High-Frequency Genetic Contents Variations in Clinical

May 2011 629
Table 5. Comparsion of Retrotransposon Copy Numbers between Control and Test Isolates as Revealed by Array CGH
Retrotransposon Orf
(orf19.111). A total of four genes RIM9, orf19.102; KAR5,
and orf19.104 encode proteins with unknown functions and
the rest four genes are annotated with molecular functions:
HAL22 is a putative phosphoadenosine-5-phosphate (PAP)
or 3-phosphoadenosine 5-phosphosulfate (PAPS) phosphatase;
orf19.107 is an RNA helicase; orf19.109 is an
tyrosin-tRNA ligase; while CAN2 is an arginine transmembrane
transporter. Consensus FDRs was less than 0.001 for
this region. qPCR confirmed the copy number variations of
the above genes (Table 4).
Functional Analysis of Genes with Copy Number Variations
We attempted to analyze the molecular functions of
158 annotated genes that were shared between at least two
isolates, with the Gene Onthology (GO) Term Finder, as well
as with GO Slim Mapper. Both tools are provided by the
Candida Genome Database (CGD). Go Term Finder searches
for GO terms significantly shared by the query genes. The
p-value was set to be 0.1. GO Slim Mapper maps annotations
of a group of genes to more general terms and/or bins
them into broad categories. We used chi-square test for significance
analysis of the GO Slim Mapper result. For Go
Term Finder, the query set was the 158 variable genes with
annotations of molecular functions, and the background set
of genes was specified as the total of C. albicans genes annotated
with molecular functions, excluding genes with unknown
function. We found no enrichment in any functional
category with the two tools.
Aneuploidy of an Entire Chromosome or a Large Portion
of Chromosome Array CGH unambiguously revealed
chromosomal aneuploidy in two isolates: the duplication of
an entire chromosome 1 in S197 and the loss of an approximately
55 kb portion of chromosome R in S241, as presented
in Fig. 3. The segmental aneuploidy in S241 extended from
479692 to 535105 bp encompassing a total of twenty genes.
One gene, orf19.3746, was excluded from array CGH analysis
because of the bad hybridization efficiency of genomic
DNA from both SC5314 and S241 with the probes on the microarray.
Ratios (S241/SC5314) of three genes, orf19.3749,
orf19.3735, and orf19.3734, were approximately 1.0, thus,
implying no change of the DNA amounts. A simple explanation
of this result is the translocation of these genes on the
other chromosome(s). Ratios of the remaining sixteen genes
were approximately 0.5, which indicated the loss of one
copy.
In addition, we found that the MTLa locus on chromosome
5 that spans approximately 9 kb is homozygous in S727. Although
our microarrays lacked the probes representing the
OBPa, PIKa, PAPa, and MTLa2 genes from the MTLa
locus, we used PCR approach to amplify these genes from
both MTL loci from the same batch of genomic DNA, which
we used for array CGH. We found MTLa, but no MTLa
locus (Fig. 4A, Table 6). Furthermore, we used qPCR to confirm
that the MTLa locus is present in two copies (Fig. 4B,
Table 3). Future research is needed to establish if the entire
chromosome 5 or, alternatively, one copy of MTL was lost
and subsequently duplicated.
DISCUSSION
Ratio (test/control)
P546 U885 F32 S241 S904 S204 S197 S727
Zorro2(0.017 a) ) Orf19.7274 0.94 1.41 1.25 1.03 1.42 1.68 0.88 1.47
Orf19.7275 1.12 1.23 1.54 1.02 1.44 1.65 0.80 1.66
Zorro3(0.19) Orf19.559 1.04 1.06 1.32 1.09 0.92 1.11 1.05 3.30
Tca2(1.2E-06) Orf19.2371 0.01 0.46 0.97 0.36 1.00 0.52 0.01 0.48
Orf19.2372 0.03 0.46 1.34 0.36 1.80 0.46 0.02 0.42
Tca3(5.1E-05) Orf19.2219 0.42 0.68 0.30 0.41 0.39 0.80 0.25 0.86
Tca8 (5.12E-05) Orf19.6078 0.42 0.39 0.58 0.97 0.30 0.33 0.75 0.81
Orf19.6079 0.29 0.34 0.29 0.91 0.28 0.32 0.81 0.81
Tca17(1) Orf19.6807 1.01 0.98 0.94 0.96 0.98 0.95 0.97 0.97
Tca4(0.0016) Orf19.2668 0.10 0.10 0.07 0.97 1.89 1.03 1.00 0.93
Orf19.2669 0.10 0.10 0.06 0.96 1.90 1.02 1.08 1.03
Tca11(1.2E-06) Orf19.6469 0.26 0.53 0.22 1.59 1.08 0.49 0.12 1.20
a) Consensus FDR across all the test samples.
Fig. 3. Segmental Loss on Chromosome R in Isolate S241
Panel A: Each row corresponds to a specific spot (gene) on the microarray. The genes
are arranged according to their chromosomal coordinates on chromosome R. Each column
corresponds to a test isolate. The status of each gene is indicated as follows: black,
present; red, amplified; green, absent. The hybridization ratios are in logarithmic scale.
Panel B presents the systemic names and array CGH profiles of these genes.
The extent of genomic variation within a species is