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

Clinical findings and ATRX gene mutations

Three male patients, ATRX-1 (age 12 years), ATRX-2 (age 12 years) and ATRX-3 (age 14 yrs), presenting peculiar features of the ATRX syndrome, were analysed in the present study after informed consent by parents or ligal guardians. All patients showed a profound mental retardation, microcephaly, facial dysmorphisms, and short stature (all below the 3^rd centile). Clinical findings and ATRX gene mutations of each patient were previously described [9, 17].

Peripheral blood mononuclear cell isolation

Peripheral blood mononuclear (PBM) cells were isolated from 5 ml of EDTA (K₃) [K₃E] blood by Ficoll gradient procedure. The whole blood was diluted with Hanks's solution (volume ratio 1:1) (Life Technologies, Paisley, Scotland) and was added to tube with Ficoll Separating Solution (volume ratio 1:1) (BIOCHROM KG, Berlin). After centrifugation for 35 min a 400 g, the mononuclear cells containing layer was carefully pipetted away and resuspended in Phosphate Buffer Saline (PBS, pH 7.4). The cellular pellet was washed in PBS solution two times.

RNA extraction and cDNA synthesis

Total RNA was extracted as described by Chomczynski and Sacchi [18]. The quantity and purity of RNA were confirmed by spectrophotometry and agarose gel electrophoresis. Reverse transcription was performed using total RNA, RNase H^- reverse transcriptase (Superscript II, Gibco BRL, Life Technologies, Gaithersburg, MD) and random primer hexamers.

cDNA microarray

cDNA microarray data was obtained using a Human UniGene 1 Microarray (IncyteGenomics, St. Louis, Missouri, USA) that contains a set of 9128 clones corresponding to 8524 unique genes/EST clusters. The analysis has been performed on pooled RNA extracted by PBMC pellet of three ATRX patients in comparison to that obtained from a pool of 42 normal males (7.6 ± 2.4 years). The results were analyzed with the GEMTOOLS Analysis Software (Incyte Genomics). Raw and processed cDNA microarray data have been submitted to public repository: "Gene Expression Omnibus-GEO" http://www.ncbi.nlm.nih.gov/geo with the accession number GSE22028.

Separation of reticulocytes and immortalized lymphoblasts of control and pathological subjects

Approximately 10 ml peripheral blood in heparin were obtained from controls and patients. Peripheral blood samples were washed 3 times in reticulocyte saline buffer (4°C) (RS: 1.3 M NaCl, 0.05 M KCl, 0.074 M MgCl₂.6H₂O pH 7.4) and then centrifuged at 10000 g for 20 minutes at 4°C and 0.5 ml was removed from the reticulocyte-rich top layer. Reticulocytes were concentrated by centrifugation at 3500 rpm for 30 min at 4°C. The supernatant was removed, and the top 0.3-0.5 ml reticulocyte-rich fraction was added to 2 ml of ice-cold RS and layered on a column consisting of 10 ml of 2 parts cellulose (C-8002, Sigma-Aldrich, Steinheim, Germany) and 1 part sigmacell type 50 microcrystallin cellulose (S-5504, Sigma-Aldrich, Steinheim, Germany) in RS. The red cells were eluted by centrifuging the column at 1200 rpm at 4°C for 2 min. The eluate was washed three times in 10 ml of ice-cold PBS and then the reticulocyte enriched fraction was recovered by centrifugation and resuspended in 2 ml of ice-cold PBS. RNA was extracted with TRI reagent (T-9424, Sigma-Aldrich, Steinheim, Germany). Lymphoblasts from EBV cell lines of controls (Immort CTRLs) and patients (Immort ATRX-1 and ATRX-2) were maintained in RPMI-1640 (Gibco, Invitrogen, Scotland, UK) media supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum and penicillin-streptomycin (50 units/50 μg/ml). The cell cultures were incubated at 37°C in a humidified 5% CO2 incubator and the culture medium was changed twice a week.

Human brain tissues and non cerebral RNAs

Brain tissue from five human subjects were studied: four males (ages 18, 26, 46, and 61) and one female (age 22) with postmortem interval between 4 and 36 hours. The specimens had been obtained at autopsy from the Forensic Medicine Department at the Karolinska Institute under guidelines approved by the ethics committee and the Swedish Board of Health and Social Welfare. The specimens were frozen in dry ice-cooled isopentane and stored at -80°C. We have only access to blocks of tissue from selected areas (spinal cord, medulla oblongata, cerebellum, striatum, hippocampus and neocortex). Total RNA was extracted as described by Chomczynski and Sacchi [18].

The human non cerebral total RNAs were commercially obtained from Ambion.Inc (Austin, TX): pancreas (cod.7954); liver (cod.7960); heart (cod.7966); lung (cod.7968); spleen (cod.7970); testis (cod.7972); ovary gland (cod.7974); kidney (cod.7976); skeletel muscle (cod.7982).

mRNA splice variants by PCR analysis

To detect GRAF1/OPHN-1-L alternative transcripts were used the following primers: F19: CCGATATGCCTCTCACCAAT; F18: GCAGCCATCATGGACATCAA; R22: AGACCGTGCCTGCTGTGAAC; R22b: CATTATCGAAGACCGTGCCTG. Amplification were performed under the following conditions: denaturation step at 95°C for 3 min; 95°C × 1 min, 67°C for F19-R22 or 60°C for F18-R22b × 2 min, 72°C × 3 min × 35 cycles. Amplification products were separated by 1,8% agarose gel electrophoresis and visualized with ethidium bromide. To confirm the identify of alternative amplified variants, DNA fragments were recovered by glass-milk method [19] and sequencing was performed by standard fluorescent dideoxy chain-termination procedure with the Abi Prism 377 automatic sequencer.

Quantitative Real Time RT-PCR

Quantitative Real Time RT-PCR (qRT-PCR) experiments were performed in the ABI PRISM 7700 Sequence Detection System from Applied Biosystems. Three sequence-specific oligonucleotides were designed using the Primer Express oligo design software (Applied Biosystems) based on the sequence of target gene. The following genes were analyzed: human Oligophrenin-1 (OPHN-1, GenBank ID: NM_002547, clone ID: 4216520), transcriptional regulator ATRX isoform-1 (XNP/ATRX, GenBank ID: NM_000489), GRAF1/Oligophrenin-1-like (GRAF1/OPHN-1-L, GenBank ID: NM_015071), Alpha hemoglobin (GenBank ID: NM_000558), Beta hemoglobin (GenBank ID: NM_000518), HAIK1 type I, intermediate filament cytocheratin or human Keratin 23 gene (HAIK1 or KRT23, clone ID: 2820372), putative lymphocyte G0/G1 switch or G0S2 gene (G0/G1 or G0S2, GenBank ID: BE873759,clone ID 1217764).

Primers and probes used for qRT-PCR are reported in Additional file 1 Table S1. Details of the conditions used for qRT-PCR are previously described [20].

Identification of the genes whose expression is altered in ATRX peripheral blood cells

In order to confirm the results obtained by cDNA microarray analysis, we performed qRT-PCR assays for some transcripts among those showing the largest changes in gene expression (see in Table 1 ranking genes down regulated in ATRX according to the ratio ATRX/control). As shown in Figure 1 the putative lymphocyte G0/G1 switch (or G0S2) gene mRNA levels (position 1 in Table 1), and the incyte EST clone ID 2820372, HAIK1 type I, intermediate filament cytocheratin or human Keratin 23 gene (KRT23, GeneID: 25984, position 4 in Table 1) were decreased in the freshly separated lymphomonocytes obtained by three different ATRX patients confirming the cDNA microarray data. On the contrary, the quantitative analysis in qRT-PCR has not confirmed the strong reduction of oligophrenin-1 (OPHN-1) mRNA (3.3 times lower in ATRX subjects) observed by cDNA microarray (Figure 1A). However, cDNA microarray data can be influenced by other transcripts with a high level of homology. With this in mind, a bioinformatics search of the OPHN-1 gene homologs has been executed. Such analysis revealed the presence of an "Oligophrenin-1-like (OPHN-1-L)" transcript deriving from the gene encoding for a previously identified "RhoGAP" protein, called "GRAF1" (GTPase regulator associated with the focal adhesion kinase pp125FAK or Rho-type GTPase-activating protein 26) [21]. The probe sequence in the microarray (Incyte clone ID 4216520) covers part of the 5' coding sequence of the oligophrenin sequence which is 65% homologue to GRAF1, a value that is enough to produce an hybridization signal. Indeed, by quantitative RT-PCR OPHN-1 mRNA levels in PBM were one order of magnitude lower than GRAF1 levels, suggesting that the latter transcript was the major determinant of the hybridization signal.

RNA from control subjects and ATRX patients was examined by qRT-PCR analysis in order to evaluate the level of GRAF1/OPHN-1-L transcript. These results confirmed a reduction of the "GRAF1/OPHN-1-L" transcript levels with values equal to 41.64% (SEM 7.48) of the controls in the three examined subjects (Figure 1A).

In order to confirm the observed transcript variations in PBM cells we examined the GRAF1/OPHN-1-L expression in immortalized lymphocyte lines of normal and ATRX subjects. As it can be observed in Figure 1B the GRAF1/OPHN-1-L mRNA levels were drastically reduced in ATRX immortalized lines compared to immortalized control cells. OPHN-1 mRNA levels were not detectable in both ATRX and control cell lines (no amplification to 40 cycles in 90% of the analyzed samples, data not shown).

Identification of a new splicing GRAF1/oligophrenin-1-like transcript, variant-3

The search in the database, using as query sequence "oligophrenin-1" (accession number NM_002547) supplied 2 sequences: the nucleotide sequence "GRAF1/OPHN-1-L, variant-1"(deposited with accession number NM_015071) and the nucleotide sequence "GRAF1/OPHN-1-L, variant-2" (accession number NM_001135608).

The comparison of the two transcript isoforms of "GRAF1/OPHN-1-L" sequences demonstrated that they were two splicing variants of the same gene (Additional file 2 Figure S1). The "GRAF1/OPHN-1-L" gene, localized on chromosome 5q31, is composed by 23 exons and the complete translation produces a protein of 814 amino acids corresponding to the sequence deposited as "GRAF1/OPHN-1-L, variant-1". In the "GRAF1/OPHN-1-L, variant-2" is present only a 5'segment of exon 21, while the "GRAF1/OPHN-1-L, variant-1" sequence contains the whole exon 21. Therefore, the whole exon 21, of 276 nucleotides, could be subdivided in two fragments: the exon 21-I (from nucleotide position 2024 to 2134 in the sequence with accession number NM_015071: 111 bases encoding a 37-amino acids sequence) and the exon 21-II (from the nucleotide position 2135 to 2299: 165 bases encoding a 55-amino acids sequence). The transcript variant-1 contains the whole exon 21 (exon21-I + exon21-II encoding a sequence of 92 amino acids; exon 21 is reported in NCBI GenBank as exon 21b), while only exon 21-I (reported in NCBI GenBank as exon 21a) is present in the transcript variant-2 (Figure 2A, Table 3, Additional file 2 Figure S1).

Name	Number of exons	GenBank ID	Exon number involved in splice site (number of base involved)	Total number of aminoacids
GRAF1/OPHN-1-L, variant-1	23	NM_015071	Exon 21b 21-I + 21-II (2024-2299) 276 nt	814
GRAF1/OPHN-1-L, variant-2	23	NM_001135608	Exon21a 21-I (2024-2134) 111 nt	759
GRAF1/OPHN-1-L, variant-3	22	HM037040	No Exon 21	641

The splicing of the exon 21-II does not modify the reading frame, so that variant-2 differs from variant-1 only for the lack of 55 amino acids close to the carboxy-terminal portion. Moreover the exon 21-II contains the canonical splice acceptor and donor sites gt...ag in the correct positions (Additional file 2 Figure S1). To investigate the presence of GRAF1/OPHN-1-L transcripts a RT-PCR analysis was performed using specific primers to amplify the zone including exon 21. In particular two forward primers (F18, F19), localized to the exon 18 and 19, and two reverse primers (R22 and R22b), localized to the exon 22, have been synthetized. RT-PCR amplification with the primers F18 and R22b, generated 3 amplification bands of 724 bp, 559 bp, and 448 bp (Figure 2B). RT-PCR analysis, performed with the primers F19 and R22 generated 3 amplification bands of 633 bp, 468 bp, and 357 bp (Figure 2C). The sequencing of the amplification products revealed that the bands of greater size (724 bp, 633 bp) contained the whole exon 21 (21-I + 21-II), the intermediate bands (559 bp, 468 bp) contained only exon 21-I, while the band of smaller size (448 bp, 357 bp) did not contain exon 21. Therefore, besides the two known transcripts (variant-1 and variant-2), one additional transcript variant (variant-3) was detected in such analysis, suggesting that GRAF1/OPHN-1-L gene generated three alternatively spliced transcripts differing in the use of exon 21 (Additional file 2 Figure S1). The translation of variant-3 sequence produces a protein of 641 amino acids. The sequence of variant-3 has been submitted to NCBI GenBank with accession number HM037040.

Tissue distribution of "GRAF1/oligophrenin-1-like" isoforms

GRAF1/OPHN-1-L expression was examined in specific brain regions, as well as in a series of other adult tissues. Figure 2C shows the tissue distribution of the GRAF1/OPHN-1-L variants obtained using the forward primer F19, localized to exon 19, and the reverse primers R22, localized to exon 22. The GRAF1/OPHN-1-L variant-3 was predominantly and highly expressed in the human cerebral areas (357 bp, Figure 2C). In the adult brain, the transcript variant-3 was expressed in all tested regions including cerebral cortex, hippocampus, medulla oblongata, cerebellum, striatum, and spinal cord. In parenchymatous organs, endocrine glands, muscular tissue and lymphomonocytes the variant-2 is predominant (468 bp, Figure 2C). On the contrary the variant-1 is weakly expressed in the cerebral tissues and in the peripheral lymphocytes.

mRNA levels for alpha-and beta-globins in reticulocytes

Since alpha-thalassemia is one of the main feature of the ATRX syndrome, changes in expression of the globin genes were also investigated. Probes for alpha-globin chains were not present in the cDNA microarray platform used for the present study, but we could detect significant increases in the levels of the beta, gamma and delta globin mRNAs in ATRX patients (values 2.76, 2.70, 2.66 times higher respectively, see Table 2). The same analysis did not reveal significant differences for the embryonic zeta-globin chains.

In order to confirm and to extend the cDNA microarray results we performed a qRT-PCR analysis for the alpha-and beta-globin transcripts. As reported in Figure 3 we found a reduction of the alpha/beta ratio (alpha globin mRNA/beta globin mRNA) in two ATRX patients (ATRX-1 and ATRX-2) with values of 0.26 and 0.24 (controls mean = 1 ± 0.43; Figure 3), while ATRX-3 had a value in the range of the controls (ATRX-3, alpha/beta ratio 0.78). It is well-known that hematologic findings vary widely among ATRX patients and in some cases the manifestation of alpha-thalassemia may be subtle [22]. Indeed, alpha/beta transcript ratios are in agreement with the presence of erythrocyte HbH inclusion bodies in ATRX-1 and ATRX-2 but not in ATRX-3. The percentage of HbH cells to normal RBCs, visualized by staining with 1% brilliant cresyl blue, were 1%, 3.7% and 0% in ATRX-1, ATRX-2 and ATRX-3, respectively.

The presence of detectable globin transcripts in RNAs extracted from lymphomonocyte samples suggested that these cell preparations contained a nonnegligible amount of contaminating reticulocytes. This hypothesis is supported by the observation that such transcripts showed a 3 times reduction in samples enriched for CD4+ lymphocytes and by the fact they were not detectable in RNA samples extracted by immortalized lymphocyte cell lines. On this basis, we decided to perform a quantitative analysis of alpha-and beta-globin transcripts in RNA samples extracted from reticulocytes isolated from the peripheral blood of control and ATRX subjects. Indeed, such analysis confirmed the reduction of the alpha/beta ratio in the ATRX subjects (Figure 4). However the absolute values of the alpha-and beta-globin transcripts showed remarkable variations between samples and did not allow to establish if the modification of the ratio is due to a decrease of alpha-globin transcripts and/or an increase of beta-globin ones.