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Table of Contents    
Year : 2016  |  Volume : 64  |  Issue : 6  |  Page : 1136-1137

A new diagnostic algorithm for an early diagnosis of patients with fragile X syndrome

United States Food and Drug Administration (USFDA), 10903 New Hampshire Avenue, Building 22, Room 4191 Silver Spring, MD 20993, USA

Date of Web Publication11-Nov-2016

Correspondence Address:
Rama S Dwivedi
United States Food and Drug Administration (USFDA), 10903 New Hampshire Avenue, Building 22, Room 4191 Silver Spring, MD 20993
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0028-3886.193774

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How to cite this article:
Dwivedi RS. A new diagnostic algorithm for an early diagnosis of patients with fragile X syndrome. Neurol India 2016;64:1136-7

How to cite this URL:
Dwivedi RS. A new diagnostic algorithm for an early diagnosis of patients with fragile X syndrome. Neurol India [serial online] 2016 [cited 2021 Jan 21];64:1136-7. Available from:

Fragile X syndrome (FXS) is the second most common cause of X-linked intellectual disability. This debilitating triplet repeat disorder is caused by hyperexpansion, and subsequently, hypermethylation of a highly polymorphic CGG repeat tract present at the 5'untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) gene. In spite of efforts made by the scientific community, the treatment for this neurodevelopmental disorder is scanty, and patient management depends at large on supportive therapy. An early diagnosis would not only provide an answer to the parents' quest for searching for the cause of their child's disorder but also could give parents an opportunity to receive genetic counseling for family planning and would allow the child to receive available interventional services.[1],[2],[3]

In the article under focus,[4] the authors have used a new diagnostic algorithm in order to provide an early diagnosis of patients suffering from FXS. Two types of advanced polymerase chain reaction (PCR) methods [triplet repeat primed (TP)-PCR and methylation-specific (MS)-PCR)] have been validated to identify the full spectrum of FMR1 alleles, i.e., normal (6–44 CGG repeats), gray zone (GZ), premutation (PM; 55–200 CGG repeats), and full mutation (FM; >200 CGG repeats). The conventional PCR fails to amplify large PM alleles and FM alleles due to the inherent complexity of the CGG repeat region; thus traditionally, southern blot (SB) is used to detect large PM and FM alleles. However, SB is technically demanding, labor intensive and a time consuming technique with requirements of large quantity of high quality DNA. Moreover, SB cannot differentiate between the borderline alleles, i.e., normal and PM, large PM, and FM alleles. These disadvantages limit the use of SB in routine FXS diagnosis and efforts have been made to improve the PCR-based methods.

In the recent past, several methods incorporating the advanced PCR technology have emerged that could detect the expanded allele accurately viz. TP-PCR, MS-PCR, reverse transcription (RT)-PCR, melting curve analysis (MCA), etc.[5],[6],[7] In addition, many commercial kits are in place for the molecular diagnosis of FXS that have replaced the SB technique. Here, the in-house TP-PCR method used in this study could accurately define the number of CGG repeats in the GZ and PM allele but not in the FM allele because the infrastructure available in the authors' institute had the basic model of Capillary Sequencer, that is, the Applied Biosystems ®[ABI] 310 genetic analyzer, which is unable to resolve the larger fragments. The authors tackled this problem by using MS-PCR whose results in combination with the former technique can confirm the FM expansion. MS-PCR will also be able to provide the methylation status of the FMR1 allele in female samples.

In my view, the protocol developed in this study seems to have a parallel potential as compared to the SB assay or the kit-based diagnostic techniques, owing to its technical handiness, rapidity, accuracy, and cost-effectiveness. This algorithm will be particularly useful in developing nations like ours because kit-based diagnosis are prohibitively expensive and families who belong to a weaker socioeconomic section might find it difficult to afford the high cost of kit based diagnosis. One of the limitations in this protocol seems its inability to deduce the accurate size of the FM expansion. I feel that it will, however, not be of much concern as the size of the FM allele is not associated with the phenotypic severity in FXS, unlike other triplet repeat disorders such as myotonic dystrophy. Furthermore, in the available commercial kits, which are used reliably worldwide for the routine diagnosis of FXS, full expansion mutations are not specifically sized but are detected as expansions of greater than 200 repeats. The methodology used by the authors can accurately size the GZ and PM alleles, which is of due importance as both these alleles can expand in successive generations to the next higher alleles, that is, PM and FM, respectively, in a size dependent manner. This information can supplement the genetic counselling that will be offered to at-risk individuals in the family of the affected patients. The authors have applied the diagnostic algorithm they have developed to provide the diagnosis in FXS suspected cases that comprise intellectually disabled cases, premature ovarian failure subjects and prenatal samples that were referred to their institute, thereby depicting the practical utility of their method.

  References Top

Centers for Disease Control and Prevention (CDC). Delayed diagnosis of fragile X syndrome - United States, 1990-1999. MMWR Morb Mortal Wkly Rep. 2002;51:740-2.  Back to cited text no. 1
Levy HL, Albers S. Genetic screening of newborns. Annu Rev Genomics Hum Genet 2000;1:139-77.  Back to cited text no. 2
Barnicoat A. Screening for fragile X syndrome: A model for genetic disorders? BMJ 1997;315:1174-5.  Back to cited text no. 3
Muthuswamy S, Dean DD, Agarwal S. Molecular screening of intellectually disabled patients and those with premature ovarian failure for CGG repeat expansion at FMR1 locus: Implication of combined triplet repeat primed polymerase chain reaction and methylation-specific polymerase chain reaction analysis. Neurol India 2016;64:1175-9.  Back to cited text no. 4
  Medknow Journal  
Zhou Y, Lum JM, Yeo GH, Kiing J, Tay SK, Chong SS. Simplified molecular diagnosis of fragile X syndrome by fluorescent methylation-specific PCR and GeneScan analysis. Clin Chem 2006;52:1492-500.  Back to cited text no. 5
Todorov T, Todorova A, Georgieva B, Mitev V. A unified rapid PCR method for detection of normal and expanded trinucleotide alleles of CAG repeats in Huntington chorea and CGG repeats in fragile X syndrome. Mol. Biotechnol 2010;45:150-4.  Back to cited text no. 6
Teo CR, Law HY, Lee CG, Chong SS. Screening for CGG repeat expansion in the FMR1 gene by melting curve analysis of combined 5' and 3' direct triplet-primed PCRs. Clin Chem 2012;58:568-79.  Back to cited text no. 7


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