Cytogenetic and molecular analysis of the acute monocytic leukemia cell line THP-1 with an MLL-AF9 translocation

Cell lines derived from patients with leukemia are used in many molecular biology studies. Here we report the cytogenetic analysis of the THP-1 cell line using G-banding, fluorescence in situ hybridization (FISH), and spectral karyotyping (SKY), and the molecular characterization of the MLL-AF9 rearrangement by RT-PCR. The THP-1 cell line was established from the peripheral blood of a 1-year-old boy with acute monocytic leukemia (AML-M5). THP-1 is near-diploid and consists of two related subclones with a number of aberrations, including the t(9;11), associated with AML M5. The use of FISH allowed us to identify and characterize otherwise hidden cytogenetic rearrangements, which include duplication of the 3' portion of MLL in the derivative 9 chromosome and a deletion of the 5' portion of the AF9 gene involved in the translocation. In addition to confirming the FISH results, SKY allowed for a more precise characterization of the karyotype of THP-1 and allowed us to identify other abnormalities in this cell line, including der(1)t(1;12), der(20)t(1;20), deletions 6p, 12p, and 17p, trisomy 8, and monosomy 10. Sequencing of the RT-PCR product showed a direct in-frame fusion product on the derivative chromosome 11 between exon 6 (exon 9) of MLL and exon 5 of AF9, which is most commonly involved in MLL-AF9 translocations. This study demonstrates that combining different techniques to achieve a more precise characterization of the THP-1 cell line provides important information that will be valuable for understanding the critical events required for leukemogenesis

Further characterization of complex chromosomal rearrangements in myeloid malignancies: spectral karyotyping adds precision in defining abnormalities associated with poor prognosis

The mixed lineage leukemia gene (MLL, also known as HRX, ALL-1 and Htrx) located at 11q23 is involved in translocations with over 40 different chromosomal bands in a variety of leukemia subtypes. Here we report our analysis of a rare but recurring translocation, t(11;15)(q23;q14). This translocation has been described in a small subset of cases with both acute myeloblastic leukemia and ALL. Recent studies have shown that MLL is fused to AF15q14 in the t(11;15). Here we analyse a sample from another patient with this translocation and confirm the presence of an MLL-AF15q14 fusion. However, we have also identified and cloned another fusion transcript from the same patient sample. In this fusion transcript, MLL is fused to a novel gene, MLL partner containing FYVE domain (MPFYVE). Both MLL-AF15q14 and MLL-MPFYVE are in-frame fusion transcripts with the potential to code for novel fusion proteins. MPFYVE is also located on chromosome 15, approximately 170 kb telomeric to AF15q14. MPFYVE contains a highly conserved motif, the FYVE domain which, in other proteins, has been shown to bind to phosphotidyl-inositol-3 phosphate (PtdIns(3)P). The MLL-MPFYVE fusion may be functionally important in the leukemia process in at least some patients containing this translocation

Fatal Injuries in the Slums of Nairobi and their Risk Factors: Results from a Matched Case-Control Study

Injuries contribute significantly to the rising morbidity and mortality attributable to non-communicable diseases in the developing world. Unfortunately, active injury surveillance is lacking in many developing countries, including Kenya. This study aims to describe and identify causes of and risk factors for fatal injuries in two slums in Nairobi city using a demographic surveillance system framework. The causes of death are determined using verbal autopsies. We used a nested case-control study design with all deaths from injuries between 2003 and 2005 as cases. Two controls were randomly selected from the non-injury deaths over the same period and individually matched to each case on age and sex. We used conditional logistic regression modeling to identity individual- and community-level factors associated with fatal injuries. Intentional injuries accounted for about 51% and unintentional injuries accounted for 49% of all injuries. Homicides accounted for 91% of intentional injuries and 47% of all injury-related deaths. Firearms (23%) and road traffic crashes (22%) were the leading single causes of deaths due to injuries. About 15% of injuries were due to substance intoxication, particularly alcohol, which in this community comes from illicit brews and is at times contaminated with methanol. Results suggest that in the pervasively unsafe and insecure environment that characterizes the urban slums, ethnicity, residence, and area level factors contribute significantly to the risk of injury-related mortality

Molecular cytogenetic characterization of breakpoints in 19 patients with hematologic malignancies and 12p unbalanced translocations

Structural rearrangements of the short arm of chromosome 12 are frequent cytogenetic findings in various hematologic malignancies. The ETV6 gene is the most common target for rearrangements in 12p13. Fluorescence in situ hybridization (FISH) investigations have shown that translocations of 12p other than t(12;21) are frequently accompanied by small interstitial deletions that include ETV6. Unbalanced translocations involving ETV6 have rarely been described, and breakpoints outside ETV6 appear to be strongly associated with complex karyotypes. We studied bone marrow samples from 19 patients known to have 12p unbalanced translocations and complex karyotypes, using FISH and spectral karyotyping. FISH analysis confirmed the hemizygous deletion of the ETV6 and CDKN1B genes in 74% of cases. We found four cases with interstitial deletions. In these four cases and in two others (6/19, 31.5%), the fusion with the partner chromosome was in the subtelomeric region of 12p13.3, confirming that there is a recurrent breakpoint in this region

Systematic review and meta-analysis: prevalence of alcohol use among young people in eastern Africa.

OBJECTIVE: Systematic review and meta-analysis of published studies of alcohol use among young people (age 15-24 years) in eastern Africa to estimate prevalence of alcohol use and determine the extent of use of standardised screening questionnaires in alcohol studies. METHODS: Five databases (MEDLINE, EMBASE, Global Health, Africa-wide, and PsycINFO) were searched for publications until 30th June 2013. Results were summarised using the guidelines on preferred reporting items for systematic reviews and meta-analyses (PRISMA) and on quality assessment using the modified quality assessment tool for systematic reviews of observational studies (QATSO). Heterogeneity was assessed using the I(2) statistic (DerSimonian-Laird). RESULTS: We identified 2785 potentially relevant studies, of which 56 were eligible for inclusion. Only two studies (4%) used the standardised Alcohol Use Disorder Identification Test (AUDIT) questionnaire, and six studies (13%) used the Cut down, Annoyed, Guilt, Eye opener (CAGE) questionnaire. The reported median prevalence of alcohol use was ever-use 52% [interquartile range (IQR): 20-58%], use in the last month 28% (IQR: 17-37%), use in the last year 26% (IQR: 22-32%), and problem drinking as defined by CAGE or AUDIT 15% (IQR: 3-36%). We observed high heterogeneity between studies, with the highest prevalence of ever use of alcohol among university students (82%; 95%CI: 79-85%) and female sex workers (66%; 95%CI: 58-74%). Current use was most prevalent among male sex workers (69%; 95%CI: 63-75%). CONCLUSIONS: Reported alcohol use and problem drinking were common among diverse groups of young people in eastern Africa, indicating the urgent need for alcohol-focused interventions in this population. Few studies have used standardised alcohol screening questionnaires. Epidemiological research to investigate alcohol-focused interventions in young people should aim to apply such questionnaires that should be validated for use in this population

Identification of new translocations involving ETV6 in hematologic malignancies by fluorescence in situ hybridization and spectral karyotyping

TEL/ETV6 is the first transcription factor identified that is specifically required for hematopoiesis within the bone marrow. This gene has been found to have multiple fusion partners; 35 different chromosome bands have been involved in ETV6 translocations, of which 13 have been cloned. To identify additional ETV6 partner genes and to characterize the chromosomal abnormalities more fully, we studied bone marrow samples from patients known to have rearrangements of 12p, using fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). FISH analysis was done with 14 probes located on 12p12.1 to 12p13.3. Nine ETV6 rearrangements were identified using FISH. The aberrations include t(1;12)(p36;p13), t(4;12)(q12;p13) (two patients), t(4;12)(q22;p13), t(6;12)(p21;p13), der(6)t(6;21)(q15;q?)t(12;21)(p13;q22), t(6;12)(q25;p13), inv(12)(p13q24), and t(2;2;5;12;17)(p25;q23;q31;p13;q12). Six new ETV6 partner bands were identified: 1p36, 4q22, 6p21, 6q25, 12q24, and 17q12. Our present data as well previous data from us and from other researchers suggest that ETV6 is involved in 41 translocations. The breakpoints in ETV6 were upstream from the exons coding for the HLH (helix-loop-helix) domain in six cases. Although cytogenetic analysis identified 12p abnormalities in all cases, FISH and SKY detected new and unexpected chromosomal rearrangements in many of them. Thus, complete characterization of the samples was achieved by using all three techniques in combination