Development and validation of a multiplex-PCR assay for X-linked intellectual disability

BMC Medical Genetics

Rosário Santos¹  Isabel Marques¹  Bárbara Oliveira²  Paula Jorge¹ 

[1] Centro de Genética Médica Dr. Jacinto Magalhães, CHP, Praça Pedro Nunes 88, 4099-028, Porto, Portugal;New address: Instituto Nacional de Saúde Dr. Ricardo Jorge INSA I.P., Lisbon, Portugal

关键词: Multiplex-PCR;    ARX;    AFF2;    FMR1;    X-linked intellectual disability (XLID);   

Others  :  1122685
DOI  :  10.1186/1471-2350-14-80

 received in 2012-05-23, accepted in 2013-06-07,  发布年份 2013

【 摘 要 】

Background

X-linked intellectual disability is a common cause of inherited cognitive deficit affecting mostly males. There are several genetic causes implicated in this condition, which has hampered the establishment of an accurate diagnosis. We developed a multiplex-PCR assay for the mutational hotspot regions of the FMR1, AFF2 and ARX genes.

Methods

The multiplex-PCR was validated in a cohort of 100 males selected to include known alleles for the FMR1 repetitive region: five full mutations (250–650 CGGs), ten premutations (70–165 CGGs) and eighty-five in the normal range (19–42 CGGs). Sequencing or Southern blotting was used to confirm the results, depending on the allele class. In this cohort, with the exception of one sample showing an AFF2 intermediate-sized allele, all other samples were normal (8–34 CCGs). No ARX variant was found besides the c.429_452dup. The validated assay was applied to 5000 samples (64.4% males and 35.6% females).

Results

The normal-allelic range of both FMR1 and AFF2 genes as well as the nature of ARX variants identified was similar in both genders. The rate of homozygosity observed in female samples, 27.5% for FMR1 and 17.8% for AFF2 alleles, is comparable to that published by others. Two FMR1 premutations were identified, in a male (58 CGGs) and a female case [(CGG)₄₇/(CGG)₆₁], as well as several FMR1 or AFF2 intermediate-sized alleles. One AFF2 premutation (68 CCGs) and two putative full expansions were picked up in male subjects, which seems relevant considering the rarity of reported AFF2 mutations found in the absence of a family history.

Conclusions

We developed a robust multiplex-PCR that can be used to screen the mutational hotspot regions of FMR1, AFF2 and ARX genes. Moreover, this strategy led to the identification of variants in all three genes, representing not only an improvement in allele-sizing but also in achieving a differential diagnosis. Although the distinction between females who are truly homozygous and those with a second pre- or full mutation sized allele, as well as a definitive diagnosis, requires a specific downstream technique, the use of this multiplex-PCR for initial screening is a cost-effective approach which widens the scope of detection.

【 授权许可】

   
2013 Jorge et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Najmabadi H, Hu H, Garshasbi M, Zemojtel T, Abedini SS, Chen W, Hosseini M, Behjati F, Haas S, Jamali P, Zecha A, Mohseni M, Püttmann L, Vahid LN, Jensen C, Moheb LA, Bienek M, Larti F, Mueller I, Weissmann R, Darvish H, Wrogemann K, Hadavi V, Lipkowitz B, Esmaeeli-Nieh S, Wieczorek D, Kariminejad R, Firouzabadi SG, Cohen M, Fattahi Z, et al.: Deep sequencing reveals 50 novel genes for recessive cognitive disorders. Nature 2011, 478:57-63.
• [2]Ropers HH, Hamel BC: X-linked mental retardation. Nat Rev Genet 2005, 6:46-57.
• [3]Chiurazzi P, Schwartz CE, Gecz J, Neri G: XLMR genes: update 2007. Eur J Hum Genet 2008, 16:422-434.
• [4]Delbridge ML, McMillan DA, Doherty RJ, Deakin JE, Graves JA: Origin and evolution of candidate mental retardation genes on the human X chromosome (MRX). BMC Genomics 2008, 9:65-70. BioMed Central Full Text
• [5]Verkerk AJMH, Pieretti M, Sutcliffe JS, Fu Y-H, Kuhl DPA, Pizzuti A, Reiner O, Richards S, Victoria MF, Zhang F, Eussen BE, van Ommen G-JB, Blonden LAJ, Riggins GJ, Chastain JL, Kunst CB, Galjaard H, Caskey CT, Nelson DL, Oostra BA, Warren ST: Identification of a gene (FMR-1) containing a [CGG] repeat coincident with a break point cluster region exhibiting length variation in fragile X syndrome. Cell 1991, 65:905-914.
• [6]Crawford DC, Acuna JM, Sherman SL: FMR1 and the fragile X syndrome: human genome epidemiology review. Genet Med 2001, 3:359-371.
• [7]Maddalena A, Richards CS, McGinniss MJ, Brothman A, Desnick RJ, Grier RE, Hirsch B, Jacky P, McDowell GA, Popovich B, Watson M, Wolff DJ: Technical standards and guidelines for fragile X: the first of a series of disease-specific supplements to the standards and guidelines for clinical genetics laboratories of the American college of medical genetics. Quality assurance subcommittee of the laboratory practice committee. Genet Med 2001, 3(3):200-205.
• [8]Bensaid M, Melko M, Bechara EG, Davidovic L, Berretta A, Catania MV, Gecz J, Lalli E, Bardoni B: FRAXE-associated mental retardation protein (AFF2) is an RNA-binding protein with high affinity for G-quartet RNA forming structure. Nucleic Acids Res 2009, 37:1269-1279.
• [9]Youings SA, Murray A, Dennis N, Ennis S, Lewis C, McKechnie N, Pound M, Sharrock A, Jacobs P: FRAXA and FRAXE: the results of a five year survey. J Med Genet 2000, 37:415-421.
• [10]Shoubridge C, Fullston T, Gécz J: ARX spectrum disorders: making inroads into the molecular pathology. Hum Mutat 2010, 31:889-900.
• [11]Laperuta C, Spizzichino L, D'Adamo P, Monfregola J, Maiorino A, D'Eustacchio A, Ventruto V, Neri G, D'Urso M, Chiurazzi P, Ursini MV, Miano MG: MRX87 family with Aristaless X dup24bp mutation and implication for polyalanine expansions. BMC Med Genet 2007, 8:25-29.
• [12]Suri M: The phenotypic spectrum of ARX mutations. Dev Med Child Neurol 2005, 47:133-137.
• [13]Shoubridge C, Gardner A, Schwartz CE, Hackett A, Field M, Gécz J: Is there a Mendelian transmission ratio distortion of the c.429_452(24bp) polyalanine tract ARX mutation? Eur J Hum Genet 2012, 20(12):1311-1314.
• [14]van Karnebeek CD, Jansweijer MC, Leenders AG, Offringa M, Hennekam RC: Diagnostic investigations in individuals with mental retardation: a systematic literature review of their usefulness. Eur J Hum Genet 2005, 13:6-25.
• [15]Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988, 16(3):1215.
• [16]Rousseau F, Heitz D, Biancalana V, Blumenfeld S, Kretz C, Boue J, Tommerup N, Van Der Hagen C, DeLozier-Blanchet C, Croquette MF, Gilgenkrantz S, Jalbert P, Voelckel MA, Oberlé I, Mandel JL: Direct diagnosis by DNA analysis of the fragile X syndrome of mental retardation. N Engl J Med 1991, 325:1673-1681.
• [17]Romero-Rubio MT, Andrés-Celma M, Castelló-Pomares ML, Roselló M, Ferrer-Bolufer I, Martínez-Castellano F: ARX mutations and mental retardation of unknown etiology: three new cases in Spain. Rev Neurol 2008, 47(12):634-637.
• [18]Nahhas FA, Monroe TJ, Prior TW, Botma PI, Fang J, Snyder PJ, Talbott SL, Feldman GL: Evaluation of the human fragile X mental retardation 1 polymerase chain reaction reagents to amplify the FMR1 gene: testing in a clinical diagnostic laboratory. Genet Test Mol Biomarkers 2012, 16(3):187-192.
• [19]Khaniani MS, Kalitsis P, Burgess T, Slater HR: An improved Diagnostic PCR Assay for identification of Cryptic Heterozygosity for CGG Triplet Repeat Alleles in the Fragile X Gene (FMR1). Mol Cytogenet 2008, 1:5. BioMed Central Full Text
• [20]Lyon E, Laver T, Yu P, Jama M, Young K, Zoccoli M, Marlowe N: A simple, high-throughput assay for Fragile X expanded alleles using triple repeat primed PCR and capillary electrophoresis. J Mol Diagn 2010, 12(4):505-511.
• [21]Elias MH, Ankathil R, Salmi AR, Sudhikaran W, Limprasert P, Bin Alwi Zilfalil BA: A New Method for FMR1 Gene Methylation Screening by Multiplex Methylation-Specific Real-Time Polymerase Chain Reaction. J Mol Diagn 2011, 15(6):387-393.
• [22]Costa A, Gao L, Carrillo F, Cáceres-Redondo MT, Carballo M, Díaz-Martín J, Gómez-Garre P, Sobrino F, Lucas M, López-Barneo J, Mir P, Pintado E: Intermediate alleles at the FRAXA and FRAXE loci in Parkinson's disease. Parkinsonism Relat Disord 2011, 17(4):281-284.
• [23]Hantash FM, Goos DN, Crossley B, Anderson B, Zhang K, Sun W, Strom CM: FMR1 premutation carrier frequency in patients undergoing routine population-based carrier screening: insights into the prevalence of fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency in the United States. Genet Med 2011, 13:39-45.
• [24]Seixas AI, Vale J, Jorge P, Marques I, Santos R, Alonso I, Fortuna AM, Pinto-Basto J, Coutinho P, Margolis RL, Sequeiros J, Silveira I: FXTAS is rare among Portuguese patients with movement disorders: FMR1 premutations may be associated with a wider spectrum of phenotypes. Behav Brain Funct 2011, 7:19. BioMed Central Full Text
• [25]Miano MG, Laperuta C, Chiurazzi P, D'Urso M, Ursini MV: Ovarian dysfunction and FMR1 alleles in a large Italian family with POF and FRAXA disorders: case report. BMC Med Genet 2007, 11:8-18.
• [26]Shinozaki Y, Osawa M, Sakuma H, Komaki H, Nakagawa E, Sugai K, Sasaki M, Goto Y: Expansion of the first polyalanine tract of the ARX gene in a boy presenting with generalized dystonia in the absence of infantile spasms. Brain Dev 2009, 31(6):469-472.
• [27]Cossée M, Faivre L, Philippe C, Hichri H, de Saint-Martin A, Laugel V, Bahi-Buisson N, Lemaitre JF, Leheup B, Delobel B, Demeer B, Poirier K, Biancalana V, Pinoit JM, Julia S, Chelly J, Devys D, Mandel JL: ARX polyalanine expansions are highly implicated in familial cases of mental retardation with infantile epilepsy and/or hand dystonia. Am J Med Genet A 2011, 155A(1):98-105.