張有成，王　和

(1.貴州省人民醫(yī)院 產(chǎn)科，貴州 貴陽　550002；2.四川大學(xué)華西第二醫(yī)院 產(chǎn)前診斷中心，四川 成都　610041)

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專家論壇

中國西南一城市智力低下人群中脆性X綜合征發(fā)病率調(diào)查

張有成1，王和2

(1.貴州省人民醫(yī)院 產(chǎn)科，貴州 貴陽550002；2.四川大學(xué)華西第二醫(yī)院 產(chǎn)前診斷中心，四川 成都610041)

[摘要]目的 探討中國西南地區(qū)智力低下人群中脆性X綜合征(fragile X syndrome，F(xiàn)XS)發(fā)病率。方法 通過一種可檢測脆性X智力低下基因1(fragile X mental retardation 1,FMR1)上CGG重復(fù)序列長度的聚合酶鏈反應(yīng)方法，對中國西南一城市智力低下人群進行脆性X綜合征的篩查，探討智力低下人群中脆性X綜合征的發(fā)病率。結(jié)果 研究樣本發(fā)現(xiàn)，頻率最高的CGG重復(fù)數(shù)是29個CGG重復(fù)，其次為30和31個CGG重復(fù)，再其次為36和37個CGG重復(fù)；CGG等位基因頻率分布形式與中國其它地區(qū)的研究結(jié)果一致；但沒有發(fā)現(xiàn)前突變和全突變病人。結(jié)論 中國西南地區(qū)智力低下人群中脆性X綜合征發(fā)病率可能低于華中及華北地區(qū)的報道人群。

[關(guān)鍵詞]脆性X綜合征；CGG重復(fù)；分子篩查；中國；智力低下病人

Fragile X syndrome (FXS) is referred to as the most common known cause of inherited mental retardation[1]. The disorder is mainly associated with the mutation of the FMR1 gene located on FRAXA, a fragile site at band Xq27.3. The most common mutation of FMR1 is the expansion of a CGG trinucleotide repeat in the 5’ untranslated region of exon 1. When the expansion exceeds the threshold of 200 repeats, the CpG island located on the upstream of the CGG repeat becomes hypermethylated, and the gene is silenced. The absence of the product transcribed from FMR1 gene eventually leads to the manifestations of the syndrome[2].

The frequency of FXS among the mentally retarded populations was estimated to range from 2.6% to 8.7% in western countries[3]. A similar frequency was observed by a research for Chinese populations in North and Central China[4]. However, other studies from South Chinese populations offered different results[5]. The incidence of FXS in mentally retarded patients in Southwest Chinese populations has not been reported.

The molecular detection for CGG expansion mutation of FMR1 has currently become the main method for diagnosis of FXS. The molecular tests for FXS are of two types: Polymerase chain reaction (PCR) analysis for screening and Southern Blot Hybridization for definite determination of premutation and full mutation[6]. Since longer CGG expansion can not be amplified by PCR, so all selected or unsuccessfully detected cases by PCR need to be confirmed by Southern Blot Hybridization. It is important for picking out each case of carrier and full mutation, so individuals with unexplained mental retardation should be screened for FXS. Based on this screening, the incidence of FXS among mentally retarded patients can also be evaluated from a crowd. Here, we adopted one PCR assay allowing to determine the CGG repeat length by capillary electrophoresis to carry out a screening test for FXS in a mentally retarded population from a Southwest Chinese city, so as to evaluate the incidence of FXS in mentally retarded patients. With the purpose of providing supplementary confirmation of the presence of full mutation allele, we added another PCR assay for detection of methylation at the CpG island upstream of the FMR1 gene.

1Materials and methods

1.1Subject EnrollmentThis study was approved by the Medical Ethical Committees of the West China Second University Hospital, Sichuan University. Informed consent was obtained from each participant before the blood draw. Subjects were recruited from Chengdu, a city of Southwest China. All 404 individuals were divided into two groups. One group was composed of 304 nonspecifically mentally retarded boys recruited from public service. By clinicians and researchers interviewing, these individuals were evaluated to be moderate to severely retarded, and their IQs ranged from 30 to 70. Another group comprised one hundred unrelated donors for detecting length variation of CGG repeats among normal individuals, these donors included 40% of women and 60% of men. Genomic DNA was extracted from peripheral blood cells by using RelaxGene Blood DNA System (TIANGEN) according to the manufacturer’s instructions.

1.2PCR assay for detecting length of CGG repeats for 404 individualsWe screened the FMR1 gene status of the 304 mentally retarded boys and detected the length variation of CGG repeats among 100 normal individuals by a PCR assay relied on the Abbott molecular fragile X ASR reagents (Abbott Molecular). Using these reagents, a sequence containing FMR1 CGG repeat region is amplified with fluorescently labeled amplicons. Each PCR fragment is labeled at the 3’ end with a fluorescent dye, and these labeled PCR fragments can then be analyzed by an Applied Biosystems capillary instrument. CGG repeats less than 645 can be detected with this method. A total reaction volume of 20 μl included:13 μl of high GC PCR buffer, 0.5 μl of gender primers, 0.7 μl of fragile X primers, 1.2 μl of TR PCR enzyme mix, 2.6 μl of DNase free water and 2 μl of DNA sample (50 ng). The PCR amplification consisted of 15 cycles of 98.5 ℃,10 seconds; 58 ℃, 1 minute;75 ℃,6 minutes;and 15 cycles of 98.5.Auto×0.1 ℃/cycle,10 seconds;56 ℃,1 minute;75 ℃,6 minutes. PCR product clean up was processed by using a Clean Up Enzyme Mix to reduce the stutter (n-1) signal observed with amplification of GC rich DNA targets. Then the sample was detected by performing capillary electrophoresis on a 3 500 Genetic Analyser (Applied Biosystems) according to the instructions of the manufacturer. The number of CGG repeats can be calculated by the following formula: Number of CGG repeats = (Fragment length-193 )÷3.

1.3Detection of FMR1 gene methylation for 304 mentally retarded boysWe also detected FMR1 methylation at the CpG island upstream of the CGG repeat by using a PCR assay. Genomic DNA samples were digested with a methylation sensitive enzyme, then followed by amplification of the sequence including the methylated site. If the site was methylated, the sequence would be protected from the cleavage with the enzyme and PCR would produce a product, but if the site was unmethylated, the sequence would be digested and no product would be amplified. The sequences of the primers for this amplification were 5′-AGTGCGACCTGTCACCGCCCTTC-3′ and 5′-GAAACCACG -TCACGTGATCAACGCTGTTCC-3′. Two tubes were set up for each sample. A total volume of 20 μl including 1 μg of genomic DNA, 2 μl of enzyme buffer (Fermentas), and 17 μl of ddH2O was added to each tube. 12 U of Eag1 enzyme (Fermentas) was added to one of the tubes, while the other contained no enzyme. Then the tubes were incubated for 16 hours. PCRs for the two tubes were then performed in a 25 μl volume containing 2.5 μl of 10× PCR buffer, 2 mM of MgCl2,20 pmol of forward and reverse primer, 0.2 mM of dNTPs, 10% DMSO, 5 U of Taq DNA polymerase (TaKaRa) and 2 μl of digestion reaction. PCR conditions were initial denaturation for 10 min at 95 ℃, followed by 30 cycles of 1 min at 95 ℃, 1.5 min at 65 ℃, 2 min at 72 ℃, and with a final extension for 5 min at 72 ℃. 10 μl of products were analyzed on a 2% agarose gel.

2 Results

2.1FMR1 gene status and CGG repeat pattern in 304 mentally retarded boysBy analyzing the fragment data from Applied Biosystems genetic analyzer, a range of CGG repeats from 21 to 39 was provided by 304 individuals with mental retardation (Table 1 ), which were normal alleles of FMR1 gene. The capillary electropherogam traces for one sample was shown in Figure 1. Premutation and full mutation alleles can not be detected. In 304 X chromosomes (Table 2 ), 16 alleles of different size were observed. The most common CGG repeat allele was 29 repeats (48.03%), followed by 30 (21.05%) and 31 (7.89%) repeats. There were two minor peaks at 36 and 37 repeats, each of which was found in 17 chromosomes (5.59%).

Tab 1CGG repeat allele distribution from 304 mentally retarded boys and one hundred normal individuals in Chengdu city

CGGrepeatsizeNumberofXchromosomes(%)mentallyretardednormal202(1.43)212(0.66)3(2.14)221(0.33)232(0.66)241(0.33)251(0.33)272(0.66)1(0.71)2811(3.62)9(6.43)29146(48.03)52(37.14)3064(21.05)32(22.86)3124(7.89)18(12.86)324(2.86)345(1.64)359(2.96)7(5)3617(5.59)12(8.57)3717(5.59)381(0.33)391(0.33)Total304(100)140*(100)

* The normal individuals included 40 women and 60 men, so the total number of X chromosomes was 140.

Tab 2Prevalence of fragile X syndrome (FXS) in mentally retarded patients in different Chinese populations

RegionNo.ofpatientsNo.ofFXSPrevalence(%)Ref-er-enceNorthandcentralChina803303.7ZhongNetal.,1999HongKong132420.6PangCPetal.,1999Chengducity230400Ourstud-y

1：One region in south China; 2：One region in Southwest China.

170 bp refers to an amplification target from Y chromosome; 203 bp refers to an amplification target from X chromosome; and 280 bp refers to the amplification segment containing CGG repeat of FMR1 gene. Number of CGG repeats = ( 280-193 )÷3 = 29. 　　Fig 1　Electropherogam traces for PCR products by using an Abbott fragile X ASR reagent to detect FMR1 gene status for one boy with mental retardation

2.2Length variation of CGG repeats in normal individualsBy analyzing the results from one hundred unrelated donors, the length of CGG repeats in normal individuals was detected to range from 20 to 36 (Table 1). The peak pattern of CGG repeat allele distribution showed no distinction to that of mentally retarded patients. The most common allele was also 29 repeats (37.14%), followed by 30 (22.86%) and 31 (12.86%) repeats. There was also a minor peak at 36 repeats, which was found in 12 chromosomes (8.57%).

2.3Methylation status of the FMR1 gene for 304 boys with mental retardationFor everyone of those 304 boys, a PCR product of about 207 bp in size was produced by amplifying the reaction in tube without Eag1 enzyme, but no PCR product was produced by amplifying the reaction in tube contained Eag1 enzyme (Figure 2). This suggested that the CpG islands upstream of the FMR1 gene in 304 boys were unmethylated.

M=100 bp ladder. Numerals 1-8 refer to samples from eight boys with mental retardation. -=no Eag1 enzyme digestion, a PCR product of 207 bp was produced; +=digestion with the Eag1 enzyme, no PCR product was produced.　　Fig 2　PCR assay for methylation of the CpG island upstream of the FMR1 gene

3Discussion

FXS is an unusual X-linked dominant inherited disease. Based on the size of the expansion, there are four genotypes for CGG repeat allele: 6-44 repeats of CGG is termed as normal allele; 45-54 repeats is termed as grey zone or intermediate allele; 55-200 repeats as permutation and more than 200 repeats as full mutation alleles[7]. The Abbott molecular fragile X ASR reagents can detect the accurate length of CGG repeats less than 645. Using these reagents, not only the CGG repeat size of normal and premutation alleles can be determined, but partial full mutation (≤ 645 CGG repeats) status of FMR1 gene can be detected as well.

It had been reported that a repeat number of 29 was by far the most frequent CGG repeat allele by analyzing 492 X chromosomes from four different races (Caucasian, Blank, Hispanic, and Asian), and the pattern of distribution among races was found no significant difference[8]. By capillary electrophoresis analyzing 444 X chromosomes from 404 individuals in Chengdu city, we found that the most frequent CGG repeat allele was the same as 29 repeats, followed by 30 and 31 repeats, and there was a secondary peak at 36 and 37 repeats. Our results were consistent with the findings of other studies for the CGG-repeat distribution pattern in Chinese populations[4-5,9].

Based on observation of cytogenetic marker, fragile site Xq27.3, the prevalence of fragile X syndrome was estimated to be 1/1 000 or 1/1 250 to 1/2 600 in males[10]. After the utilization of molecular diagnostic test for this disorder, a revised incidence of 1/4000 in males was generally estimated by published studies[11]. A relatively recent study, by surveying children with special education in the metropolitan area of Atlanta, GA, USA, estimated a prevalence of FXS at 1 in 2 545 among African-American males and 1 in 3 717 among Caucasian males[12]. A large population screening for FXS measured 36 124 newborn males by detecting FMR1 methylation in DNA isolated from the dried blood spots, and demonstrated an incidence of 1/5 161 in males. This probably represented the true male incidence of FXS[13]. Only two large-scale FXS screenings for Chinese populations had been reported[9,14]. One study from Central China reported that 88 patients with mental retardation were selected from 172 600 individuals, and six of these patients were found to have a full mutation by testing FMR1 CGG repeat sizes. Then according to that the incidence of Chinese children with mental retardation was 1.2%, a prevalence of FXS was calculated to be about 1/1 250 (6÷88×1.2%)[14]. But another research from Taiwan estimated an incidence of about 1/10 046 in males by screening of 10 046 newborn boys[5]. The prevalence of FXS in Southwest Chinese populations has not been reported.

The frequency of FXS among the mentally retarded populations was estimated to range from 2.6% to 8.7% of moderate to severely retarded males[3]. A study by analyzing 803 mentally retarded individuals with special education needs in five mainland cities of Central, Northwest and Northeast China, estimated that the prevalence of FXS among moderate to severely retarded males was 3.7%, and ranged from 3.1% to 6.8%. This result is close to the prevalence of FXS in Caucasian mentally retarded populations[4]. But another study from south China, Hong Kong, by surveying 324 patients with mild mental retardation, obtained a fragile X prevalence of 0.6%[5], which was very lower than that of Central and Northern region of China.

We tested 304 subjects with mental retardation from Chengdu city, but none subject was examined to have a full mutation or premutation by detecting the CGG repeat in FMR1 gene. The CpG islands upstream of the FMR1 gene in 304 boys were also detected to be unmethylated. This suggests that a very low prevalence of FXS may be existed in southwest Chinese populations. Based on our estimating result and other explorations for screening of FXS in southern Chinese people[5,15], we think that the prevalence of FXS in southwest China should be less than that of previously reported in north and central China ( Table 2). It had been reported that the fragile X syndrome mutation may be associated with founder chromosome haplotypes[4,16], so we consider that the prevalence of FXS in one region may be associated with the genetic background of the local population. To address this, a large-scale screening program including different subpopulations will be needed to determine the prevalence of the fragile X syndrome in Chinese populations.

Conflict of interest

The authors have no conflicts of interest to disclose.

[References]

[1] Mclennan Y, Polussa J, Tassone F, et al. Fragile X syndrome[J]. Curr Genomics，2011, 12(3): 216-224.

[2] Bagni C, Tassone F, Neri G, et al. Fragile X syndrome: causes, diagnosis, mechanisms, and therapeutics[J]. J Clin Invest, 2012, 122(12): 4314-4322.

[3] Daneberga Z, Pronina N, Lace B, et al. FMR1 linked haplotype analysis in a mentally retarded male population[J]. Cent Eur J Med, 2011, 6(6): 750-757.

[4] Zhong N, Ju W, Xu W, et al. Frequency of the fragile X syndrome in Chinese mentally retarded populations is similar to that in Caucasians[J]. Am J Med Genet, 1999, 84(3): 191-194.

[5] Pang C P, Poon P M K, Chen Q L, et al. Trinucleotide CGG repeat in the FMR1 gene in Chinese mentally retarded patients[J]. Am J Med Genet, 1999, 84(3): 179-183.

[6] Spector E B, Kronquist K E. ACMG standards and guidelines for clinical genetics laboratories, 2005〔EB/OL〕. 2005, available at: http://www.acmg.net.

[7] Fu Y, Kuhl D P A, Pizzuti A, et al. Variation of the CGG repeat at the FragileX site results in genetic instability: resolution of the Sherman paradox[J]. Cell, 1991, 67(6): 1047-1058.

[8] Peprah E. Fragile X syndrome: the FRM1 CGG repeat distribution among world populations[J]. Ann Hum Genet, 2012, 76(2): 178-191.

[9] Tzeng C C, Tsai L P, Hwu W L, et al. Prevalence of the FMR1 mutation in Taiwan assessed by large-scale screening of newborn boys and analysis of DXS548-FRAXAC1 haplotype[J]. Am J Med Genet, 2005, 133(1): 37-43.

[10] Gustavson K H, Blomquis T H K, Holmgren G. Prevalence of the fragile-X syndrome in mentally retarded boys in a Swedish county[J]. Am J Med Genet， 1986, 23(1): 581-587.

[11] Turner G, Webb T, Wake S, et al. Prevalence of fragile X syndrome[J]. Am J Med Genet，1996, 64(1): 196-197.

[12] Crawford D C, Meadows K L, Newman J L, et al. Prevalence of the fragile X syndrome in African-Americans[J]. Am J Med Genet, 2002, 110(3): 226-233.

[13] Coffee B, Keith K, Albizua I, et al. Incidence of fragile X syndrome by newborn screening for methylated FMR1 DNA[J]. Am J Hum Genet, 2009, 85(9): 503-514.

[14] Shen Y, Zhu N, Fan Y. Screening and genetic analysis of fragile X syndrome in Tongling Anhui province of China[J]. Natl Med J China, 1997, 77(4): 260-262.

[15] Chen T A, Lu X F, Che P K,et al. Variation of the CGG repeat in FMR1 gene in normal and fragile X Chinese subjects. Ann Clin Biochem，1997, 34(5): 517-520.

[16] Zhong N, Ye L L, Dobkin C, et al. Fragile X founder chromosome effects: Linkage diseaquilibrium or microsatellite heterogeneity? [J]. Am J Med Genet，1994, 51(4): 405-411.

[收稿2016-04-12;修回2016-05-21]

(編輯：譚秀榮)

[基金項目]國家科技支撐計劃項目(NO：2014BAI06B03)。

[通信作者]王和 ,男，博士生導(dǎo)師,四川大學(xué)華西第二醫(yī)院教授、四川省產(chǎn)前診斷中心主任、國家產(chǎn)前診斷技術(shù)專家組專家、中華預(yù)防醫(yī)學(xué)會出生缺陷預(yù)防與控制專委會常務(wù)委員，產(chǎn)前診斷學(xué)組副組長、擔(dān)任《中華婦產(chǎn)科雜志》《四川大學(xué)學(xué)報》《中華婦幼臨床醫(yī)學(xué)雜志》《中華醫(yī)學(xué)遺傳學(xué)雜志》等雜志編委。畢業(yè)于原華西醫(yī)科大學(xué)。曾受國家教育部等派遣到荷蘭Erasmus大學(xué)、萊頓大學(xué)、美國密西根大學(xué)、杜克大學(xué)等進修與工作。負(fù)責(zé)國家自然科學(xué)基金6項，部省級科研項目多項；在國內(nèi)外發(fā)表論文百余篇；獲得國家發(fā)明專利6項；培養(yǎng)博、碩士研究生多名。E-mail:wanghe2008_cd@126.com。

[中圖法分類號]R749.93

[文獻(xiàn)標(biāo)志碼]A

[文章編號]1000-2715(2016)03-0223-06

An investigation for the incidence of fragile X syndrome in mentally retarded patients in a Southwest Chinese city

ZhangYoucheng1,WangHe2

(1.Department of Obstetrics, People’s Hospital of Guizhou Province, Guiyang Guizhou 550002, China；2.Prenatal Diagnosis Center, West China Second University Hospital, Sichuan University, Chengdu Sichuan 610041, China)

[Abstract]Objective To investigate the incidence of fragile X syndrome (FXS) in mentally retarded patients in Southwest China.Methods We adopted one polymerase chain reaction (PCR) assay allowing to determine the CGG repeat length of the FMR1 gene to carry out a screening test for FXS in mentally retarded patients in a Southwest Chinese city, so as to evaluate the incidence of FXS in mentally retarded patients.Results We found that the most frequent CGG repeat allele was 29 repeats, followed by 30 and 31 repeats, and there was a secondary peak at 36 and 37 repeats. These findings were consistent with those of other studies for the CGG-repeat distribution pattern in Chinese populations. But none subject was examined to have a full mutation or premutation in the present studies.Conclusion The prevalence of FXS in mentally retarded patients in southwest China is estimated to be less than that of previously reported in North and Central China.

[Key words]fragile X syndrome; CGG repeat; molecular screening; Chinese; mentally retarded patients