in determining which patients are at risk for developing clinically significant prostate carcinoma.

QTL mapping in a mouse model of cardiomyopathy reveals an ancestral modifier allele affecting heart function and survival.

The progression from myocardial hypertrophy to heart failure is a complex process, involving genetic and environmental factors. Elucidating the genetic components contributing to heart failure has been difficult, largely because of the heterogeneity of human populations. We have employed a strategy to map genetic loci that modify the heart failure phenotype in a transgenic mouse model of cardiomyopathy caused by cardiac-specific overexpression of calsequestrin. Strain-specific differences in both cardiac function and survival are observed when the transgene is moved into different inbred mouse strains. We have previously reported linkage results from mapping in reciprocal backcrosses between C57/BL6 (BL6) and DBA/2J (DBA) and a backcross between DBA/AKR and AKR. Here we report the results of a genome-wide linkage scan in the reciprocal backcross between DBA/AKR and DBA. We identified one novel locus on Chromosome (Chr) 18 that affects heart function and a second on Chr 3 that shows significant linkage to both survival and heart function. Intriguingly, the Chr 3 allele of AKR shows a susceptibility effect on phenotype, whereas the overall effect of the AKR genetic background is protective. The Chr 3 locus also completely overlaps the Hrtfm2 locus, which was previously mapped in crosses between DBA and BL6. Mapping the same QTL in two different crosses allowed us to use ancestral haplotypes to narrow the candidate gene interval from 9 to 2 Mb. Identification of the genes at these QTLs in the mouse will provide novel candidate genes that can be evaluated for their role in human heart failure.