Hong Kong Med J 2017;23:Epub 3 Mar 2017
Identification of fragile X pre-mutation carriers in the Chinese obstetric population using a robust FMR1 polymerase chain reaction assay: implications for screening and prenatal diagnosis
Yvonne KY Cheng, MRCOG, FHKAM (Obstetrics and Gynaecology)1; Christina SW Lin, MSc1; Yvonne KY Kwok, PhD1; YM Chan, MRCOG, FHKAM (Obstetrics and Gynaecology)1; TK Lau, FRCOG, FHKAM (Obstetrics and Gynaecology)2; TY Leung, FRCOG, FHKAM (Obstetrics and Gynaecology)1; KW Choy, PhD1
1 Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Shatin, Hong Kong
2 Fetal Medicine Centre, Paramount Medical Centre, Hong Kong
Corresponding author: Dr KW Choy (email@example.com)
Introduction: There is significant morbidity associated with fragile X syndrome. Unfortunately, most maternal carriers are clinically silent during their reproductive years. Because of this, many experts have put forward the notion of preconception or prenatal fragile X carrier screening for females. This study aimed to determine the prevalence of fragile X syndrome pre-mutation and asymptomatic full-mutation carriers in a Chinese pregnant population, and the distribution of cytosine-guanine-guanine (CGG) repeat numbers using a robust fragile X mental retardation 1 (FMR1) polymerase chain reaction assay.
Methods: This was a cross-sectional survey in prospectively recruited pregnant women from a university hospital in Hong Kong. Chinese pregnant women without a family history of fragile X syndrome were recruited between April 2013 and May 2015. A specific FMR1 polymerase chain reaction assay was performed on peripheral blood to determine the CGG repeat number of the FMR1 gene. Prenatal counselling was offered to full-mutation and pre-mutation carriers.
Results: In 2650 Chinese pregnant women, two individuals with pre-mutation alleles (0.08%, one in 1325) and one asymptomatic woman with full-mutation (0.04%, one in 2650) alleles were identified. The overall prevalence of pre-mutation and full-mutation alleles was 0.11% (1 in 883). Furthermore, 30 (1.1%) individuals with intermediate alleles were detected. In the 2617 women with normal CGG repeats, the most common CGG repeat allele was 30.
Conclusions: The overall prevalence of pre-mutation and asymptomatic full-mutation carriers in the Chinese pregnant population was one in 883, detected by a new FMR1 polymerase chain reaction assay.
New knowledge added by this study
- This study reports the prevalence of fragile X pre-mutation carriers in Chinese pregnant women.
- The prevalence of pre-mutation and asymptomatic full-mutation carriers was one in 883 and disproves the belief that carrier rates in Chinese are extremely low.
- Maternal fragile X carriers are not rare in a Chinese population. Women should be offered the option of carrier screening during the preconception period or prenatally.
Fragile X syndrome (FXS) is the second leading genetic cause of intellectual disability after Down syndrome,1 affecting one in 4000 males and one in 8000 females.2 The typical phenotypes include behavioural abnormalities, autism, cognitive impairment, and dysmorphism such as large protruding ears, elongated face, and macroorchidism in male patients. This syndrome is caused by a defective fragile X mental retardation 1 (FMR1) gene located on the X chromosome, where there is an unstable cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5’ untranslated region.3 Normally the number of CGG repeats is less than 44, but if it is more than 200 (full mutation), the FMR1 gene expression will be ‘shut down’ due to methylation of its promoter. The protein product, which is essential for normal neurodevelopment, is thus not produced. When the repeat number is between 55 and 200 (pre-mutation),4 the FMR1 gene can be expressed but the repeat number is potentially expandable to full mutation during its transmission to the next generation. Such risk of expansion is increased with the size of the repeat number, and is close to 100% when the size of CGG repeats is 100 or more. In addition, pre-mutation carriers are at risk of developing fragile X–associated primary ovarian insufficiency (FXPOI) and fragile X–associated tremor/ataxia syndrome (FXTAS) in late adult life, although they are mentally normal.5 6 Intermediate alleles are repeat numbers between 45 and 54, and individuals carrying theses alleles are at risk of expanding into pre-mutation but not into full mutation.7 8 9
Because of the significant morbidity associated with FXS, and since most maternal carriers are clinically silent during their reproductive years, many experts have put forward the notion of preconception or prenatal fragile X carrier screening for females.10 The prevalence of pre-mutation carriers will directly affect the efficacy and cost-effectiveness of screening, but this varies widely between different ethnic groups and countries. While it is well known that Caucasians and Jews have high carrier rates of 1 in 100-250,10 many studies in Chinese populations report an extremely low carrier rate.11 12 13 Among these studies, the largest included 10 046 newborn boys, but identified only six pre-mutation carriers (1 in 1674).13 These studies, however, were limited by the fact that the screening methods used were polymerase chain reaction (PCR) assays that were not accurate or were unable to amplify long CGG repeats.14 In addition, the screening of a low-risk Chinese pregnant population has not been studied.
Recently, we have validated a new fragile-X-related–specific PCR assay that utilises a low-cost capillary electrophoresis instrument and the FragilEase reagent kit (PerkinElmer Inc, Waltham [MA], US), and is able to detect CGG repeat numbers at a level as high as 1000.14 The repeat numbers analysed by this new assay were highly concordant with those obtained from the conventional reference method (PCR + Southern blot) in 112 archived samples, including 25 samples of full mutation (the largest allele size measured at 1380 repeats). The intra-assay (coefficient of variation <2.5%) and inter-assay imprecision was within 1 CGG repeat.14 The objectives of this study were to determine the prevalence of FXS pre-mutation carriers and the distribution of repeat numbers in the Chinese pregnant population in Hong Kong, using this FXS-specific PCR assay.
This was a prospective observational study conducted at a university hospital in Hong Kong between April 2013 and May 2015. Chinese pregnant women between 4 and 41 weeks of gestation, at or above the age of 18 years who could understand English or Chinese and give informed consent were eligible for the study. Eligible women were approached in the antenatal clinic or the antenatal ward by the research assistant at one convenient time-point once per day and invited to participate in the study. Women with a known family history of FXS were excluded to avoid an over-representation of the pre-mutation carrier rate in the general population, so that data obtained would be more useful in determining whether population-based screening is beneficial. Pre-test counselling was given by a research assistant with a bachelor’s and master’s degree in human genetics. Printed information about fragile X carrier testing was provided and included information about the clinical features and maternal inheritance of the disease. It was also explained to participants that genetic counselling would be offered if they were found to have an increased CGG repeat number of ≥45. Testing was entirely voluntary, and no payment was received by the participants. Written informed consent was obtained. Two millilitres of maternal blood was collected in an EDTA tube from each participant. The FMR1 CGG repeat result could be obtained within 1 day but study samples were processed in batches so results were available between 1 day and 7 months later. Women were informed prior to consenting that the result might not be available before delivery.
The FMR1 CGG repeat status of each sample was tested at a screen cut-off value of ≥45.14 The details of the PCR method are described below. All participants had the right to access personal data and study results. Positive test results (≥45 CGG repeats) would be made known to the participants, and genetic counselling would be provided. Where indicated, prenatal and postnatal diagnoses were offered by means of chorionic villus sampling or amniocentesis and cord blood or neonatal blood respectively, with analysis of CGG repeats in the extracted DNA from the sample using the same PCR method.
Approval for the study was obtained from the institutional review board of the Joint-Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee (CRE-2013.055).
Polymerase chain reaction–only assay for detection of CGG repeats in FMR1 gene
Genomic DNA was extracted from peripheral blood samples using DNeasy Blood & Tissue Kit according to the manufacturer’s protocol (Qiagen, Hilden, Germany) or using the automatic system, chemagic Prepito-D, following the manufacturer’s protocol (PerkinElmer, Turku, Finland).
Polymerase chain reaction analysis of fragile X mutations
The FMR1 repeat region of each DNA sample was amplified using the FragilEase PCR reagent kit following the manufacturer’s protocol (PerkinElmer). It involved a forward (TCA GGC GCT CAG CTC CGT TTC GGT TTC A) and a reverse primer (FAM-AAG CGC CAT TGG AGC CCC GCA CTT CC) anneal to FMR1-specific sequence upstream and downstream of the trinucleotide repeat region, respectively. Thermal cycle amplification of the highly GC-rich trinucleotide repeat region produced fragments whose size was directly related to the number of trinucleotide repeats present in the DNA sample. Two female reference DNA samples (one pre-mutation carrier [30/80 repeats] and one individual with full mutation [20/200 repeats]) for evaluating the analytical performance of the assay were obtained from the Coriell Institute for Medical Research (Camden, US). The known repeat sizes of the reference samples were concurrently amplified and used to calculate the CGG repeat numbers of the unknown samples.
Purification of the polymerase chain reaction product
Polymerase chain reaction products were purified using NucleoFast 96 PCR plate (MACHEREY-NAGEL GmbH & Co KG, Germany) or the PureLink PCR Micro Kit (Invitrogen, Carlsbad [CA], US). Purification procedures were performed according to the manufacturer’s instructions with a final elution volume of 20 µL.
Fragment sizing with microfluidic capillary electrophoresis
The fragment size for the sample was analysed using 2100 Bioanalyzer (Agilent Technologies, Santa Clara [CA], US). In this study, 3 µL of the purified PCR product and 3 µL of the 7500-size marker reagent (from an Agilent DNA 7500 kit) were loaded into each of the 12 wells of the Bioanalyzer chip. A standard curve was constructed from the two female reference samples (Coriell NA20240 [30/80 CGG repeats] and NA20239 [20/200 CGG repeats]) with known repeat size. This allowed the determination of CGG repeat size with higher accuracy.
Report of FMR1 fragment size
FraXsoft analysis software (PerkinElmer) was used to calculate the CGG repeat lengths by utilising the base pair size data exported from the bioanalyser. Fragment sizes that were below 200 CGG repeats were interpolated from their base-pair electrophoresis result using a linear regression constructed between the four allele values of the two Coriell female reference samples. Larger repeat sizes (>200 repeats) were calculated by extrapolation along the same regression line.14
A convenient sample of 2650 Chinese pregnant women was recruited between 4 and 41 weeks of gestation. FMR1 allelic expansion was screened in each subject, and two pre-mutation carriers (0.08%, one in 1325) and one asymptomatic individual with full mutation (0.04%, one in 2650) who were unrelated were identified. The overall prevalence was therefore one in 883 (0.11%) or 11 per 10 000 (95% confidence interval, 3-36 per 10 000 using the Wilson method with continuity correction). These three women are described in detail below. There were also 30 (1.1%) women with CGG repeats who fell into the intermediate category. The remaining 2617 women screened were found to have normal CGG repeats, and the most common CGG repeat allele was 30. This distribution of allele frequencies for CGG repeats in the FMR1 gene in the population with normal CGG repeat numbers is shown in the Figure.
Figure. Distribution of allele frequencies for the number of CGG repeats in FMR1 gene in the 2617 normal pregnant women
One asymptomatic subject with full-mutation allele
The woman had FMR1 gene testing carried out during the third trimester and was found to have a full mutation with CGG repeat number of 35/401. She was phenotypically normal. She had completed junior high school education and was working in a fast food restaurant. Upon detailed questioning, she suspected that her brother and one of her maternal male cousins might have some autistic features, but she was not aware of any mental retardation, or any formal genetic diagnosis in either of these two relatives. Genetic counselling was given. Prenatal diagnosis was not performed since the woman had been tested during the third trimester and termination of pregnancy was not an option. She subsequently delivered a healthy baby girl. The parents were counselled about testing the baby for FXS but they declined and preferred to observe the development of their child first. The child was referred for follow-up of her development. The woman’s other family members also declined fragile X screening because they were phenotypically normal with no current reproductive plans.
Two subjects with pre-mutation allele
The first pre-mutation carrier was a 31-year-old nulliparous female with no features associated with FXS or family history of intellectual disability or autism. Testing of the FMR1 gene was done at 13 weeks and 4 days, and the result was available at 14 weeks and 4 days indicating a CGG repeat number of 30/70. The woman requested prenatal diagnosis for her female fetus following genetic counselling. Amniocentesis was performed at gestation 16 weeks and 4 days. The PCR analysis result was available at 17 weeks and 2 days, and revealed that the maternal pre-mutation allele had been transmitted to the fetus and expanded to CGG repeat number of 30/579, indicating the female fetus carried a full-mutation allele. Genetic counselling was provided and the couple opted for termination of pregnancy and planned for pre-implantation genetic diagnosis in future.
The second carrier was a 39-year-old female with normal phenotype and no family history of intellectual disability or autism. She had testing of the FMR1 gene at 20 weeks and 6 days of gestation. The result was available at 21 weeks and 2 days of gestation showing a CGG repeat number of 31/64, and her fetus was female. Following genetic counselling, the parents decided not to have any prenatal or postnatal diagnosis owing to the variable and unpredictable phenotype in full-mutation females.
This is the largest study of the prevalence of fragile X pre-mutation carriers in Chinese pregnant women, as well as the largest one using this new FXS-specific PCR assay (FragilEase) in the Chinese population. The combined prevalence of pre-mutations and full mutations of FXS in normal asymptomatic pregnant Hong Kong Chinese women was as high as 0.11% (1 in 883). We included also one case of full mutation in our estimation of prevalence because some women with full mutations are apparently asymptomatic but are at risk of transmitting FXS to their offspring. Therefore, the combined prevalence would reflect more precisely the overall risk of transmitting FXS in the general population. Our finding is consistent with the recent publication by Huang et al,15 who identified one pre-mutation carrier in their population of 1113 Han Chinese (534 males and 579 non-pregnant females). Our finding also refutes the belief that FXS pre-mutations are extremely rare in Chinese.11 12 13 In fact, the incidence in Chinese is comparable with some of the incidences reported from Korea (1 in 1090).16
A standard capillary analyser is only capable of detecting and sizing FMR1 PCR products with less than 200 CGG repeats. Thus differentiating full mutations with greater than 200 CGG repeats from apparently homozygous normal female samples, and confirming full mutations, has historically required the Southern blot reflex test. The Southern blotting assay, however, is expensive, labour intensive, and requires a large amount of DNA making its use in screening impractical.13 The advantage of this FragilEase PCR assay is the ability to detect up to 1000 CGG repeats, so that even asymptomatic full-mutation individuals can be identified during routine screening, as shown in one of our cases. It has high throughput and high sensitivity of 99%.14 The cost for each fragile X assay is estimated to be only US$44, deduced from a parallel run of a minimum of four samples plus two reference standards on each Bioanalyzer chip, and this cost includes that of FragilEase reagent kit, DNA extraction kit, PCR-related consumables, Bioanalyzer kit, equipment maintenance, and staff costs. Processing of each chip takes 1 hour and a maximum of nine samples per chip can run. The low cost of this test is beneficial as a screening test.
Our study showing an incidence of one pre-mutation or full mutation of FXS in 883 pregnant Chinese women has important implications for counselling and implementation of a FXS carrier screening programme in Hong Kong and in China, as well as in countries where Chinese immigrants are numerous. It remains controversial whether FXS should be screened for, and which model should be adopted. Some experts advocate universal prenatal screening17 as it is much more effective in identification of pre-mutation carriers compared with case finding followed by cascade screening. The latest UK Health Technology Assessment (HTA) review18 indicated that the maximal rate of detection of female pre-mutation carriers by population screening is 60% whereas only 6% of carriers will be identified by active cascade screening. In their simulation model, the additional number of births of FXS children that could be avoided each year was estimated to be 15 with cascade screening compared with 39 with prenatal screening. Since family size tends to be small in many developed countries now, the effectiveness of cascade screening has become very limited. In Hong Kong, the average number of children per household is only 1.24.19 In mainland China, until 2015, families were allowed to have only one child. The chance of revealing a positive family history with affected siblings or close relatives is thus low. Furthermore, even though parents might be planning their second child, the first affected child would not have been diagnosed with FXS if very young. Such diagnosis is particularly difficult and is delayed in Hong Kong, China, or other Asia-Pacific regions where clinical genetic services are inadequate.20 The variable phenotypes of FXS may also be masked by the mixed education levels of the population in different geographical regions. Indeed, the patient in our study who carried a full mutation is a very good example of the limitation of cascade screening with an uncertain family history or without a formal genetic assessment.
Unlike first-trimester combined Down syndrome screening that requires intensive training and effort in ultrasound measurement and a stringent algorithm in risk calculation to achieve a 90% detection rate with a 5% false-positive rate,21 22 23 24 screening for fragile X carriers is relatively simple by a maternal blood test and is thus acceptable to most women. It can also be done before conception. Furthermore, once a carrier is identified, other carriers may be found through family screening. Hence the potential utility of this screening can be profound. In the validation study of FragilEase by Kwok et al,14 78 samples tested positive, of which one was classified as false positive. This sample was tested to have a CGG repeat number of 55 (pre-mutation) by FragilEase whereas Southern blot analysis determined the repeat number to be 53 (intermediate). This gives a false-positive rate of 1.3%. The false-positive result occurred because the CGG repeat number was at the lower limit of the pre-mutation range, such that a difference of 2 repeats led to an intermediate allele being classified in the pre-mutation range. We believe that this false-positive rate is overestimated, as the majority of the pre-mutation carriers do not have a repeat number at this lower limit that could lead to such a false-positive result. Although the positive predictive value calculated was 8.0%, assuming a fragile X pre-mutation carrier prevalence of one in 883, the performance of the test should be better because the positive predictive value was underestimated due to the overestimated false-positive rate.
There are no current data on the health care costs of caring for a FXS patient in Hong Kong or Asia. In the UK, the lifetime cost for each FXS patient is estimated to be UK$380 000 and the HTA model expects population screening to be a cost-effective strategy.18 In fact, it has been shown that health care professionals and families of patients with FXS are in favour of preconception or prenatal screening.25 26 Detection of pre-mutation alleles also offers information about the women’s own health, as they are at increased risk of FXPOI and FXTAS.27 The above factors may affect a woman’s fertility planning and allow informed choices not only in this pregnancy but also subsequent pregnancies.
Despite this, the UK National Screening Committee and the American College of Obstetricians and Gynecologists do not advocate universal screening,28 29 but rather screening in those with a family history of congenital intellectual disability, autism, or premature ovarian failure. There are concerns over the counselling about complex genetic mechanisms and the psychological impact of FXS when population screening is offered. The difficulties in counselling include (1) the variable phenotypes (eg female fetuses with full mutation) associated with FXS, and (2) identification of individuals with pre-mutation allele may lead to anxiety in these individuals because both FXPOI and FXTAS have no specific treatment. Another factor that limits population screening for fragile X is the access to prenatal care and screening, especially in rural areas of mainland China.
The strength of this study lies in its size. It is the largest study of the prevalence of fragile X pre-mutation carriers performed in the Chinese pregnant women. This study also demonstrated the feasibility of this validated FXS-specific PCR-based method (FragilEase) in the Chinese population.14 One limitation is that not all pregnant women were approached for the study and the study participants were recruited by convenient sampling. During the study period, approximately 13 600 Chinese women attended our hospital but only 2650 women were recruited. Women were recruited each day at one convenient time-point by the research assistant in the antenatal ward or clinic. This was not a true random sample and hence has doubtful representativeness. Nonetheless, as the largest obstetric hospital in Hong Kong with participants recruited from both antenatal clinic and obstetric wards, and a large sample size of 2650, we aimed to include a group most typical of the general obstetric population. Another limitation is that our cohort represented mainly the Southern Chinese population and not the entire Chinese population. Despite this, the findings of our study should provide a strong foundation for further large-scale national studies that may benefit our understanding of the carrier frequencies in different parts of China and the Asia-Pacific region. Further studies are also required to look into the different models of carrier screening programmes and their cost-effectiveness in our locale to determine which screening strategy is the most appropriate in the Chinese pregnant population.
The prevalence of pre-mutation and full-mutation alleles altogether in the asymptomatic Southern Chinese population was one in 883, and that for pre-mutation alleles alone was one in 1325. These figures are higher than those reported previously in small-scale studies, and indicate that FXS is a clinical condition not to be overlooked in our locale. Further studies of the prevalence in different areas of Asia may be beneficial to direct future screening strategies.
We would like to thank the research and laboratory staff of the Fetal Medicine Team, Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong. We would also like to thank all the pregnant women and their families who participated in the study.
This work was supported partially by funding from the Hong Kong Obstetrical and Gynaecological Trust Fund. PerkinElmer has supported some of the PCR study reagents, but has no role in the design of the study; collection, analysis, or interpretation of the data; writing, review, or approval of the manuscript; or the decision to submit the manuscript for publication.
1. Sherman S. Epidemiology. In: Hagerman RJ, Silverman AC, editors. Fragile X syndrome: diagnosis, treatment, and research. Baltimore: The Johns Hopkins University Press; 1991: 69-97.
2. Coffee B, Keith K, Albizua I, et al. Incidence of fragile X syndrome by newborn screening for methylated FMR1 DNA. Am J Hum Genet 2009;8:503-14. CrossRef
3. Verkerk AJ, Pieretti M, Sutcliffe JS, et al. Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 1991;65:905-14. CrossRef
4. Kronquist KE, Sherman SL, Spector EB. Clinical significance of tri-nucleotide repeats in fragile X testing: a clarification of American College of Medical Genetics guidelines. Genet Med 2008;10:845-7. CrossRef
5. Jacquemont S, Leehey MA, Hagerman RJ, Beckett LA, Hagerman PJ. Size bias of fragile X premutation alleles in late-onset movement disorders. J Med Genet 2006;43:804-9. CrossRef
6. Uzielli ML, Guarducci S, Lapi E, et al. Premature ovarian failure (POF) and fragile X premutation females: from POF to fragile X carrier identification, from fragile X carrier diagnosis to POF association data. Am J Med Genet 1999;84:300-3. CrossRef
7. Nolin SL, Glicksman A, Ding X, et al. Fragile X analysis of 1112 prenatal samples from 1991 to 2010. Prenat Diagn 2011;31:925-31. CrossRef
8. Nolin SL, Brown WT, Glicksman A, et al. Expansion of the fragile X CGG repeat in females with premutation or intermediate alleles. Am J Hum Genet 2003;72:454-64. CrossRef
9. Fernandez-Carvajal I, Lopez Posadas B, Pan R, Raske C, Hagerman PJ, Tassone F. Expansion of an FMR1 grey-zone allele to a full mutation in two generations. J Mol Diagn 2009;11:306-10. CrossRef
10. Hill MK, Archibald AD, Cohen J, Metcalfe SA. A systematic review of population screening for fragile X syndrome. Genet Med 2010;12:396-410. CrossRef
11. Chiang SC, Lee YM, Wang TR, Hwu WL. Allele distribution at the FMR1 locus in the general Chinese population. Clin Genet 1999;55:352-5. CrossRef
12. Huang KF, Chen WY, Tsai YC, et al. Original article pilot screening for fragile X carrier in pregnant women of southern Taiwan. J Chin Med Assoc 2003;66:204-9.
13. Tzeng CC, Tsai LP, Hwu WL, et al. Prevalence of the FMR1 mutation in Taiwan assessed by large-scale screening of newborn boys and analysis of DXS548-FRAXAC1 haplotype. Am J Med Genet A 2005;133A:37-43. CrossRef
14. Kwok YK, Wong KM, Lo FM, et al. Validation of a robust PCR-based assay for quantifying fragile X CGG repeats. Clin Chim Acta 2016;456:137-43. CrossRef
15. Huang W, Xia Q, Luo S, et al. Distribution of fragile X mental retardation 1 CGG repeat and flanking haplotypes in a large Chinese population. Mol Genet Genomic Med 2015;3:172-81. CrossRef
16. Han SH, Heo YA, Yang YH, Kim YJ, Cho HI, Lee KR. Prenatal population screening for fragile X carrier and the prevalence of premutation carriers in Korea. J Genet Med 2012;9:73-7. CrossRef
17. Musci TJ, Moyer K. Prenatal carrier testing for fragile X: counseling issues and challenges. Obstet Gynecol Clin North Am 2010;37:61-70. CrossRef
18. Song FJ, Barton P, Sleightholme V, Yao GL, Fry-Smith A. Screening for fragile X syndrome: a literature review and modelling study. Health Technol Assess 2003;7:1-106. CrossRef
19. The Hong Kong Family Planning Association. The report on the Survey of Family Planning Knowledge, Attitude and Practice in Hong Kong 2012. Available from: http://www.famplan.org.hk. Accessed 21 Apr 2016.
20. Chopra M, Duan T. Rare genetic disease in China: a call to improve clinical services. Orphanet J Rare Dis 2015;10:140. CrossRef
21. Leung TY, Chan LW, Law LW, et al. First trimester combined screening for trisomy 21 in Hong Kong: outcome of the first 10,000 cases. J Matern Fetal Neonat Med 2009;22:300-4. CrossRef
22. Sahota DS, Leung TY, Fung TY, Chan LW, Law LW, Lau TK. Medians and correction of biochemical and ultrasound markers in Chinese undergoing first-trimester screening for trisomy 21. Ultrasound Obstet Gynecol 2009;33:387-93. CrossRef
23. Leung TY, Chan LW, Leung TN, et al. First-trimester combined screening for trisomy 21 in a predominantly Chinese population. Ultrasound Obstet Gynecol 2007;29:14-7. CrossRef
24. Sahota DS, Leung WC, To WK, Chan WP, Lau TK, Leung TY. Quality assurance of nuchal translucency for prenatal fetal Down syndrome screening. J Matern Fetal Neonatal Med 2012;25:1039-43. CrossRef
25. Skinner D, Sparkman KL, Bailey DB Jr. Screening for fragile X syndrome: parent attitudes and perspectives. Genet Med 2003;5:378-84. CrossRef
26. Acharya K, Ross LF. Fragile X screening: attitudes of genetic health professionals. Am J Med Genet A 2009;149:626-32. CrossRef
27. Jacquemont S, Hagerman RJ, Hagerman PJ, Leehey MA. Fragile-X syndrome and fragile X–associated tremor/ataxia syndrome: two faces of FMR1. Lancet Neurol 2007;6:45-55. CrossRef
28. Antenatal screening for fragile X syndrome: external review against programme appraisal criteria for the UK National Screening Committee (UK NSC). UK National Screening Committee; Oct 2014.
29. ACOG Committee Opinion No. 469: Carrier screening for fragile X syndrome. The American College of Obstetricians and Gynecologists Committee; Oct 2010.