Standardization of capillary electrophoresis for diagnosis of fragile X syndrome in the Brazilian public health system

Abstract

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability. The most common etiology of the syndrome is expansion and methylation of a CGG trinucleotide at chromosome region Xq27.3 involving FMR1 (fragile X mental retardation 1 gene). This disorder is commonly underdiagnosed in children and adolescents, given the high clinical variability. In Brazil, molecular diagnosis of FXS by CE does not exist in the public health system. The current standard for separation and identification of DNA fragment sizes is 50 cm CE, which is uncommon in public genotyping laboratories. This study describes the standardization of 36 cm CE for fragment analysis of samples from patients with intellectual disability suggestive of FXS. Genomic dsDNA was isolated from patients and amplified by PCR using the FMR1 AmplideX Kit. It was then possible to detect changes in repeat length of FMR1, such as full mutation and premutation. Thus, the proposed standardization proved to be effective for the diagnosis of FXS, permitting suitable genetic counseling for families. Inclusion of molecular testing such as this in the Brazilian public health service bridges the gap between available technologies and effective diagnosis, universalizing access to genetic testing in central Brazil.

Research Article Molecular Diagnosis of X-Fragile Syndrome: Perspectives for the Public Health System in the Central Region of Brazil

“…Molecular separation methods proposed by capillary electrophoresis for the identification of mutations in the FMR1 promoter region have been shown to be more efficient than other methodologies, such as by Southern blot, by accurately categorizing mutation ranges and mainly by detecting mutation amplicons which are generally present in very low amounts to be identified by conventional electrophoresis. (Gigonzac et al, 2016) The fact that the sample presents more male patients than the female one is justified because it is a disease with dominant inheritance linked to the X chromosome, affecting more men than women. The phenomenon of Genetics and Molecular Research 16 (4): gmr16039848 random inactivation of this chromosome, in the female sex, also inactivates part of the mutated X chromosomes, with consequent reduction of clinical manifestations of the disease (Willemsen et al, 2001).…”

“…The amplification products were subjected to capillary electrophoresis in an ABI3500 automatic genetic analyzer (Applied Biosystems, USA), according to the protocol established by Gigonzac et al (2016) by adjusting the polymer density of the capillary and all conditions for separating the fragments. The obtained electropherograms were analyzed using GeneMapper® software, version ID-X 1.4 (Applied Biosystems, Vermon Hills, Illinois, USA), allowing the determination of the size of the fragments of each patient.…”

“…It is estimated that 3% of the world population has some kind of cognitive deficit, 40% of which is associated with X chromosome abnormalities. FXS is the main cause of mental deficiency inherited in the world, with an estimated frequency of 1: 4000 men and 1: 6000 women (France et al, 2011, Gigonzac et al, 2016. Despite its high prevalence, FXS remains poorly understood and underdiagnosed by health professionals, mainly pediatricians and family physicians (Goméz and Alonso, 2016).…”

“…Until 2014, there was no routine for the molecular diagnosis for FXS in the state of Goiás under the Unified Health System (SUS), which implied a lack of regional epidemiological data on the syndrome, besides a serious problem for all patients who depend of the public health service. By mid-2015, the state pioneered the implementation of a new methodology for a rapid, accurate, and effective diagnosis of FXS (Gigonzac et al, 2016). Despite this great step for laboratory diagnosis, there is still a great difficulty in the identification and referral of patients due to the wide phenotypic variability of the disease, which shares characteristics with several other disorders.…”