Decreased expression of GRAF1/OPHN-1-L in the X-linked alpha thalassemia mental retardation syndrome

Abstract

Background: ATRX is a severe X-linked disorder characterized by mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassemia. The disease is caused by mutations in ATRX gene, which encodes a protein belonging to the SWI/SNF DNA helicase family, a group of proteins involved in the regulation of gene transcription at the chromatin level. In order to identify specific genes involved in the pathogenesis of the disease, we compared, by cDNA microarray, the expression levels of approximately 8500 transcripts between ATRX and normal males of comparable age.

Methods: cDNA microarray was performed using total RNA from peripheral blood mononuclear cells of ATRX and normal males. Microarray results were validated by quantitative real-time polymerase chain reaction.

Results: cDNA microarray analysis showed that 35 genes had a lower expression (30-35% of controls) while 25 transcripts had a two-fold higher expression in comparison to controls. In the microarray results the probe for oligophrenin-1, a gene known for its involvement in mental retardation, showed a decreased hybridization signal. However, such gene was poorly expressed in blood mononuclear cells and its decrease was not confirmed in the quantitative real-time RT-PCR assay. On the other hand, the expression of an homologous gene, the GTPase regulator associated with the focal adhesion kinase 1/Oligophrenin-1-like (GRAF1/OPHN-1-L), was relatively high in blood mononuclear cells and significantly decreased in ATRX patients. The analysis of the expression pattern of the GRAF1/OPHN-1-L gene in human tissues and organs revealed the predominant brain expression of a novel splicing isoform, called variant-3.

Conclusions: Our data support the hypothesis of a primary role for altered gene expression in ATRX syndrome and suggest that the GRAF1/OPHN-1-L gene might be involved in the pathogenesis of the mental retardation. Moreover a novel alternative splicing transcript of such gene, predominantly expressed in brain tissues, was identified.

Figure 1

Quantitative mRNA expression in ATRX patients. A. qRT-PCR analysis of the OPHN-1, GRAF1/OPHN-1-L, G0/G1 and HAIK1 transcript levels in ATRX patients (n = 3, ATRX-1, ATRX-2, ATRX-3) and in normal controls (n = 8). Results are expressed as percentage of control average. Each bar represents the mean ± SEM. Statistical significance was determined by Student t test and indicated by asterisks: * p < 0.05; ** p < 0.01. B. qRT-PCR analysis showing a reduction of the GRAF1/OPHN-1-L transcript levels in the immortalized cell lines belonging to two ATRX patients, ATRX-1, ATRX-2. Results are expressed as percentage of control average (n = 4 immortalized cell lines from normal subjects). Each column is the average of three determination ± SD: * p < 0.01.

Figure 2

Drawing and tissue distribution of GRAF1/OPHN-1-L isoforms. A. Schematic representation of the exon 20-to-22 of GRAF1/OPHN-1-L variant-1 -2 and -3. Variant-1 contains the whole exon 21 reported in NCBI GenBank as exon 21b, variant-2 has exon 21-I (reported in NCBI GenBank as exon 21a) while exclusion of exon 21 results in the variant-3. B. RT-PCR amplification of the GRAF1/OPHN-1-L transcripts (variant-1: 724 bp; variant-2: 559 bp; variant-3: 448 bp) was confirmed in neural e non neural tissues (Lm: lymphomonocytes, Cx: cerebral cortex, K: kidney) with the other following primers: F18-R22b. C. Tissue distribution of GRAF1/OPHN-1-L isoforms. RT-PCR amplification of the GRAF1/OPHN-1-L transcripts (variant-1: 633 bp; variant-2: 468 bp; variant-3: 357 bp) from various human organs and tissues with following primers: F19-R22. L: 100 bp ladder; Lm: lymphomonocytes, He: heart, Cx: cerebral cortex, S: spleen, SM: skeletal muscle, Li: liver, Lu: lung, K: kidney, T: testis, O: ovary, P: pancreas, H: hippocampus, MO: medulla oblongata, Cb: cerebellum, St: striatum, SC: spinal cord.

Figure 3

Alpha/beta ratio in ATRX lymphomonocytes. Alpha-globin/beta-globin mRNA ratio measured by qRT-PCR in lymphomonocytes from the peripheral blood of patients, ATRX-1, ATRX-2, ATRX-3 compared to control subjects (CTRLs).

Figure 4

Alpha/beta ratio in ATRX reticulocytes. qRT-PCR analysis showing a reduction of alpha/beta ratio in reticulocytes purified from the peripheral blood of patients (ATRX-1 and ATRX-2) compared to control (pooled samples of six normal subjects). Data are expressed as % of control and represent mean ± SD of two independent experiments. Statistical significance was determined by Student t test and indicated by asterisk: * p < 0.05.