The ASXL1 Gene Alterations in Patients with Myeloid Diseases and Chromosomal 20q Deletion

Deletion of the long arm of chromosome 20 - del(20q) - is a recurrent abnormality observed in various myeloid disorders, including myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), or acute myeloid leukemia (AML). It is a primary cytogenetic aberration, occurring often as a sole abnormality or the first abnormality in complex karyotypes, therefore it is assumed to play a key role in pathogenesis of myeloid malignancies. The proximal breakpoints of the deletion are consistently located in the 20q11.21 band, and the distal breakpoints span from 20q13.13 to band 20q13.33. In our previous study we showed a fusion of the ASXL1 and TSHZ2 genes resulting from an isochromosome of a deleted 20q in a patient with MDS (Brezinova et al Br J Haematol 2014). The ASXL1 gene is one of the most frequently mutated genes in myeloid disorders, mutations are generally associated with more aggressive course of the disease and poor clinical outcome. The aim of this study was to determine the frequency of ASXL1 breakpoints and/or ASXL1/TSHZ2 fusion in del(20q) cases. Fluorescence in situ hybridizations (FISH) with locus specific probes for 20q11 and 20q12 regions (Abbott, Des Plaines, IL; Kreatech Diagnostics, Amsterdam, The Netherlands) confirmed the cytogenetically observed deletions of 20q in a cohort of 20 patients with myeloid malignancies (MDS 13x, MPN 3x, AML 2x, myelofibrosis 1x, thrombocytopenia 1x). In 15 patients deletion of 20q was a sole aberration, in 2 patients a variant of del(20q), an isochromosome of deleted 20q, was detected by FISH with a subtelomeric probe, Vysis ToTel 20p/20q (Abbott). Metaphase FISH mapping with a set of 7 bacterial artificial chromosome (BAC) probes (BlueGnome, Cambridge, UK) distributed in 20q11.21 and 20q13.2, with a chromosome-20-specific centromeric probe (Kreatech Diagnostics) as a control was used for determination of the breakpoints. Array comparative genomic hybridization (CytoChip Cancer 180K, BlueGnome) was performed on DNA samples of bone marrow cells of 11 patients with suspected ASXL1 gene deletion to find out the gene copy number variation. A weak signal of RP11-358N2 BAC probe (30.93-31.12 Mb from telomere on the short arm, 20q11.21) was observed in 6 patients(30%), suggesting the proximal breakpoint of the deletion in the ASXL1 gene (30.95-31.03 Mb). However, the distal breakpoint in the TSHZ2 gene (51.80-52.11 Mb; 20q13.2) was found in one patient only, as previously reported. In 6 patients (30%) the signal of RP11-358N2 BAC probe was not present on the derivative chromosome confirming the ASXL1 gene deletion. In the remaining 8 patients the proximal breakpoint of the deletion was determined distally to the ASXL1 gene, in 3 patients into 9.3 Mb region between the MAPRE and the PTPRT genes. None but 1 patient had the distal breakpoint localized in the TSHZ2 gene. Three patients died in a group of 12 patients with ASXL1 gene alteration (deletion or partial deletion). Using combination of molecular cytogenetic methods we proved that the extent of 20q deletions varied among the patients with a frequent proximal breakpoint in the ASXL1 gene. In our cohort, the ASXL1 gene was altered (deleted or partially deleted) in totally 60% of patients. The determination of the ASXL1 gene alteration in del(20q) cases may have a clinical and prognostic impact, but the relations with clinical data should be studied in a larger cohort of patients. Supported by MHCR project for conceptual development of research organization 00023736, RVO-VFN64165/2012, and GACR P302/12/G157/1. Disclosures No relevant conflicts of interest to declare.

Selective sweep near the In(2L)t inversion breakpoint in an African population of Drosophila melanogaster

Chromosomal inversions largely inhibit recombination and may be associated with selective forces, such as hitch-hiking effects: the effect of positive selection on linked loci. A West African population of Drosophila melanogaster showed a high frequency (0·61) of the In(2L)t inversion. Departure from neutrality statistically associated with the inversion polymorphism was previously recorded at Su(H), a locus distant from the proximal breakpoint of the inversion. These results were consistent with hitch-hiking effects with recombination. The present sequence polymorphism survey involves a 1 kb fragment of the Vha68-1 locus located closer to the proximal breakpoint of the inversion. It shows a significant deficit of polymorphism with respect to divergence when compared with other loci studied in the same population, thus suggesting selective effects. Only 11 polymorphic sites are present in a sample of 20 chromosomes and these sites present a significant excess of rare-frequency variants. The major haplotype shows an unexpectedly high frequency. Our estimate of the background selection effect is not sufficient to account for the observed reduction of polymorphism. Intraspecific variation is structured between inverted and standard chromosomes; there are no shared polymorphisms but also no fixed differences between them. This pattern, together with that found on other loci previously studied near this inversion breakpoint, suggests hitch-hiking effects enhanced by the inversion.

Molecular Variation at the In(2L)t Proximal Breakpoint Site in Natural Populations of Drosophila melanogaster and D. simulans

A previous study of nucleotide polymorphism in a Costa Rican population of Drosophila melanogaster found evidence for a nonneutral deficiency in the number of haplotypes near the proximal breakpoint of In(2L)t, a common inversion polymorphism in this species. Another striking feature of the data was a window of unusually high nucleotide diversity spanning the breakpoint site. To distinguish between selective and neutral demographic explanations for the observed patterns in the data, we sample alleles from three additional populations of D. melanogaster and one population of D. simulans. We find that the strength of associations among sites found at the breakpoint varies between populations of D. melanogaster. In D. simulans, analysis of the homologous region reveals unusually elevated levels of nucleotide polymorphism spanning the breakpoint site. As with American populations of D. melanogaster, our D. simulans sample shows a marked reduction in the number of haplotypes but not in nucleotide diversity. Haplotype tests reveal a significant deficiency in the number of haplotypes relative to the neutral expectation in the D. simulans sample and some populations of D. melanogaster. At the breakpoint site, the level of divergence between haplotype classes is comparable to interspecific divergence. The observation of interspecific polymorphisms that differentiate major haplotype classes in both species suggests that haplotype classes at this locus are considerably old. When considered in the context of other studies on patterns of variation within and between populations of D. melanogaster and D. simulans, our data appear more consistent with the operation of selection than with simple demographic explanations.

Unusual Haplotype Structure at the Proximal Breakpoint of In(2L)t in a Natural Population of Drosophila melanogaster

The existence of temporally stable frequency clines for In(2L)t in natural populations of Drosophila melanogaster suggests a role for selection in the maintenance of this polymorphism. We have collected nucleotide polymorphism data from the proximal breakpoint junction regions of In(2L)t to infer its evolutionary history. The finding of a novel LINE-like element near the In(2L)t breakpoint junction in sampled inverted chromosomes supports a transposable element-mediated origin for this inversion. An analysis of nucleotide variation in a Costa Rican population sample of standard and inverted chromosomes indicates a unique and relatively recent origin for In(2L)t. Additional In(2L)t alleles from three geographically diverse populations reveal no detectable geographic differentiation. Low levels of In(2L)t nucleotide polymorphism suggest a recent increase in the inversion's frequency in tropical populations. An unusual feature of our sample of standard alleles is a marked heterogeneity in levels of linkage disequilibrium among polymorphic sites across the breakpoint region. We introduce a test of neutral equilibrium haplotype structure that corrects both for multiple tests and for an arbitrarily chosen window size. It reveals that an ~1.4-kb region immediately spanning the breakpoint has fewer haplotypes than expected under the neutral model, given the expected level of recombination in this genomic region. Certain features of our data suggest that the unusual pattern in standard chromosomes is the product of selection rather than demography.

Behavior and cytogenetics of fruitless in Drosophila melanogaster: different courtship defects caused by separate, closely linked lesions.

The fruitless (fru) courtship mutant was dissected into three defects of male reproductive behavior, which were separable as to their genetic etiologies by application of existing and newly induced chromosomal aberrations. fru itself is a small inversion [In(3R) 90C; 91B] on genetic and cytological criteria. Uncovering the fru distal breakpoint with deletions usually led to males with two of the fru courtship abnormalities: no copulation attempts with females (hence, behavioral sterility) and vigorous courtship among males, including the formation of "courtship chains." However, certain genetic changes involving region 91B resulted in males who formed courtship chains but who mated with females. Uncovering the fru proximal breakpoint led to males that passively elicit inappropriately high levels of courtship. This elicitation property was separable genetically from the sterility and chain formation phenotypes and provisionally mapped to the interval 89F-90F, which includes the fru proximal breakpoint. Behavioral sterility and chaining were also observed in males expressing certain abnormal genotypes, independent of the fru inversion. These included combinations of deficiencies, each with a breakpoint in 91B, and a transposon inserted in 91B.

MATERNAL AND ZYGOTIC INTERACTIONS BETWEEN THE ABNORMAL OOCYTE MUTATION AND THE SCUTE4 INVERSION IN DROSOPHILA MELANOGASTER

ABSTRACT The authors have studied the interaction between the abnormal oocyte mutation and an inversion of the X chromosome, In(1)sc 4, which has a proximal breakpoint in or near the heterochromatic region (ABO) that maternally interacts with the abo product. It has been demonstrated that the presence of X chromosomes carrying this inversion, besides a marked increase in the severity of the maternal effect of the abo mutation, produces a zygotic effect resulting in the lethality of the progeny of stocks homozygous for abo and sc 4. These results indicate that the sc 4 inversion carries an abnormal region indispensable for the development of abo zygotes from sc 4;abo mothers.