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|NI FEATURE: THE EDITORIAL DEBATE-- PROS AND CONS
|Year : 2016 | Volume
| Issue : 6 | Page : 1138-1139
CGG repeat expansion at FMR1 locus – A new molecular diagnostic algorithm in fragile X syndrome
Neeraj Kumar, Hardeep Singh Malhotra, Ravindra Kumar Garg
Department of Neurology, King George Medical University, Lucknow, Uttar Pradesh, India
|Date of Web Publication||11-Nov-2016|
Ravindra Kumar Garg
Department of Neurology, King George Medical University, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Kumar N, Malhotra HS, Garg RK. CGG repeat expansion at FMR1 locus – A new molecular diagnostic algorithm in fragile X syndrome. Neurol India 2016;64:1138-9
The Fragile X Mental Retardation 1 (FMR1) gene encoding the FMR1 protein is essentially needed for normal brain functioning, development, and synaptic plasticity. Synaptic plasticity refers to the strengthening or weakening of synaptic transmission over time in response to changing synaptic activity. Dynamic changes in neuronal synaptic efficacy is essential for learning, information processing, and memory storage. These dynamic changes in the neural circuits of human brain, in fact, form a molecular basis responsible for the experience-dependent alterations in adaptive behaviors.
FMR1 gene-related disorders are commonly encountered by psychiatrists, gynecologists, and neurologists. Mutations in the FMR1 gene, located on chromosome Xq27.3, may lead to fragile X syndrome (FXS), premature ovarian failure, and fragile X-associated tremor/ataxia syndrome (FXTAS). FMR1 gene expansion leads to methylation and inhibition of the FMR protein (FMRP), which provides RNA stabilization and subcellular transportation. It also plays a role in the translation process of neural messenger RNAs, thus helping in synapsis development and neural plasticity. On the basis of GCG repeats, four FMR1 allelic categories can be formed, namely, (1) normal alleles with 6 to 44 CGG repeats; (2) gray zone with 45 to 54 CGG repeats; (3) premutation alleles with 55 to 200 CGG repeats; and (4) full mutation alleles having greater than 200 CGG repeats.
Owing to a repeat-dependent expression of the phenotype, it becomes important to correctly estimate the number of repeats, which can have both diagnostic as well as therapeutic implications. In order to address this issue, Muthuswamy et al., have assessed the utility of two different types of polymerase chain reaction processes to screen for CGG repeat expansion at the FMR1 locus in the affected patients. They combined triplet repeat primed polymerase chain reaction (TP-PCR) and methylation-specific polymerase chain reaction (MS-PCR) to diagnose patients with FXS and primary ovarian failure. Validation of results was done with genotyped samples available for estimation.
FXS is the most common heritable cause of intellectual disabilities with an incidence of 1 in 5000 in males and 1 in 2500–8000 in females. Patients may present with mental retardation, seizures, anxiety, autistic behavior, attention deficit hyperactivity disorder, large ears, a long face, macrocephaly, cleft palate, flat foot, scoliosis, macro-orchidism, mitral valve prolapse, etc., Importantly, the clinical presentation varies according to the size of CGG repeats and methylation, and characteristic features of FXS are found in patients with greater than 200 CGG repeats. Similarly, fragile X-associated primary ovarian insufficiency occurs due to a premutation involving the FMR1 gene, presenting as premature ovarian failure (loss of menstruation before 40 years of age). In the same vein, FXTAS is a manifestation of premutation of FMR1 gene, which typically presents after 50 years of age with tremor, ataxia, Parkinsonism More Details, and cognitive decline. In addition, peripheral neuropathy, endocrine dysfunction, and autonomic abnormalities might be seen.
At present, the diagnosis is established on PCR-based techniques. The gold standard for the assessment of large CGG repeat alleles is DNA-based southern blotting technique. PCR is said to be good for intermediate size and premutation range CGG repeats. Nowadays, reverse transcription (RT)-PCR is used for diagnosis. This technique utilizes two primers, which target both within and outside the CGG repeat region. This gives an advantage of detecting premutation and full mutation in both sexes. Another benefit of the PCR method is the detection of the number and location of AGG interruptions within the CGG locus. The AGG interruptions determine the expansion and, therefore, the risk of developing FXS in a child born from a mother with CGG repeats in the premutation range. This knowledge is very helpful in the preconceptual genetic counseling in premutation carrier women. The clinical presentation varies significantly with the amount of methylation, which the routine PCR techniques fail to measure. New methods utilizing bisulfite modification in PCR are helpful in the diagnosis of FXS. Quantification of methylation can be done using PCR based assay, followed by bisulfite treatment and melting curve analysis (MCA). This technique provides promising result for screening populations with CGG allele repeat greater than 55. Muthuswamy et al., utilized both the techniques to build an algorithm to diagnose patients with varying number of CGG repeats where even the newer techniques fail to differentiate between normal, permutated, and fully-mutated females. The initial application of TP-PCR was used to screen all samples and then the selected abnormal samples were processed using MS-PCR. They could diagnose 6 of 59 patients having FXS and 1 of 25 patients with primary ovarian failure. By means of reducing the load for MS-PCR, and per se by the combined usage of these two PCR forms, the cost and time entailed may be brought down and the technique may be applied in centers where advanced hardware is not available.
Screening and early identification of the carrier and permutation states help in diagnosing a person with defect in the FMR1 gene. Genetic counseling at the preconceptional stage may prevent the birth of FXS affected child, and even though treatments in FMR1 gene-related disorders are still supportive, early treatment may be offered depending on the manifestation. Genetic modification is not yet possible even after recognition of the culprit gene. Valproic acid is hypothesized to be reactivating FM alleles in FXS, and is, therefore, helpful in attention deficit hyperactivity disorder (ADHD). Gamma aminobutyric acid (GABA) mediated inhibition is useful in seizures and bipolar disorders. L-acetyl-carnitine inhibits the fragile sites and is useful in ADHD. Methylphenidate is also used for ADHD. Clonidine and melatonin can be given for sleep disturbances. Anxiety and other mood disorders benefit by the administration of minocycline, selective serotonin reuptake inhibitors, and lithium. Cognitive and behavior therapy plays a vital role in such patients. A National Institutes of Health Toolbox Cognitive Battery has also been designed for patients with FXS so that proper evaluation of such patients with good reproducibility can be done.
Future research should focus on cost-effective and rapid diagnostic tests. Differentiation in pre-mutation and full mutation in both sexes, with an ability to detect very large CGG repeats is expected.
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