Statistical Analysis

Statistical analysis was performed using procedures implemented in the Statistical Product and Service Solution package (SPSS, Chicago, IL). A one-way analysis of variance was used to assay the association of genotype classes and YTHDF2 relative mRNA levels. The test was applied on log-transformed relative expression data, which presented a normal distribution. Monte-Carlo chi-square and Fisher’s Exact Tests were applied to test the differences in genotype and allele frequency distributions between age classes, and to test the differences between observed and expected homozygosity and/or heterozygosity and genotype distribution in the two age classes. Proportion of expected (under Hardy–Weinberg conditions) homozygotes and heterozygotes in each age group were, respectively, calculated as P (p )2 and 1   P (p )2, where p are the fre- i i i quencies of single alleles in each group.

Electronic Database Information

Genomic sequences and chromosomal positions cited in this article are referred to in the July 2003 human reference sequence (UCSC version hg16) and are available at the UCSC Web site (http://www.genome.ucsc.edu). The Censor program for the analysis of repetitive elements and Repbase

database of repetitive elements are both available at the Genetic Information Research Institute Web site (http:// www.girinst.org). The Repeat Masker program is available at http://www.repeatmasker.org. The count of Expressed Sequence Tags (ESTs) relative to the YTHDF2 gene is available at the UniGene section of the National Center for Biotechnology Information (NCBI) Web site (http:// www.ncbi.nlm.nih.gov). Sequences deposited in GenBank are available at http://www.ncbi.nlm.nih.gov/Genbank, and the Web site of the single nucleotide polymorphism data- base is http://www.ncbi.nlm.nih.gov/SNP. The Swiss-Prot database of protein sequences and the Protein Family Data- base are available, respectively, at http://www.expasy.org/ sprot and http://www.sanger.ac.uk/Software/Pfam. Guide- lines for Human Gene Nomenclature are available at the Human Genome Variation Society Web site (http://www. genomic.unimelb.edu.au/mdi).

       

R ESULTS

Figure 1. Alu density in a genomic region (chromosome 1: 27,500,001– 30,100,000) corresponding to 1p35.3 and neighboring 100 kb portions of 1p36.11 and 1p35.2. Alu density (represented by height of histograms) was calculated in nonoverlapping windows (each 100 kb long) along the chromosome. Below each histogram is indicated the position of the corresponding sequence of chromosome 1. Black histogram represents 100 kb sequence containing the YTHDF2 gene; empty space between the 9th and 10th histograms (from left)¼gap in the human reference sequence. Black horizontal line indicates average Alu density in the human genome (10.8%).

Cloning and Sequencing

the QM376-400 polymorphism and having an estimated length of 384 bp was isolated and cloned. The DNA inserts from four clones showed a restriction pattern identical to that of the DNA fragment used for the cloning, and were sequenced. The DNA fragments had an identical sequence (GenBank accession no. CL526225), with an effective length of 382 bp and containing a TG microsatellite with 16 repeats. A BLAST (Basic Local Alignment Search Tool) search against the human genome revealed that this sequence corresponds to a portion of the fourth intron of the YTHDF2 gene, on chromosome 1p35.3 (but with a different number of TG repeats: 16 in our sequence, 22 in the UCSC human reference sequence). The repeat element composition of the sequence corresponding to the QM376- 400 fragment and to the surrounding portion of YTHDF2 fourth intron (chr. 1: 28,773,429–28,775,861), was analyzed using the Censor program (27) and the Repbase database of repetitive elements (28). As expected, the cloned QM376- 400 fragment is flanked by two Alu sequences positioned in inverted orientation: an Alu Sx of 302 bp, and an Alu Y of 278 bp. The tail of Alu Y is followed by the (TG)n microsatellite. The remaining part of the sequence is con- stituted by a 290 bp portion of a MER element (medium reiterated frequency repeat) (34,35) that, based on the com- parison with the Repbase database of repetitive elements (28), is a Golem-B/MER7B type DNA nonautonomous transposon. The region surrounding the locus is almost entirely constituted, for roughly 2.5 kb, by three fragments of a MER transposon separated by intervening Alu elements. The reciprocal positions of these repetitive elements could be explained assuming that, in the evolutionary history of the locus, an ancient MER transposon became the target for successive insertions of new elements.