A Gradual Transition Toward Anaplasia in Wilms Tumor Through Tolerance to Genetic Damage.

Patients with Wilms tumor (WT) in general have excellent survival, but the prognosis of patients belonging to the subgroup of WT with diffuse anaplasia (DA) is poor due to frequent resistance to chemotherapy. We hypothesized that DA WT cells might undergo changes, such as acquiring a persistent tolerance to DNA damage and copy number aberrations (CNAs), which could eventually lead to their resistance to chemotherapy treatment. Tissue sections from chemotherapy-treated DA WTs (n = 12) were compared with chemotherapy-treated nonanaplastic WTs (n = 15) in a tissue microarray system, enabling analysis of 769 tumor regions. All regions were scored for anaplastic features and immunohistochemistry was used to quantify p53 expression, proliferation index (Ki67), and DNA double-strand breaks (γH2AX). CNAs were assessed by array-based genotyping and TP53 mutations using targeted sequencing. Proliferation index and the frequency of DNA double-strand breaks (γH2AX dot expression) increased with higher anaplasia scores. Almost all (95.6%) areas with full-scale anaplasia had TP53 mutations or loss of heterozygosity, along with an increased amount of CNAs. Interestingly, areas with wild-type TP53 with loss of heterozygosity and only one feature of anaplasia (anaplasia score 1) also had significantly higher proliferation indices, more DNA double-strand breaks, and more CNAs than regions without any anaplastic features (score 0); such areas may be preanaplastic cell populations under selective pressure for TP53 mutations. In conclusion, we suggest that chemoresistance of DA WTs may be partly explained by a high proliferative capability of anaplastic cells, which also have a high burden of double-stranded DNA breaks and CNAs, and that there is a gradual emergence of anaplasia in WT.

Validation of a targeted next-generation sequencing panel for pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDAC) is reported to be amongst the cancers with the lowest survival rate at 5 years. In the present study we aimed to validate a targeted next-generation sequencing (tNGS) panel to use in clinical routine, investigating genes important for PDAC diagnostic, prognostic and potential theragnostic aspect. In this NGS panel we also designed target regions to inquire about loss of heterozygosity (LOH) of chromosome 18 that has been described to be possibly linked to a worse disease progression. Copy number alteration has also been explored for a subset of genes. The last two methods are not commonly used for routine diagnostic with tNGS panels and we investigated their possible contribution to better characterize PDAC. A series of 140 formalin-fixed paraffin-embedded (FFPE) PDAC samples from 140 patients was characterized using this panel. Ninety-two % of patients showed alterations in at least one of the investigated genes (most frequent KRAS, TP53, SMAD4, CDKN2A and RNF43). Regarding LOH evaluation, we were able to detect chr18 LOH starting at 20% cell tumor percentage. The presence of LOH on chr18 is associated with a worse disease- and metastasis-free survival, in uni- and multivariate analyses. The present study validates the use of a tNGS panel for PDAC characterization, also evaluating chr18 LOH status for prognostic stratification.

The Importance of Confirming False Homozygosity in Pretransplant HLA Typing Results of Patients with Hematologic Malignancies.

Loss of heterozygosity (LOH) on chromosome 6p, where the HLA genes are located, can result in incorrect homozygosity findings during HLA genotyping in patients with hematologic malignancies. The degree of HLA compatibility between donor and recipient is crucial in hematopoietic stem cell transplantation. Therefore, we present a case of false homozygosity in HLA genotyping due to LOH on chromosome 6p in a patient diagnosed with acute myeloid leukemia (AML). HLA molecular typing was conducted on both peripheral blood and buccal swab samples. The analysis included sequence-based typing (SBT) and next-generation sequencing-based typing. Additionally, chromosomal microarray analysis (CMA) was performed. A 68-year-old male presented with anemia and thrombocytopenia. Subsequent bone marrow examination confirmed AML. High-resolution HLA genotyping of Peripheral blood during blast crisis revealed homozygosity at the -A, -B, and -C loci. Conventional karyotyping showed a normal karyotype, 46,XY[20]. Retesting of HLA genotyping one week later confirmed the homozygous results. Subsequently, HLA typing was repeated using buccal swab specimens, confirming heterozygosity at all 4 HLA loci. CMA on peripheral blood samples during blast crisis revealed a large terminal region of copy-neutral LOH spanning approximately 43.5 Mb in the chromosome region 6p25.3p21.1. LOH at the HLA gene locus can significantly impact donor selection, potentially leading to the selection of mistakenly identified homozygous donors. Clinicians and laboratory personnel should be aware of these issues to prevent erroneous HLA typing results in patients with hematologic malignancies. It is advisable to confirm the HLA typing of recipients with hematologic malignancies whenever homozygosity is detected at any locus. This can be achieved through careful interpretation of low peaks in SBT, and by using buccal swab samples or peripheral blood collected after achieving remission.

An overlooked subset of Cx3cr1wt/wt microglia in the Cx3cr1CreER-Eyfp/wt mouse has a repopulation advantage over Cx3cr1CreER-Eyfp/wt microglia following microglial depletion.

BACKGROUND: Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-Eyfp/wt mouse strain for studies of microglia. METHODS: Immunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1CreER-Eyfp/wt mouse brains. Genetically mediated microglia depletion using Cx3cr1CreER-Eyfp/wtRosa26DTA/wt mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies. RESULTS: We unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia in Cx3cr1CreER-Eyfp/wt mouse brains, thus termed Cx3cr1highCre-Eyfp- microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1highCre-Eyfp- microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1highCre-Eyfp- microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1highCre-Eyfp- microglia are Cx3cr1wt/wtCre-Eyfp-. Finally, we demonstrated that CX3CL1-CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro. CONCLUSIONS: Our results raise a cautionary note regarding the use of Cx3cr1CreER-Eyfp/wt mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.

Vitamin B6 Deficiency Promotes Loss of Heterozygosity (LOH) at the Drosophila warts (wts) Locus

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is a cofactor for more than 200 enzymes involved in many metabolic pathways. Moreover, PLP has antioxidant properties and quenches the reactive oxygen species (ROS). Accordingly, PLP deficiency causes chromosome aberrations in Drosophila, yeast, and human cells. In this work, we investigated whether PLP depletion can also cause loss of heterozygosity (LOH) of the tumor suppressor warts (wts) in Drosophila. LOH is usually initiated by DNA breakage in heterozygous cells for a tumor suppressor mutation and can contribute to oncogenesis inducing the loss of the wild-type allele. LOH at the wts locus results in epithelial wts homozygous tumors easily detectable on adult fly cuticle. Here, we found that PLP depletion, induced by two PLP inhibitors, promotes LOH of wts locus producing significant frequencies of wts tumors (~7% vs. 2.3%). In addition, we identified the mitotic recombination as a possible mechanism through which PLP deficiency induces LOH. Moreover, LOH of wts locus, induced by PLP inhibitors, was rescued by PLP supplementation. These data further confirm the role of PLP in genome integrity maintenance and indicate that vitamin B6 deficiency may impact on cancer also by promoting LOH.

Homozygosity for a Novel DOCK7 Variant Due to Segmental Uniparental Isodisomy of Chromosome 1 Associated with Early Infantile Epileptic Encephalopathy (EIEE) and Cortical Visual Impairment.

Early infantile epileptic encephalopathy (EIEE) is a severe neurologic and neurodevelopmental disease that manifests in the first year of life. It shows a high degree of genetic heterogeneity, but the genetic origin is only identified in half of the cases. We report the case of a female child initially diagnosed with Leber congenital amaurosis (LCA), an early-onset retinal dystrophy due to photoreceptor cell degeneration in the retina. The first examination at 9 months of age revealed no reaction to light or objects and showed wandering eye movements. Ophthalmological examination did not show any ocular abnormalities. The patient displayed mildly dysmorphic features and a global developmental delay. Brain MRI demonstrated pontine hypo-/dysplasia. The patient developed myoclonic epileptic seizures and epileptic spasms with focal and generalized epileptiform discharges on electroencephalogram (EEG) at the age of 16 months. Genetic screening for a potentially pathogenic DNA sequence variant by whole-exome sequencing (WES) revealed a novel, conserved, homozygous frameshift variant (c.5391delA, p.(Ala1798LeufsTer59)) in exon 42 of the DOCK7 gene (NM_001271999.1). Further analysis by SNP array (Karyomapping) showed loss of heterozygosity (LOH) in four segments of chromosome 1. WES data of the parents and the index patient (trio analysis) demonstrated that chromosome 1 was exclusively inherited from the mother. Four LOH segments of chromosome 1 alternately showed isodisomy (UPiD) and heterodisomy (UPhD). In WES data, the father was a noncarrier, and the mother was heterozygous for this DOCK7 variant. The DOCK7 gene is located in 1p31.3, a region situated in one of the four isodisomic segments of chromosome 1, explaining the homozygosity seen in the affected child. Finally, Sanger sequencing confirmed maternal UPiD for the DOCK7 variant. Homozygous or compound heterozygous pathogenic variants in the DOCK7 (dedicator of cytokinesis 7) gene are associated with autosomal recessive, early infantile epileptic encephalopathy 23 (EIEE23; OMIM #615,859), a rare and heterogeneous group of neurodevelopmental disorders diagnosed during early childhood. To our knowledge, this is the first report of segmental uniparental iso- and heterodisomy of chromosome 1, leading to homozygosity of the DOCK7 frameshift variant in the affected patient.

The genetic basis of oral leukoplakia and its key role in understanding oral carcinogenesis.

Oral leukoplakia (OL) is the most common oral potentially malignant disorder, with a global prevalence of 2%-3%, variable malignant transformation rate and incompletely understood aetiology. Considering the subjectivity in oral dysplasia grading, other evaluation methods have been tested as predictors of malignant transformation. DNA ploidy status and loss of heterozygosity signatures have been shown to be good predictive markers of malignant transformation. However, effective markers to predict which lesions will progress to invasive carcinoma and by which mechanisms remain unclear. Recent evidence suggests that dysplasia progression to carcinoma occurs through neutral clonal evolution (i.e. randomly). We focus on the genetic basis of OL, encompassing the gross chromosomal alterations and single-gene mutations, and discuss such alterations in the context of aetiology, clinical presentation and progression. The deeper we understand the genetic basis of OL, the more we approach a better comprehension of the complex and poorly understood process of oral carcinogenesis.

Role of miR-24 in Multiple Endocrine Neoplasia Type 1: A Potential Target for Molecular Therapy.

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominant inherited multiple cancer syndrome of neuroendocrine tissues. Tumors are caused by an inherited germinal heterozygote inactivating mutation of the MEN1 tumor suppressor gene, followed by a somatic loss of heterozygosity (LOH) of the MEN1 gene in target neuroendocrine cells, mainly at parathyroids, pancreas islets, and anterior pituitary. Over 1500 different germline and somatic mutations of the MEN1 gene have been identified, but the syndrome is completely missing a direct genotype-phenotype correlation, thus supporting the hypothesis that exogenous and endogenous factors, other than MEN1 specific mutation, are involved in MEN1 tumorigenesis and definition of individual clinical phenotype. Epigenetic factors, such as microRNAs (miRNAs), are strongly suspected to have a role in MEN1 tumor initiation and development. Recently, a direct autoregulatory network between miR-24, MEN1 mRNA, and menin was demonstrated in parathyroids and endocrine pancreas, showing a miR-24-induced silencing of menin expression that could have a key role in initiation of tumors in MEN1-target neuroendocrine cells. Here, we review the current knowledge on the post-transcriptional regulation of MEN1 and menin expression by miR-24, and its possible direct role in MEN1 syndrome, describing the possibility and the potential approaches to target and silence this miRNA, to permit the correct expression of the wild type menin, and thereby prevent the development of cancers in the target tissues.

Pitfalls in mononucleotide microsatellite repeats instability assessing (MSI) in the patients with B-cell lymphomas.

Analysis of microsatellite instability (MSI) is a routine study in the diagnostics of solid malignancies. The standard for determining MSI is a pentaplex PCR panel of mononucleotide repeats: NR-21, NR-24, NR-27, BAT-25, BAT-26. The presence of MSI is established based on differences in the length of markers in the tumor tissue and in the control, but due to the quasimonomorphic nature of standard mononucleotide loci the use of a control sample is not necessary in the diagnosis of MSI-positive solid tumors. The significance of the MSI phenomenon in oncohematology has not been established. This paper presents the results of a study of MSI in B-cell lymphomas: follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBL). We have shown that aberrations of mononucleotide markers occur in these diseases, but the nature of the changes does not correspond to the classical MSI in solid neoplasms. This fact requires further study of the pathogenesis of such genetic disorders. Due to the possibility of ambiguous interpretation of the results of the MSI study for previously uncharacterized diseases, strict compliance with the methodology of parallel analysis of the tumor tissue and the control sample is mandatory.

Tumor genetic alterations and features of the immune microenvironment drive myelodysplastic syndrome escape and progression.

The transformation and progression of myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (sAML) involve genetic, epigenetic, and microenvironmental factors. Driver mutations have emerged as valuable markers for defining risk groups and as candidates for targeted treatment approaches in MDS. It is also evident that the risk of transformation to sAML is increased by evasion of adaptive immune surveillance. This study was designed to explore the immune microenvironment, immunogenic tumor-intrinsic mechanisms (HLA and PD-L1 expression), and tumor genetic features (somatic mutations and altered karyotypes) in MDS patients and to determine their influence on the progression of the disease. We detected major alterations of the immune microenvironment in MDS patients, with a reduced count of CD4+ T cells, a more frequent presence of markers related to T cell exhaustion, a more frequent presence of myeloid-derived suppressor cells (MDSCs), and changes in the functional phenotype of NK cells. HLA Class I (HLA-I) expression was normally expressed in CD34+ blasts and during myeloid differentiation. Only two out of thirty-six patients with homozygosity for HLA-C groups acquired complete copy-neutral loss of heterozygosity in the HLA region. PD-L1 expression on the leukemic clone was also increased in MDS patients. Finally, no interplay was observed between the anti-tumor immune microenvironment and mutational genomic features. In summary, extrinsic and intrinsic immunological factors might severely impair immune surveillance and contribute to clonal immune escape. Genomic alterations appear to make an independent contribution to the clonal evolution and progression of MDS.

Detection of Loss of Heterozygosity in cfDNA of Advanced EGFR- or KRAS-Mutated Non-Small-Cell Lung Cancer Patients.

Liquid biopsy is currently approved for management of epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) patients. However, one unanswered question is whether the rate of cell-free DNA (cfDNA)-negative samples is due to technical limitations rather than to tumor genetic characteristics. Using four microsatellite markers that map specific chromosomal loci often lost in lung cancer, we conducted a pilot study to investigate whether other alterations, such as loss of heterozygosity (LOH), could be detected in EGFR-negative cfDNA. We analyzed EGFR-mutated NSCLC patients (n = 24) who were positive or negative for EGFR mutations in cfDNA and compared the results with a second cohort of 24 patients bearing KRAS-mutated cancer, which served as a representative control population not exposed to targeted therapy. The results showed that in EGFR-negative post-tyrosine-kinase-inhibitor (TKI) cfDNAs, LOH frequency was significantly higher than in both pre- and post-TKI EGFR-positive cfDNAs. By contrast, no association between KRAS status in cfDNA and number of LOH events was found. In conclusion, our study indicates the feasibility of detecting LOH events in cfDNA from advanced NSCLC and suggests LOH analysis as a new candidate molecular assay to integrate mutation-specific assays.

Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini.

BACKGROUND: Rust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. Plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. Identifying avirulence genes is crucial for understanding how virulence evolves in the field. RESULTS: To facilitate avirulence gene cloning in the flax rust fungus, Melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated DNA sequencing (RADseq) data. The map comprises 13,412 RADseq markers in 27 linkage groups that together span 5860 cM and contain 2756 recombination bins. The marker sequences were used to anchor 68.9 % of the M. lini genome assembly onto the genetic map. The map and anchored assembly were then used to: 1) show that M. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the AvrL2 and AvrM14 avirulence genes by map-based cloning. AvrM14 is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. AvrL2 is located in the region of the M. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein. CONCLUSIONS: The M. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes.

An integrated analysis tool for analyzing hybridization intensities and genotypes using new-generation population-optimized human arrays.

BACKGROUND: Affymetrix Axiom single nucleotide polymorphism (SNP) arrays provide a cost-effective, high-density, and high-throughput genotyping solution for population-optimized analyses. However, no public software is available for the integrated genomic analysis of hybridization intensities and genotypes for this new-generation population-optimized genotyping platform. RESULTS: A set of statistical methods was developed for an integrated analysis of allele frequency (AF), allelic imbalance (AI), loss of heterozygosity (LOH), long contiguous stretch of homozygosity (LCSH), and copy number variation or alteration (CNV/CNA) on the basis of SNP probe hybridization intensities and genotypes. This study analyzed 3,236 samples that were genotyped using different SNP platforms. The proposed AF adjustment method considerably increased the accuracy of AF estimation. The proposed quick circular binary segmentation algorithm for segmenting copy number reduced the computation time of the original segmentation method by 30-67 %. The proposed CNV/CNA detection, which integrates AI and LOH/LCSH detection, had a promising true positive rate and well-controlled false positive rate in simulation studies. Moreover, our real-time quantitative polymerase chain reaction experiments successfully validated the CNVs/CNAs that were identified in the Axiom data analyses using the proposed methods; some of the validated CNVs/CNAs were not detected in the Affymetrix Array 6.0 data analysis using the Affymetrix Genotyping Console. All the analysis functions are packaged into the ALICE (AF/LOH/LCSH/AI/CNV/CNA Enterprise) software. CONCLUSIONS: ALICE and the used genomic reference databases, which can be downloaded from http://hcyang.stat.sinica.edu.tw/software/ALICE.html , are useful resources for analyzing genomic data from the Axiom and other SNP arrays.

The c.*229C > T gene polymorphism in 3'UTR region of the topoisomerase IIß binding protein 1 gene and LOH in BRCA1/2 regions and their effect on the risk and progression of human laryngeal carcinoma.

Topoisomerase IIß binding protein 1 (TopBP1), a multiple-BRCT-domain, protein plays crucial roles in chromosome replication, DNA damage repair, apoptosis, and cell cycle checkpoint signalling. The aim of this study was to identify five SNPs at loci potentially located in the 3'UTR region of the TopBP1 gene (rs185903567, rs116645643, rs115160714, rs116195487, rs112843513), their relationship with the risk of squamous cell laryngeal cancer (SCLC), tumor invasiveness, and prognosis. Genotyping was performed in 323 genetically unrelated individuals with SCLC and 418 randomly selected healthy volunteers. Allele-specific TopBP1 mRNA and protein expressions were determined by using real-time PCR and Western blotting techniques, respectively. LOH in BRCA1/BRCA2 was determined by using microsatellite markers. Compared to homozygous common allele carriers, heterozygosity for the T variant was associated with increased risk of SCLC (adjusted odds ratio [OR] = 9.83, 95 % confidence interval [CI]: 3.12-22.16, p dominant < 0.0001). The presence of risk allele at rs115160714 TopBP1 determined a higher incidence of nodal metastases (OR = 7.98, 95 % CI: 3.94-16.00, p = 0.001) and higher tumor grade (OR = 6.48, 95 % CI: 0.86-48.01, p = 0.03). The heterozygotes displayed diffuse tumor growth with no distinct borderline (OR = 3.10, 95 % Cl: 0.92-10.62, p = 0.049) and higher depth of invasion (OR = 2.66, 95 % Cl: 0.78-9.03, p = 0.04). Relationships were also identified between TopBP1 mRNA/protein expression and overall survival (p < 0.0001). The incidence of LOH in BRCA1/BRCA2 was significantly related to higher tumor grade and TFG (p < 0.05). The results of this study suggest that rs115160714 TopBP1 may be a genetic marker of etiology and progression in laryngeal cancer.

Analytical Validation of Loss of Heterozygosity and Mutation Detection in Pancreatic Fine-Needle Aspirates by Capillary Electrophoresis and Sanger Sequencing.

Pancreatic cystic disease, including duct dilation, represents precursor states towards the development of pancreatic cancer, a form of malignancy with relatively low incidence but high mortality. While most of these cysts (>85%) are benign, the remainder can progress over time, leading to malignant transformation, invasion, and metastasis. Cytologic diagnosis is challenging, limited by the paucity or complete absence of cells representative of cystic lesions and fibrosis. Molecular analysis of fluids collected from endoscopic-guided fine-needle aspiration of pancreatic cysts and dilated duct lesions can be used to evaluate the risk of progression to malignancy. The basis for the enhanced diagnostic utility of molecular approaches is the ability to interrogate cell-free nucleic acid of the cyst/duct and/or extracellular fluid. The allelic imbalances at tumor suppressor loci and the selective oncogenic drivers are used clinically to help differentiate benign stable pancreatic cysts from those progressing toward high-grade dysplasia. Methods are discussed and used to determine the efficacy for diagnostic implementation. Here, we report the analytical validation of methods to detect causally associated molecular changes integral to the pathogenesis of pancreatic cancer from pancreatic cyst fluids.

Tumor Predisposing Post-Zygotic Chromosomal Alterations in Bladder Cancer-Insights from Histologically Normal Urothelium.

Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.

Identification of a novel 91.5 kb-deletion (αα)FJ in the α-globin gene cluster using single-molecule real-time (SMRT) sequencing.

OBJECTIVES: To present a novel 91.5-kb deletion of the α-globin gene cluster (αα)FJ identified by genetic assay and prenatal diagnosis in a Chinese family. SUBJECTS AND METHODS: The proband was a 34-year-old G3P1 (Gravida 3, Para 1) female at the gestational age of 21+ weeks with a history of an edematous fetus. A routine genetic assay (reverse dot blot hybridization, RDB) was performed to detect common thalassemia mutations. Multiplex ligation-dependent probe amplification (MLPA) and single-molecule real-time technology (SMRT) were used to detect rare thalassemia mutations. RESULTS: The hematological phenotypes of the proband, her mother, elder sister, husband, daughter, and nephew were consistent with the phenotype of α-thalassemia trait. No mutations were found in these family members by RDB, except for the proband's husband who carried an α-globin gene deletion --SEA/αα. MLPA results showed that the proband and other α-thalassemia-suspected relatives had heterozygous deletions around the POLR3K-3-463nt, HS40-178nt, and HBA-HS40-382nt probes. The 5'-breakpoint was out of probe scope and could not be determined. SMRT was performed and a 91.5-kb deletion (NC_000016.10: g.39268_130758del) in the α-globin gene cluster (αα)FJ was identified in the proband and other suspected relatives, which could explain their phenotypes. At the proband's gestational age of 22+ weeks, an amniotic fluid sample was collected and analyzed. As only the 91.5-kb deletion (αα)FJ was identified in the fetus with RDB, MLPA, and SMRT. The proband was suggested to continue the pregnancy. CONCLUSION: We first reported a 91.5-kb deletion (NC_000016.10: g.hg38-chr16:39268-_130758del) of the HS-40 region in the α-globin gene cluster (αα)FJ identified in a Chinese family. Since the HS-40 loss of heterozygosity in combination with the heterozygous deletion --SEA might result in Hb Bart's hydrops fetalis, routine genetic assay, and SMRT were recommended to individuals at risk for prenatal diagnosis.

Genome diversity of Leishmania aethiopica.

Leishmania aethiopica is a zoonotic Old World parasite transmitted by Phlebotomine sand flies and causing cutaneous leishmaniasis in Ethiopia and Kenya. Despite a range of clinical manifestations and a high prevalence of treatment failure, L. aethiopica is one of the most neglected species of the Leishmania genus in terms of scientific attention. Here, we explored the genome diversity of L. aethiopica by analyzing the genomes of twenty isolates from Ethiopia. Phylogenomic analyses identified two strains as interspecific hybrids involving L. aethiopica as one parent and L. donovani and L. tropica respectively as the other parent. High levels of genome-wide heterozygosity suggest that these two hybrids are equivalent to F1 progeny that propagated mitotically since the initial hybridization event. Analyses of allelic read depths further revealed that the L. aethiopica - L. tropica hybrid was diploid and the L. aethiopica - L. donovani hybrid was triploid, as has been described for other interspecific Leishmania hybrids. When focusing on L. aethiopica, we show that this species is genetically highly diverse and consists of both asexually evolving strains and groups of recombining parasites. A remarkable observation is that some L. aethiopica strains showed an extensive loss of heterozygosity across large regions of the nuclear genome, which likely arose from gene conversion/mitotic recombination. Hence, our prospection of L. aethiopica genomics revealed new insights into the genomic consequences of both meiotic and mitotic recombination in Leishmania.

Accumulation of STR-Loci Aberrations in Subclones of Jurkat Cell Line as a Model of Tumor Clonal Evolution.

Many genetic markers are known to distinguish tumor cells from normal. Genetic lesions found at disease onset often belong to a predominant tumor clone, and further observation makes it possible to assess the fate of this clone during therapy. However, minor clones escape monitoring and become unidentified, leading to relapses. Here we report the results of in vitro study of clonal evolution in cultured tumor cell line (Jurkat) compared to the cell line of non-tumor origin (WIL2-S). Cell lines were cultured and cloned by limiting dilutions. Subclones were tested by short tandem repeats (STR) profiling. Spontaneous STR aberrations in cells of non-tumor origin occur in less than 1 of 100 cultured cells. While in the cells of tumor origin, new aberrations appear in 1 or even more of 3 cultured cells. At the same time, a significant relationship was found between the accumulation of aberrations in the pool of subclones and the rate of cell growth. One can speculate that this approach could be applied for the analysis of primary patient tumor cell culture to obtain information concerning the evolutionary potential of the tumor cells that may be useful for the selection of a therapy approach.