Identification of a unique Alu-based polymorphism and its use in human population studies

Alu elements represent a family of short interspersed DNA elements (SINEs) found in primate genomes. These are members of a group of transposable elements that integrate into the genome by the process of retrotransposition. Recent integrations of Alu elements within the human genome have generated presence/absence variants useful as DNA markers in human population studies as well as in forensic and paternity analyses. Besides the ease of use, this type of marker is unique because the absence of the Alu represents the ancestral form. We have identified an Alu-based polymorphism that consists of four alleles in which we can predict the evolutionary order. Additionally, we have developed a simple PCR plus restriction endonuclease assay to readily distinguish the four alleles. We have thus far analyzed DNA from a small set of samples comprising ten different ethnic groups. The three populations of African descent exhibited a relatively low frequency of the absence allele in contrast to the other populations, as well as being the only populations in which all four alleles were identified. One presence allele was not found in both European Caucasian and South American populations that were sampled, whereas a different presence allele was not observed among the sampled Asian populations. Additionally, the four-allele system identified variations among populations not observed by simply scoring as presence/absence variants. Therefore, extending beyond the two-allele dimorphic Alu system further elucidates population variations. These features afford this marker as a unique tool in the study of both global and regional analyses of human populations.     

Most human Alu and murine B1 repeats are unique

Alus and B1s are short interspersed repeat elements (SINEs) indirectly derived from the 7SL RNA gene. While most researchers recognize that there exists extensive variability between individual elements, the extent of this variability has never been systematically tested. We examined all Alu elements over 200 nucleotides and all B1 elements over 100 nucleotides in the human and mouse genomes, and analyzed the number of copies of each element at various stringencies from 22 nucleotides to full length. Over 98% of 923,277 Alus and 365,377 B1s examined were unique when queried at full length. When the criterion was reduced to half the length of the repeat, 97% of the Alus and 73% of the B1s were still found to be a single copy. All single and multi-copy sequences have been mapped and documented. Access to the data is possible using the AluPlus website http://www.ibr.hawaii.edu.     

Alu and LINE1 distributions in the human chromosomes: evidence of global genomic organization expressed in the form of power laws

Spatial distribution and clustering of repetitive elements are extensively studied during the last years, as well as their colocalization with other genomic components. Here we investigate the large-scale features of Alu and LINE1 spatial arrangement in the human genome by studying the size distribution of interrepeat distances. In most cases, we have found power-law size distributions extending in several orders of magnitude. We have also studied the correlations of the extent of the power law (linear region in double-logarithmic scale) and of the corresponding exponent (slope) with other genomic properties. A model has been formulated to explain the formation of the observed power laws. According to the model, 2 kinds of events occur repetitively in evolutionary time: random insertion of several types of intruding sequences and occasional loss of repeats belonging to the initial population due to "elimination" events. This simple mechanism is shown to reproduce the observed power-law size distributions and is compatible with our present knowledge on the dynamics of repeat proliferation in the genome.     

Effect of alcohol consumption on peripheral blood Alu methylation in Korean men

Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create a substantial public health problem worldwide. To date, no therapeutic can effectively solve these problems. They are complex diseases characterized by both genetic and environmental factors. DNA methylation can act as a downstream effector of environmental signals and account for multi-factorial nature of the disease. Global DNA methylation of peripheral blood cells has recently been proposed as a potential biomarker for disease risk. Alu elements host one-quarter of CpG dinucelotides in the genome to function as proxies for global DNA methylation. In this study, we evaluated the Alu methylation in the peripheral blood DNA of healthy volunteers and AUD patients using the pyrosequencing technology. The Alu methylation level is significantly higher in AUD compared to healthy controls (23.4?±?1.6 versus 22.1?±?1.0, t?=?7.83, p?<?0.0001). Moreover, significant correlation was found between Alu methylation and alcohol use disorders identification test score (r?=?0.250, p?<?0.0001), alcohol problem (r?=?0.294, p?<?0.0001), and life position (r?=?–0.205, p?=?0.0005). Overall, these novel findings indicate that alcohol-related increase in Alu methylation might play a complex role in the etiology and pathogenesis of AUD. Further studies are required to elucidate the mechanisms underlying this relationship.     

Evolutionary evidence suggests that CpG island-associated Alus are frequently unmethylated in human germline

Most Alu elements are considered to belong to the methylated fraction of the genome that has undergone CpG depletion, whereas CpG islands are characteristically unmethylated. By analysing the CpG content of >12,000 autosomal CpG island-Alu Sx pairs we wanted to study what happens when an Alu is situated close to a CpG island. We have found that many Alus located close to CpG islands have retained a high proportion of CpG sites, which is consistent with these Alus being unmethylated in the human germline.     

Diagnostic Role of Cell-free DNA Integrity in Thyroid Cancer Particularly for Bethesda IV Cytology

BackgroundThe cell-free DNA integrity index (cfDI) is promising for the differentiation between malignant and benign tumors, but little data has been reported on thyroid cancer (TC). We explored its diagnostic role in TC, mainly in cases of Bethesda category IV.MethodscfDI was evaluated by quantitative real-time polymerase chain reaction using 2 primer sets to identify cell-free DNAs (cfDNAs) Alu83 and Alu244. Blood samples were collected from 85 patients with thyroid nodules (18 papillary [PTC], 21 follicular [FTC], 21 medullary, and 25 benign thyroid nodules [BTN]) before fine-needle aspiration cytology and surgical treatment and also from 25 patients with autoimmune thyroid disease (ATD) and 25 healthy subjects (HS).ResultscfDNA Alu244 concentration ≥6.95 ng/mL and cfDI ≥0.3 were excellent sensitive and specific tests to discriminate TC particularly cytologically indeterminate thyroid nodules (Bethesda IV) from the control groups (BTN, ATD, and HS). The levels of both cfDNA Alu83 and Alu244 were decreased while cfDI was increased significantly in medullary compared with FTC and PTC, with a nonsignificant difference between the latter subgroups. There was a significantly positive correlation between both cfDNA Alu83 and Alu244 with the T-classification of TNM staging and capsular invasion among PTC and FTC patients and between cfDI with Bethesda categories. Additionally, ATD had significantly higher cfDNA Alu83 and lower cfDI than HS.ConclusioncfDI is a useful noninvasive molecular biomarker in TC that correlates with the Bethesda classification and histopathology. Tumor size and capsular invasion were correlated with quantitative cfDNA among PTC and FTC.     

Characterization of DNA rearrangements of N-myc gene amplification in three neuroblastoma cell lines by pulsed-field gel electrophoresis

We characterized N-myc gene amplification in three human neuroblastoma cell lines (IMR-32, TGW, GOTO). Rearrangements in long-range regions surrounding amplified N-myc genes were examined by pulsed-field gel electrophoresis. Since rare-cutting enzymes completely digested DNA at the middle of the N-myc gene, we were able to construct a physical map upstream and downstream of the germline N-myc gene, and to obtain information on restriction sites surrounding amplified N-myc genes. This method enables us to envisage the organization of amplified units over a long range. Digestion patterns differed considerably among the germline and the three cell lines, but were simple in each case. We estimated that the minimal distance between neighboring N-myc genes is at least several hundred kilobases. Our data suggest that amplification units contain several DNA fragments derived from ditterent loci, but that they are homogeneous.