INTRODUCTION — Fragile X syndrome is an X-linked disorder and the most common inherited cause of intellectual disability [1]. It is also the most common single-gene cause of autism spectrum disorder. Both males and females can be affected, although in general, males are more severely affected and some females are asymptomatic.
This topic will discuss preconception/prenatal screening and prenatal diagnosis for fragile X syndrome. The epidemiology, pathogenesis, clinical features, postnatal diagnosis, and postnatal management of the disorder are reviewed separately:
●(See "Fragile X syndrome: Clinical features and diagnosis in children and adolescents".)
●(See "Fragile X syndrome: Management in children and adolescents".)
OVERVIEW
FMR1 gene and protein — The fragile X messenger ribonucleoprotein 1 gene (FMR1) is located on the X chromosome at Xq27.3 and most commonly has approximately 30 cytosine-guanine-guanine (CGG) trinucleotide repeats, with a "normal" range of approximately 5 to 44 CGG repeats. FMR1 produces the fragile X messenger ribonucleoprotein (FMRP).
As long as FMR1 has ≤44 CGG repeats, gene transcription produces an adequate level of FMRP (ie, a level not associated with adverse phenotypic effects) and this region of the X chromosome remains stable, passing from generation to generation without significant alteration.
Pathogenesis of fragile X syndrome — Fragile X syndrome is primarily caused by expansion in the number of CGG repeats within FMR1; deletions or point mutations within FMR1 account for only 1 percent of cases [2]. As repeat size increases, stability decreases, thereby facilitating further increases in the number of repeats in the FMR1 region during gametogenesis (discussed below). The lower and upper boundaries of repeat length are variably defined, but generally described as:
●Normal – 5 to 44 CGG repeats
●Intermediate expansion – 45 to 54 CGG repeats
●Premutation – 55 to 200 CGG repeats
●Full mutation – >200 CGG repeats
Expansion of CGG repeats within the premutation range (55 to 200 CGG repeats) increases the quantity of mRNA with toxic effects [3]. Expansion of CGG repeats to a full mutation (>200 CGG repeats) promotes hypermethylation of FMR1, resulting in impaired transcription and reduced production of FMRP, which may adversely impact prenatal and postnatal brain development.
The appropriate distinction between intermediate and premutation length is uncertain, but clinically relevant because intermediate expansion does not have a clinical phenotype and does not expand directly to a full mutation, whereas females (but not males) with a premutation are at risk of expansion to a full mutation in their offspring [4].
Risk for FMR1 expansion during gametogenesis — The risk for FMR1 expansion when the gene is passed from parent to child depends on the repeat size, sex of the parent, and frequency of adenine-guanine-guanine (AGG) trinucleotide interspersion.
Oocytes — In females (46,XX), expansion of the FMR1 region during meiosis is supported by the finding of full FMR1 expansions (not just premutation expansion) in the ovaries. The distinction of whether this occurs during meiosis 1 or 2 remains unresolved [5].
Expansion in the number of CGG repeats within the FMR1 gene may be as few as one repeat or over 100 repeats. Conservatively, the upper limit of the intermediate range may be considered 54 repeats, where 55 is a "potential" premutation and 56 to 200 is a premutation. In one study, females with 45 to 54 repeats had a 6.6 percent risk of expansion, but none expanded to a full mutation in one generation [6]. There have been very rare reports of expansion to a full mutation among females with repeats of 56 CGG (one case) and 59 CGG (two cases). (See 'Post-test counseling' below.)
When a premutation expands to >90 CGG repeats, the likelihood of further expansion to a full mutation (ie, over 200 repeats) during oocyte meiosis is at least 90 percent (table 1) [7,8]. In addition to the number of CGG repeats, variables that affect frequency of expansion to a full mutation include a pedigree with full mutations; the risk is lower when the family history is negative (ie, females who are newly identified premutation carriers as a result of routine screening) [8].
Another factor affecting the frequency of expansion to a full mutation is the frequency of AGG trinucleotide interspersion in the variable region because AGG acts as a stabilizer for the region. Therefore, fewer AGG interspersions allows destabilization of the region and increases the likelihood of expansion [9-13]. In one study, 53 percent of full mutation expansions occurred from maternal alleles with no AGG interruptions, 43 percent occurred from maternal alleles with one AGG, and only 4 percent occurred from maternal alleles with two AGGs [10].
Contraction from a premutation to a normal allele is rare, but has been reported in mother-to-female offspring transmission. The role of AGG regions, which mitigate the expansion risk of the FMR1, do not appear to affect contraction rates [10]. Postzygotic mitotic CGG length changes have also been reported; discordance for CGG repeat number between identical twins is assumed to be due to this mechanism [14].
Sperm — In males, sperm cells only carry premutation alleles, even when the male has a full mutation. The reason for this is unclear. In contrast to oocytes, expansion of a premutation to a full mutation does not occur in sperm [15]. Males with a premutation ("transmitting males") pass sperm with a premutation to all of their female offspring (who always inherit their father's X chromosome) and to none of their male offspring (who never inherit their father's X chromosome). In contrast to mother-to-female offspring transmission where contraction of the premutation is rare, one-third of female offspring who inherit a premutation from their father have contraction in the size of the expansion.
The molecular processes behind contraction of CGG repeats within the FMR1 gene remain unclear. Strand mismatch and DNA polymerase slippage are considered two likely contenders, and other molecular changes may play a role. The timing of these changes remains unknown, but evidence suggests the contraction can occur during both meiosis and mitosis [5,16-18].
Factors affecting phenotype — The phenotype depends on several factors, including number of CGG repeats, FMR1 methylation, sex, and somatic cell mosaicism, as shown in the table (table 2). Each of these factors play a role in the production of FMRP and lower protein levels result in a more severe phenotype.
Sex is an important factor because males have only one X chromosome and thus have the expanded CGG region in each of their cells. Females have two X chromosomes, and the expanded CGG region is on only one of their two X chromosomes. Since one of their X chromosomes is randomly inactivated, the overall number and distribution of cells with an expanded region is variable in females and results in a wide range of effects (no discernable effects to classic fragile X characteristics). There is no accurate way to predict the phenotype in females with a full mutation fragile X. It is theoretically possible for a female to have an expanded CGG region on each of their X chromosomes if both of parents carried a premutation or full mutation. At most, such females would carry a full mutation and premutation since males with full mutations only have premutations in their sperm.
A detailed description of the drivers of phenotype and the spectrum of clinical features of fragile X syndrome is available separately. (See "Fragile X syndrome: Clinical features and diagnosis in children and adolescents".)
PRECONCEPTION AND PRENATAL SCREENING
Rationale — Preconception or prenatal screening for fragile X premutations and full mutations is performed to identify individuals at risk for conceiving a child with clinical manifestations of the disorder. When this information is available before pregnancy, carriers have an opportunity to seek interventions for avoiding pregnancy with an affected fetus, if desired. (See 'Preconception reproductive options' below.)
After pregnancy is established, carriers may choose to undergo prenatal diagnosis for fetal fragile X premutations and full mutations so they can prepare for the birth of an affected child or terminate a pregnancy with an affected fetus. (See 'Prenatal (fetal) diagnosis' below.)
Candidates for screening — The American College of Medical Genetics and Genomics (ACMG) and the American College of Obstetricians and Gynecologists (ACOG) guidelines for screening for fragile X syndrome differ slightly and are described in the tables (table 3A-B) [19,20].
A careful assessment of individual and family history is performed to identify persons with sufficient likelihood of premutation or full mutation carrier status to warrant screening. We use the following criteria for offering fragile X screening or referral for diagnostic testing:
●Individuals seeking reproductive counseling who have a family history of fragile X syndrome (confirmed premutation or full mutation of FMR1 gene) or intellectual disability of undetermined etiology.
●Individuals of either sex with intellectual disability or developmental delay of undetermined etiology, or autism.
●Young females with elevated levels of follicle-stimulating hormone, especially with a family history of premature ovarian insufficiency (menopause before age 40), fragile X syndrome, or a relative of either sex with intellectual disability of undetermined etiology.
●Individuals with late-onset intention tremor or ataxia (usually after age 50), especially with a history of infertility, family history of movement disorders, fragile X, or intellectual disability of undetermined etiology.
Some authorities suggest routinely offering fragile X carrier laboratory screening to all females, given the high test sensitivity (99 percent), the potential impact of the full mutation in offspring, and the relatively high premutation carrier rate (ranging from 1 in 257 for females with a negative family history of intellectual disability, developmental problems, or autism, to 1 in 86 for females with a positive family history [21]) [6,22]. Concerns about general population reproductive screening for fragile X include the complexities of pre- and post-test counseling, the challenges in predicting expansion of the premutation, counseling regarding health risks of premutation carriers, and the broad phenotype in the full mutation female fetus. Each of these concerns is undergoing continued review as reproductive genetic carrier screening expands for other conditions. ACOG has acknowledged that its criteria for screening will not detect the majority of premutation fragile X carriers [20]. For those individuals who request fragile X screening, pretest counseling and consent should be provided. (See 'Pre-test counseling' below.)
Universal fragile X carrier laboratory screening is not recommended for males. Low-risk males (negative personal and family history of intellectual disability, developmental problems, or autism) are not screened since their male offspring will not inherit their X chromosome and, if they carry a premutation, their female offspring will be no worse than premutation carriers since CGG expansion does not occur in sperm and some evidence suggests premutations become smaller when passed from a father to his female offspring [16,17].
Pre-test counseling — Pre-test counseling remains an essential component of any reproductive genetic carrier screening and can be performed virtually, in person, and with written materials, with validation of ongoing education of health care providers and their patients. Increasingly, patients are active participants in their health care with access to their medical records, shared decision-making, and a desire to be provided options rather than recommendations.
Inclusion of the adenine-guanine-guanine (AGG) segments helps predict expansion from a premutation to full mutation and thus disease prognostication for premutation carriers. However, patients need to understand that the prognosis of a female fetus with a full mutation remains difficult to predict, given the spectrum of presentation from normal to classic fragile X syndrome and lack of available phenotypic predictors. Pretest counseling should also include the possibility of identifying a maternal condition, as for all reproductive genetic testing. In patients undergoing fragile X carrier screening, this means discussing the risk for premature ovarian failure and the smaller risk for fragile X-associated tremor/ataxia syndrome [20].
Laboratory testing — The gold standard for fragile X testing is polymerase chain reaction (PCR) followed by Southern blot:
●PCR can determine the size of the CGG region. It can accurately identify a normal allele and premutation and assess size differences between large premutations and small full mutations. However, as the expanded region becomes a larger full mutation, PCR often is unable to discern the margins of the expansion accurately. In such cases, Southern blot is performed to further refine size and, importantly, the methylation status. Southern blot is not performed as the first-line test because it requires substantially more DNA than PCR and is more time consuming to accomplish.
Some laboratories have developed modifications of the PCR process, such as triplet repeat-primed PCR (TP-PCR), which provides comparable detection to the two-step PCR and Southern blot testing process. In 2020, a commercial PCR-based interpretation of methylation test (AmplideX Fragile X Dx and Carrier Screen Kit) became available for screening for fragile X premutations in various populations [23-25]. This PCR-based methylation assessment has been reported to be as effective as Southern blot analysis. At this time, however, it is intended only for screening for premutations in at-risk females and males, and not for fetal diagnosis, preimplantation genetic testing, or fragile X syndrome diagnosis. In these situations, confirmation by Southern blot analysis is warranted.
AGG trinucleotide genotyping may be performed, as it may be useful for counseling females with intermediate and premutation expansions [8]. As discussed above, no AGG interspersion permits destabilization and increases the likelihood of expansion, whereas two to four AGG interspersions appear to decrease the risk [10]. (See 'Risk for FMR1 expansion during gametogenesis' above.)
It is important to note that routine investigations for developmental delay/intellectual disability, such as genomic microarray and routine whole exome sequencing, do not detect fragile X. The diagnosis is usually only made when a specific FMR1 assay is requested.
Post-test counseling — Referral to a genetic counselor and other clinicians with expertise in the genetics and clinical findings in fragile X syndrome is usually appropriate, given the complexity of the disorder.
●Females with a premutation are informed of the risk of expansion to a full mutation in offspring. Females with an intermediate number of repeats may have a 6.6 percent risk for expansion to a premutation in offspring [6], and expansion to a full mutation has been described in two cases where the mothers had CGG repeat length of 59 and one case where the CGG repeat length was 56 [6,7,26].
AGG determination may further refine the risk of disease in offspring of females with intermediate or premutations. However, even the maximum number of AGG repeats does not eliminate the risk that a premutation will expand to a full mutation [27].
●Females with a premutation or full mutation are informed of the potential phenotypes of their male and female offspring (table 2) and potential sequelae in future generations. These phenotypes depend on whether the fetus inherits a premutation or full mutation, the sex of the fetus, and the methylation status of the FMR1 gene. Rarely, the number of repeats and the methylation status are discordant, which makes prediction of the phenotype difficult.
●Males are informed that they will transmit their X chromosome only to their female offspring. No expansion of a premutation to a full mutation is expected in the female offspring, and contraction may occur. A full mutation is passed on as a premutation.
While a female offspring with a full expansion is not anticipated, essentially all female offspring are expected to have a premutation and thus are at increased risk for developing premature ovarian insufficiency and tremor-ataxia syndrome as adults. They are also at risk for CTG repeat expansion in their offspring.
●Males and females with a premutation are also informed of their own risk for developing tremor-ataxia syndrome in late adult life (usually after age 50) and, in females, premature ovarian insufficiency (menopause before age 40). In addition, implications for family members should be discussed as they are also at risk of being carriers. (See "Fragile X syndrome: Clinical features and diagnosis in children and adolescents" and "Fragile X syndrome: Management in children and adolescents".)
PRENATAL (FETAL) DIAGNOSIS
Candidates for prenatal (fetal) diagnosis — We offer prenatal (fetal) diagnosis for fragile X syndrome to prospective parents in whom the mother or father has >55 CGG repeats and choose this approach after risk counseling with a genetic counselor or other clinician with expertise in the genetics and clinical findings in fragile X syndrome. As discussed above, the degree of risk in offspring depends on several factors, including the absolute CGG repeat size, the number and dispersion of AGG repeats, whether the mother or father is the carrier, and the sex of the offspring. Offering prenatal (fetal) diagnosis to females with >55 CGG repeats is a conservative approach since few cases (at least three) of expansions to full mutations arising from repeats at this level in females have been reported. (See 'Prenatal (fetal) diagnosis' above.)
There are potential clinical sequelae to offspring and future generations when either parent is a carrier, although the most severe phenotype only occurs when the mother is the carrier. Prenatal (fetal) diagnosis when the father is a premutation carrier is rarely performed since, as discussed above, his male offspring will not inherit his X chromosome. His female offspring will be premutation carriers since CGG expansion does not occur in sperm and some evidence suggests premutations may become smaller when passed from a father to his female offspring.
The ACMG recommends offering prenatal (fetal) diagnosis of fragile X when the mother has a premutation or full mutation, whereas ACOG recommends offering prenatal (fetal) diagnosis to maternal or paternal known carriers of the fragile X premutation or full mutation (table 3A-B).
Procedure — Molecular diagnostics of fragile X can be applied to fetal DNA, whether obtained from chorionic villus samples (CVS) at 11 to 13 weeks of gestation or amniocytes obtained by amniocentesis at ≥15 weeks of gestation. A disadvantage of CVS is that assessment of methylation may not be possible at an early gestational age; the methylation of the expanded region is a secondary event and its exact timing is controversial. Even in individuals with a full mutation, there can be mosaicism within their tissues for the extent of methylation. Follow-up amniocentesis later in gestation may be required to determine methylation status to better predict the offspring phenotype if a large premutation or small full mutation is identified.
Determining the exact size of the expansion region by analyzing cell-free DNA in a sample of maternal blood (ie, noninvasive prenatal testing [NIPT]), is unreliable, but may be possible in the future as advances in NIPT methodology evolve.
Interpretation of findings at prenatal diagnosis — Given the complexity of this disorder, the results of prenatal testing and offspring prognosis should be provided to parents in consultation with a genetic counselor or other clinicians with expertise in the genetics and clinical findings in fragile X syndrome (table 2). (See "Fragile X syndrome: Clinical features and diagnosis in children and adolescents".)
The National Fragile X Foundation is an adjunctive resource that provides detailed information on fragile X on its website.
PRECONCEPTION REPRODUCTIVE OPTIONS
●Donor gametes – Prospective parents may avoid conceiving a pregnancy with an affected fetus by:
•Using donor gametes from an unaffected donor
•Avoiding pregnancy
●Preimplantation genetic testing – Prospective parents in whom the female carries a premutation or a full mutation may avoid implantation of a pregnancy with an affected male offspring or carrier female offspring by undergoing preimplantation genetic testing (PGT), but PGT for this condition has numerous technical challenges [28]. The polymerase chain reaction (PCR) and Southern blot techniques used for carrier testing and prenatal diagnosis are not always informative in PGT because premutations and full mutations of the FMR1 allele are highly refractory to PCR amplification of DNA from only a single cell, and a single cell does not contain sufficient DNA for Southern blot analysis. In addition, female carriers may have impaired ovarian reserve, which hinders the ability to harvest an adequate number of oocytes for in vitro fertilization and subsequent embryo transfer, all of which are necessary components of PGT [29,30]. However, examination of maternal alleles for markers closely linked to the FMR1 allele can provide helpful information for determining which of the X chromosomes has passed to her male or female offspring. By examining each maternal X chromosome for variations in gene markers (short tandem repeats [STR]) closely linked to FMR1, the STR marker results of the mother become a surrogate for the fragile X site. Such an approach can be used before methylation changes occur, such as in preimplantation embryos and chorionic villus sampling (CVS) analysis of first trimester trophoblast tissues [31]. (See "Preimplantation genetic testing".)
●Noninvasive fetal sex determination – Some females with a premutation may choose noninvasive fetal sexing (eg, cell-free DNA screening, ultrasound of fetal genitalia) to inform their decision about whether to proceed to invasive testing by CVS, given that the effects of a full mutation are so variable, covering the full spectrum from no discernible phenotype through mild cognitive impairment to full fragile X syndrome. If the risk of expansion to a full mutation is low and only a minority of females with a full mutation would be severely affected, some prospective parents may elect not to proceed to invasive testing by CVS/amniocentesis if the fetus is female.
SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Prenatal screening and diagnosis" and "Society guideline links: Fragile X syndrome".)
INFORMATION FOR PATIENTS — UpToDate offers two types of patient education materials, "The Basics" and "Beyond the Basics." The Basics patient education pieces are written in plain language, at the 5th to 6th grade reading level, and they answer the four or five key questions a patient might have about a given condition. These articles are best for patients who want a general overview and who prefer short, easy-to-read materials. Beyond the Basics patient education pieces are longer, more sophisticated, and more detailed. These articles are written at the 10th to 12th grade reading level and are best for patients who want in-depth information and are comfortable with some medical jargon.
Here are the patient education articles that are relevant to this topic. We encourage you to print or e-mail these topics to your patients. (You can also locate patient education articles on a variety of subjects by searching on "patient info" and the keyword(s) of interest.)
●Basics topic (see "Patient education: Fragile X syndrome (The Basics)")
SUMMARY AND RECOMMENDATIONS
●Genetic basis – Fragile X syndrome is the most common inherited cause of intellectual disability and the most common single-gene cause of autism. Changes in a region of the X chromosome, known as the fragile X messenger ribonucleoprotein 1 gene (FMR1), lead to the characteristic clinical features of fragile X syndrome. (See 'Introduction' above and 'FMR1 gene and protein' above.)
●Phenotype – Fragile X syndrome is associated with a variety of physical, behavioral, and cognitive abnormalities that vary according to the patient's age, sex, degree of CGG elongation, mosaicism, and FMR1 methylation (table 2). (See 'Factors affecting phenotype' above.)
●Pathogenesis of the phenotype
•Fragile X syndrome is primarily caused by expansion in the number of cytosine-guanine-guanine (CGG) repeats within the FMR1 gene; deletions or point mutations within FMR1 account for only 1 percent of cases. Expansion of CGG repeats allows hypermethylation of FMR1, resulting in impaired transcription and reduced production of the fragile X messenger ribonucleoprotein (FMRP), which adversely impacts prenatal and postnatal brain development. (See 'Pathogenesis of fragile X syndrome' above.)
•The lower and upper boundaries of repeat length are variably defined, but generally described as (see 'Pathogenesis of fragile X syndrome' above):
-Normal – 5 to 44 CGG repeats
-Intermediate expansion – 45 to 54 CGG repeats
-Premutation – 55 to 200 CGG repeats
-Full mutation – >200 CGG repeats
•As repeat size increases above the normal range, stability decreases and further increases in the number of repeats in the FMR1 region become likely. The risk for FMR1 expansion when the gene is passed from parent to child depends on the repeat size (table 1), sex of the parent (expansion only occurs from mother to child transmission), and frequency of adenine-guanine-guanine (AGG) trinucleotide interspersion. (See 'Risk for FMR1 expansion during gametogenesis' above.)
●Candidates for preconception/prenatal screening – We suggest offering preconception or prenatal screening for fragile X syndrome to females at increased risk of carrying a premutation or full mutation, rather than universal screening (Grade 2C). (See 'Candidates for screening' above.)
•Individuals at increased risk of an abnormality of the FMR1 gene include (see 'Candidates for screening' above):
•Individuals of either sex with intellectual disability, developmental delay, or autism.
•Individuals seeking reproductive counseling who have a family history of fragile X syndrome (confirmed premutation or full mutation of FMR1 gene) or undiagnosed intellectual disability or autism.
•Young females with elevated levels of follicle-stimulating hormone, especially with a family history of premature ovarian insufficiency, fragile X syndrome, or a relative of either sex with undiagnosed intellectual disability.
•Individuals with a family history of late-onset intention tremor or ataxia, especially with a family history of movement disorders, fragile X, or undiagnosed intellectual disability.
●Counseling patients about test results – After screening, referral to a genetic counselor or another clinician with expertise in the genetics and clinical findings in fragile X syndrome is appropriate.
•Females with a premutation are informed of the risk of expansion to a full mutation in offspring. This risk may be altered by the presence of AGG regions interspersed within the expanded region.
•Males with a premutation or full mutation are informed that their female offspring will have a premutation.
•Males and females with a premutation or full mutation are informed of the potential phenotypes of their male and female offspring (table 2) and potential sequelae in future generations.
•Males and females with a premutation are also informed of their own risk for developing tremor-ataxia syndrome in late adult life (usually after age 50) and, in females, premature ovarian insufficiency (menopause before age 40). In addition, implications for family members should be discussed as they are also at risk of being carriers. (See 'Post-test counseling' above.)
●Prenatal (fetal) diagnosis – Prenatal (fetal) diagnosis for fragile X is offered when the mother has a premutation or full mutation. Fetal testing is performed on cells obtained by chorionic villus sampling or amniocentesis. For males with either a premutation or full mutation, fetal testing is rarely done as all female offspring are expected to be premutation carriers. (See 'Candidates for prenatal (fetal) diagnosis' above and 'Prenatal (fetal) diagnosis' above.)
The results of fetal testing and offspring prognosis should be provided to parents in consultation with a genetic counselor. (See 'Interpretation of findings at prenatal diagnosis' above.)
●Reproductive options – Preimplantation genetic testing is an option to enable transfer only of embryos at lowest risk of fragile X syndrome. (See 'Preconception reproductive options' above.)
