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The absence of FRAXE pathognomonic features hampers early recognition, delaying testing and molecular confirmation. Hence, our laboratory uses a multiplex PCR-based strategy to genotype both FRAXA and FRAXE. However,\n                    <jats:italic>AFF2<\/jats:italic>\n                    expansions are missed giving rise to an uninformative result in around 20% of female samples. To rule out undetected expansions and confirm homozygosity Southern blot analysis is performed being labour- and resource-intensive. The aim of this study is to develop a timely and economic triplet-primed amplification (TP-PCR) screening strategy to size the\n                    <jats:italic>AFF2<\/jats:italic>\n                    GCC repeat and accurately assess homozygosity as well as pinpoint multiplex-PCR false negatives in female samples. In order to achieve this, validation was performed in a cohort of 500 females with a previous uninformative FRAXE PCR result. Interestingly, the presence of a T\u2009&gt;\u2009C SNP (rs868949662), contiguous to the GCC repetitive tract, allows triplet primer binding in two additional repeats, increasing the discrimination power of the TP-PCR assay in heterozygous and homozygous samples. Twelve alleles outside the normal range were recognized: eight intermediate and four premutated, which seems relevant considering the rarity of the\n                    <jats:italic>AFF2<\/jats:italic>\n                    expansions. All genotypes are concordant with that obtained by Southern blotting, confirming this as a strict, reproducible and low-cost homozygosity screening strategy that enables the identification of small expanded alleles missed by the routine multiplex-PCR due to allele dropout. Overall, this assay is capable of spotting multiplex-PCR false negatives besides identifying alleles up to\u2009&gt;\u200980 GCC repeats. Furthermore, the occurrence of intermediate repeat sizes with unexpected frequency, introduces new areas of clinical research in this cohort in understanding these less explored\n                    <jats:italic>AFF2<\/jats:italic>\n                    repeat sizes and newly associated phenotypes.\n                  <\/jats:p>","DOI":"10.1038\/s41598-021-93473-5","type":"journal-article","created":{"date-parts":[[2021,7,19]],"date-time":"2021-07-19T06:03:18Z","timestamp":1626674598000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Development and validation in 500 female samples of a TP-PCR assay to identify AFF2 GCC expansions"],"prefix":"10.1038","volume":"11","author":[{"given":"Cec\u00edlia","family":"Silva","sequence":"first","affiliation":[]},{"given":"Nuno","family":"Maia","sequence":"additional","affiliation":[]},{"given":"Fl\u00e1via","family":"Santos","sequence":"additional","affiliation":[]},{"given":"B\u00e1rbara","family":"Rodrigues","sequence":"additional","affiliation":[]},{"given":"Isabel","family":"Marques","sequence":"additional","affiliation":[]},{"given":"Ros\u00e1rio","family":"Santos","sequence":"additional","affiliation":[]},{"given":"Paula","family":"Jorge","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2021,7,19]]},"reference":[{"key":"93473_CR1","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1038\/ng0596-105","volume":"13","author":"J Gecz","year":"1996","unstructured":"Gecz, J., Gedeon, A. K., Sutherland, G. R. & Mulley, J. C. Identification of the gene fmr2, associated with fraxe mental retardation. Nat. Genet. 13, 105\u2013108. https:\/\/doi.org\/10.1038\/ng0596-105 (1996).","journal-title":"Nat. Genet."},{"key":"93473_CR2","doi-asserted-by":"publisher","first-page":"415","DOI":"10.1002\/(SICI)1096-8628(19960809)64:2<415::AID-AJMG36>3.0.CO;2-G","volume":"64","author":"N Zhong","year":"1996","unstructured":"Zhong, N. et al. A survey of fraxe allele sizes in three populations. Am. J. Med. Genet. 64, 415\u2013419. https:\/\/doi.org\/10.1002\/(SICI)1096-8628(19960809)64:2%3c415::AID-AJMG36%3e3.0.CO;2-G (1996)","journal-title":"Am. J. Med. Genet."},{"key":"93473_CR3","doi-asserted-by":"publisher","first-page":"201","DOI":"10.1006\/geno.1997.4867","volume":"44","author":"J Gecz","year":"1997","unstructured":"Gecz, J., Bielby, S., Sutherland, G. R. & Mulley, J. C. 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