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Denaturing HPLC-based assay for detection of ATRX gene mutations.

To the Editor:

In 2003, we described (1) a broad-range denaturing gradient gel electrophoresis method for mutation scanning of the entire open reading frame and canonical splice sites of the ATRX gene (OMIM 300032), a zinc finger transcriptional regulator undergoing X inactivation and probably involved in chromatin remodeling, DNA methylation, and gene expression in mammalian development (2,3). Mutations affecting the ATRX gene lead to the [alpha]-thalassemia/mental retardation syndrome (ATR-X syndrome; OMIM 301040).

We now propose a simpler, rapid mutational approach based on denaturing HPLC (DHPLC) (4), with which we were able to confirm all of the nucleotide variations described in our first report (1) and to detect 5 other mutations in 7 of 15 unrelated Italian patients with a clinical suspicion of ATR-X syndrome. Segregation of the syndrome was sporadic in all but 2 individuals. X-inactivation status at the human androgen receptor locus was tested in all patients' mothers as described previously (5).

PCR primers (Table 1 of the Data Supplement that accompanies the online version of this letter at http:// issue7/) were designed to amplify all 35 exons and the consensus splicing sites of the ATRX gene (Entrez Gene ID 546). A total of 44 reactions were performed at a single annealing temperature (57[degrees]C) with Optimase DNA polymerase in 1 x buffer (both from Transgenomic); exon 9 was amplified as 10 overlapping fragments. Hetero-duplex formation was obtained by mixing together, denaturing, and gradually reannealing equimolar quantities of PCR products for patients and controls. DHPLC analysis was performed with the WAVE[TM] 3500HT System (Transgenomic). Each crude PCR product (50 [micro]L) was eluted by a 2.5-min run at 3 different analysis temperatures, and the buffer gradients were chosen as suggested by the Navigator software (Transgenomic; Table 1 of the online Data Supplement). Wild-type controls were included in each run. For the heterozygous elution profiles, genomic DNA was reamplified with the DHPLC primers and sequenced using Big-Dye sequencing chemistry (Applied Biosystems). No false-positive results were reported. In the 8 patients negative by DHPLC analysis, direct sequencing failed to detect any deleterious variations. The DHPLC results are summarized in Table 1 of the online Data Supplement.

The new p.L195P (c.584T>C) mutation identified in this study was not found in 200 apparently healthy females; moreover, this amino acid belongs to the zinc finger domain, was conserved during evolution (data not shown), and is segregated in a 3-generation pedigree with a classic ATR-X phenotype and a skewed X-inactivation status in carrier females.

The chromatograms for all mutations found in our patients, including those described in our first report (1), are shown in Fig. 1.

Taking into account the time needed to set up and run the PCR and to perform DHPLC analysis, we were able to screen the entire coding region of the ATRX gene in ~16 h compared with 1 week for denaturing gradient gel electrophoresis analysis.


Mutations in the ATRX gene have also been found in other X-linked mental retardation syndromes (6) and recently in the rare [alpha]-thalassemia associated with multilineage myelodysplasia (7). This evidence suggests that, particularly in familial cases in which haplotype segregation analysis does not exclude the ATRX gene as disease locus, mutational screening should be enlarged to cover a broad spectrum of X-linked mental retardation phenotypes. This emphasizes the importance of a sensitive, rapid mutational analysis approach to this gene.


(1.) Borgione E, Sturnio M, Spalletta A, Angela Lo Giudice M, Castiglia L, Galesi O, et al. Mutational analysis of the ATRX gene by DGGE: a powerful diagnostic approach for the ATRX syndrome. Hum Mutat 2003;21: 529-34.

(2.) Gibbons RJ, Picketts DJ, Villard L, Higgs DR. Mutations in a putative global transcriptional regulator cause X-linked mental retardation with [alpha]-thalassemia (ATR-X syndrome). Cell 1995;80: 837-45.

(3.) Gibbons RJ, McDowell TL, Raman S, O'Rourke DM, Garrick D, Ayyub H, et al. Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. Nat Genet 2000;24: 368-71.

(4.) Xiao W, Oefner PJ. Denaturing high-performance liquid chromatography: a review. Hum Mutat 2001;17: 439-74.

(5.) Kubota T, Nonoyama S, Tonoki H, Masuno M, Imaizumi K, Kojima M, et al. A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR. Hum Genet 1999; 104: 49-55.

(6.) Yntema HG, Poppelaars FA, Derksen E, Oudakker AR, Van Roosmalen T, Jacobs A, et al. Expanding phenotype of XNP mutations: mild to moderate mental retardation. Am J Med Genet 2002;110: 243-7.

(7.) Gibbons RJ, Pellagatti A, Garrick D, Wood WG, Malik N, Ayyub H, et al. Identification of acquired somatic mutations in the gene encoding chromatin-remodeling factor ATRX in the [alpha]-thalassemia myelodysplasia syndrome (ATMDS). Nat Genet 2003;34: 446-9.

Michele Falco [1]

Daniela Luciano [1]

Maurizio Sturnio [1]

Angela Spalletta [1]

Domenico Scionti [1]

Mariangela Lo Giudice [1]

Corrado Romano [2]

Marco Ficheral [1] *

[1] Laboratorio di Diagnosi Genetica and

[2] Unita Operativa Complessa di Pediatria e Genetica Medica Istituto di Ricovero e Cura a Carattere Scientifico Oasi Maria Santissima Troina, Italy

* Address correspondence to this author at: Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Oasi Maria Santissima, via Conte Ruggero 73, 94018 Troina, Italy. Fax 39-0935-653327; e-mail

DOI: 10.1373/clinchem.2005.052407
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Title Annotation:Letters
Author:Falco, Michele; Luciano, Daniela; Sturnio, Maurizio; Spalleta, Angela; Scionti, Domenico; Giudice, M
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Jul 1, 2005
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