A Cohort Study of Intellectual Disability Focusing on Fragile X Syndrome in Indonesia

Background: Intellectual disability (ID) is a major public health problem because the defect, treatment and rehabilitation require long life both medical and socio-economic assessment. Fragile X syndrome (FXS) is the most common cause of inherited X-linked intellectual disabilities (ID) with reduced penetrance. With regards to behavioral and emotional phenotype, FXS commonly mixed up with idiopathic autism. The prevalence is found higher in males compared to females. In accordance with rapid development of diagnosis technique, the prevalence of FXS is defining worldwide including Indonesia using, currently, simple molecular method.Objectives: This study was aimed to diagnose genetic cause of ID and to establish the prevalence of FXS among ID population in Central Java, and Yogyakarta Province.Method: Screening has been performed since 1994 continuously in high risk population (special school with and without autism) using clinical, cytogenetic, and FMR1 gene PCR-based molecular approach. Cascade testing was subjected to the family members with positive result of FXS and many new cases were disclosed in our cohort study.Results: The prevalence of FXS among ID population was calculated to be 1.9% (5/262) in 1994 and 1.7% (9/527) in 2011. Among autism population it was determined to be 6.15% (4/65). Trisomy 21 was found in 14% (74/527) as a major cause of ID.Conclusion: The prevalence of FXS among screened ID population overtime is comparable.


INTRODUCTION
ID is a public health problem and becoming important consideration due to some reasons: 1) high prevalence in general population, 2) lifelong disorder, 3) expensive cost of management, include direct cost of long term care, rehabilitation, special education and indirect cost of increased morbidity and mortality since individual with ID is more vulnerable in health problem 1 , 4) High economic and society dependences, and 5) Prevention of ID for improving prognosis outcome is absolutely needed to be implemented even it is very challenging in low and middle income countries. 2 The prevalence of ID across the world is around 1% and almost two times higher rates in low and middle income countries. 3 Fragile X syndrome (FXS) is the most common cause of inherited mental retardation and a single gene disorder associated with autism spectrum disorders (ASD). [4][5][6] The frequency of FXS due to full mutation allele is approximately 1 in 2500 in males and 1 in 4000 in females. [7][8][9] The FXS clinical phenotype is caused by imbalance of excitatory glutamatergic and inhibitory GABAergic neurotransmitters. 10 It is mostly due to the expansion of CGG repeats more than 200 (full mutation) in the promotor region of the FMR1 gene that leads transcriptional silencing and results in lack of fragile X mental retardation protein (FMRP). 11 Fragile X syndrome is the first pathogenic mutations that affect trinucleotide repeat expansions (TREs) diseases involving non-coding repeats sequences. 12,13 Until the late 1990s, fragile X site cytogenetic analysis was used to diagnose FXS. Subsequently, molecular techniques has been used to identify the FMR1 alleles since the sequence of FMR1 gene has been discovered in the beginning of 1990s. 14 However, in developing countries where advanced laboratory equipment and services are very minimal and unaffordable, cytogenetic techniques are still an option to accomplish the FXS diagnosis, 15 more overly, clinical symptoms of FXS especially in younger age is quite challenging to be differentiated with other nonsyndromic intellectual disabilities (ID).
The prevalence of FXS varies across ethnic groups, 16 it is important to provide fragile X allele frequencies for all race and across the world, so that general (low-risk) and high-risk population screening the incidence of FMR1 gene mutation is considered necessarily. Population-based screening is the most favorable method to identify new FXS cases both clinically and laboratory assessment. General population screening to determine the diseases and carriers prevalence in both male and female have been reported worldwide. [17][18][19][20][21] Based on the American College of Medical Genetics (ACMGs) guideline, screening for FXS in high-risk population such as ID and autism population-based FXS screening have also been done to established FXS prevalence. 5,[22][23][24] Various simple and cost-effective DNA-based assay are developing with higher sensitivity and specificity to meet the requirement of population screening study. [25][26][27][28] Recently, prevalence studies were also reported from other developing countries although mostly done by scientist in Saudi Arabia, Malaysia, and Sri Lanka. 25,29,30 This study was aimed to define the prevalence of FXS in the high-risk population in Indonesia.

MATERIALS AND METHODS
These were cross sectional study designs which were done in special school in Central Java and Yogyakarta provinces. Individuals who were diagnosed with ASD using the criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, Text Revision (DSM IV-TR) for ASD and ID by experienced pediatricians were recruited. 31 This research was approved by the Institutional Review Board of the Faculty of Medicine, Diponegoro University and dr. Kariadi Hospital Semarang, Indonesia. All participants gave their consent in written/signed a consent form to participate in this study. This study was conducted in six special schools in Central Java and Yogyakarta Province.
Heparinized peripheral blood vein was collected for cytogenetic analysis of the fragile site at Xq27.3 and chromosomal abnormalities as done previously. 32 Deoxyribonucleic acid (DNA) was extracted from ethylene diamine tetra acetic acid (EDTA) blood using modified salting out method. 33 The fragile site was identified using solid staining followed by Giemsa staining for fragile X site confirmation. The FMR1 gene was analyzed using a PCR-based method to determine the CGG repeat length in the promotor region as described previously. 34 Males with no bands and females with only one band were assumed to have alleles consisting of high pre-mutation or full mutation alleles, therefore, Southern blot analysis was used to confirm the diagnosis. 34

RESULTS
This study was dedicated to determine the prevalence of FXS in the high risk population (ID and ASD). The first population study in 1997 among ID population in special school, the overall prevalence of was 5/262 (1.9%). In all, five children from three unrelated families were found to have FMR1 CGGrepeat expansions. All came from the population attending type C (ID) special schools. 22 The second population study in 2007-2010 among ID population in special school, of the 527 individuals with ID, chromosomal abnormalities were found in 87 (16.5%). Trisomy 21 was the major chromosomal abnormality, identified in 74 patients (14%). In the remaining of 445 individuals (162 females and 283 males), 607 alleles were reported (male has 1 allele, female has 2 alleles) i.e. 593 alleles were within the normal range (15-44 CGG repeats), 3 alleles in the intermediate range (45-55 CGG repeats), 2 alleles in the premutation range (between 55 and 200 CGG repeats) and 9 alleles in the full mutation range (> 200 CGG repeats). The prevalence of FXS is 9/527 (1.7%). The prevalence in males and females is 1.5% (5/329) and 2% (4/198), respectively. 35 The study in high risk (autism) population study, the fragile X site and FMR1 full mutation allele were identified in 3 out of 65 (4.6%) and 4 out of 65 (6.15 %) children aged 3 to 17 years (57 boys, 8 girls) respectively. 36 Impairment of social communication and interaction domains have been found to be dominant in four FXS children with ASD. The level of cognitive impairment in FXS usually ranging from learning disabilities to severe ID is associated with the FMRP level. 37 In addition to the cognitive impairment, behavioral and emotional impairment have also been observed as a major problem in individuals with FXS. [38][39][40][41][42] Early intervention has been reported in many centers worldwide shows efficacy decreasing the risk of severe cognitive impairment, improving behavioral problems, emotional, and psychosocial abilities in later life. There are numerous psycho-pharmacological agents that being used to improve behavioral and emotional disorders in FXS whose may have impact on cognitive function 43 ,behavioral and emotional abilities. 44,45 Psychopharmacological agents may support the effectiveness of non-psychopharmacological intervention such as behavior, physical, and speech therapy; and both intervention result the improvement of cognitive function. The similarity and overlapping clinical phenotype, especially in behavioral impairments, and neuroanatomical background between FXS and ASD encouraging the same approach in FXS targeted treatment. 46,47 The first FXS population-based study on 262 subjects at schools with special need in Indonesia showed a prevalence of 1.9%. 22 New cohort of high-risk population screening of FXS has been conducted among ASD and ID population using fragile X site method combine with molecular one. 48,49 The fragile X site expression has been able to detect 3 out of 4 FXS cases among autism population that eventually confirmed using PCR-based method followed by Southern blot method. The prevalence of FXS is 6.15% in ASD population in Indonesia. 36 Similar result also reported from a number of studies that have been carried out in ASD population varying from 4-7%. 5,23 The prevalence of FXS is 1.7% (considered equal in males and females) in ID population. Surprising result had been reported in the prevalence of FXS in male and female is equal in Indonesia, suggested that the prevalence of FXS females had been underestimated. 49 The FXS is an Xlink disorders with reduced penetrance (79% in males, 35% in females), females usually less affected due to the X-inactivation. 50,51 This study was a continuation of the first FXS population-based study with broader approach to identify FXS cases, cascade testing to identify carriers, and finally, to offer early intervention among FXS children. Population-based screening method in FXS has been developed related to Indonesian condition which is contained more than 17.000 islands indweled by 237.6 million populations with very minimal access to genetic facilities and centers. In Java island, the most populated area where 58 percent of Indonesian population live, only one FXS center conducts fragile X diagnosis both cytogenetic and molecular diagnosis that is Center for Biomedical Research (CEBIOR) Faculty of Medicine Diponegoro University.
With regards to the prevalence and the mode of inheritance, FXS screening high-risk population program should be performed routinely and initiate by the government in Indonesia. On the contrary, health care provider, government, and public awareness is inadequate with regard to identify FXS and to provide basic special need services in order to get better outcome.
Seminar, workshops, dissemination information of fragile X syndrome should also be conducted regularly using/ leaflet distribution and/or online media to educate clinicians, health care professionals, families, researchers and broader community to increase awareness the role of genetic especially FXS in ID. Using a comprehensive approach to whole Indonesian data of FXS overtime and other cause of ID, our studies will give a great contribution to science, medical community, and stake holders in increasing awareness of FXS in Indonesia. In a contrary, in developed countries such as European and Unites Stated, clinical and molecular guidelines have been established and revised continuously for fragile X and fragile X-associated disorders in order to identify FXS cases. 52,53 With regard to the prevalence of FXS, similar study is extremely needed to be done in other area of Java Island where the rest of 40 percent Indonesian population live.