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Book |
These chapters were published with modifications by Oxford University Press, 2004 |
Human Genome Epidemiology: A Scientific Foundation for Using Genetic Information to Improve Health and Prevent Disease |
Book Cover | Preface | Acknowledgments | Contents | Contributors |
PART IV CASE STUDIES: Using Human Genome Epidemiology Information to Improve Health |
Chapter 23 |
Fragile
X Syndrome: Application of Gene Identification to Clinical Diagnosis
and Population Screening Background The fragile X syndrome, an X-linked dominant disorder with reduced penetrance, is one of the most common forms of inherited mental retardation. The disorder-causing mutation is the amplification of a cytosine-guanine-guanine (CGG) repeat in the 5’ untranslated region of FMR1 located at Xq27.31. The fragile X mutation in affected persons results in the loss of the FMR1 gene product fragile X mental retardation protein (FMRP), an RNA-binding protein (2). Although its precise function is not yet understood, FMRP is thought to regulate the translation of other proteins (3). Thus, the loss of FMRP and possibly the loss of regulation of other as-yet-unidentified proteins result in the clinical phenotype of the fragile X syndrome. The clinical phenotype associated with the fragile X syndrome is wide and includes a variety of cognitive, physical, and behavioral characteristics (4). With regard to cognitive function, affected males often exhibit developmental delay early in childhood. By the age of 3 years, most males will test in the mentally retarded range (5). Ultimately, mental retardation is diagnosed in almost all males with the fragile X syndrome, with severity ranging from profound (IQ <20) to mild mental retardation (IQ 50-70), with most being moderately retarded (IQ 40-54)(6). Physically, adult males often have a long narrow face, prominent ears, a prominent jaw, and increased testicular volume (6). Other common physical features include a high arched palate, hyper-extensible finger joints, double jointed thumbs, single palmar crease, hand calluses, velvet-like skin, flat feet, and mitral valve prolapse (7). Males with the fragile X syndrome also tend to exhibit behavioral features such as hyperactivity, social anxiety, tactile defensiveness, stereotypies (e.g., hand-flapping), and hand biting (6). Language delay and perseverative speech are also commonly observed among males with the fragile X syndrome. Compared with other children who have developmental delay without the fragile X syndrome, males with the fragile X syndrome exhibit some autistic-like features, such as social avoidance, at a very young age(8). As many as 25% of males with the fragile X syndrome meet the diagnostic criteria for autism9. The association of fragile X with autism, however, is not clear because the proportion of males with the fragile X syndrome meeting the diagnostic criteria for autism seems to diminish with age(9). Females with the full mutation are often less affected than males, presumably because of X-inactivation10;(11). Approximately 30% to 50% of females with the full mutation have an IQ of <70, and 50% to 70% of females with the full mutation have an IQ of <8512. With regard to the specific cognitive and neuropsychological profile, deficits in specific IQ sub-tests (such as the arithmetic score), executive function, and visual information processing also have been noted for females with the full mutation4;(13-15). The pattern of autistic-like behavior among males also occurs among a proportion of females with the full mutation(16). Interestingly, autistic-like behavior is not correlated with IQ in females with the full mutation, which suggests that the degree of mental retardation is not the underlying cause of this behavior(16). Epidemiology of the fragile X syndrome Genetics of the fragile X syndrome In summary, the fragile X syndrome is transmitted as an X-linked dominant mutation with reduced penetrance. The reduced penetrance is related to the form of the mutation; that is, only full mutation alleles lead to the syndrome. The increased occurrence of the syndrome in succeeding generations of an identified family, anticipation, results from the instability of the repeat mutation and the bias for expansion. There is a parent-of-origin effect in that only premutation alleles carried by females are at risk for expansion to the full mutation in the next generation. Premutation alleles carried by males are relatively stable. Epidemiologic findings In contrast to the populations of northern European descent, few estimates exist for other racial/ethnic groups. There have been only two other population-based estimates, both for African-derived populations. Elbaz et al.(31) examined an Afro-Caribbean population in the French West Indies, and Crawford et al.(28) examined an African-American population in metropolitan Atlanta, Georgia, USA. Surprisingly, both studies suggested that the point estimate in these admixed, African-derived populations is approximately 1 in 2,500 males, which is higher than that observed in white populations (Table 22-1). Further studies are needed to explore this possible higher general population prevalence as the confidence intervals for both studies overlap with estimates in populations of northern European descent. Even less is known about the prevalence of the full mutation among
females in the general population. On the basis of the prevalence of
the full mutation among white males in the general population (approximately
1 in 4,000) and the transmission only by females of the full mutation
to their offspring, the expected prevalence of the full mutation allele
among females is 1 in 4,000. Assuming that 50% of females with the full
mutation will be affected with the fragile X syndrome phenotype, the
expected prevalence of females affected by the fragile X syndrome is
approximately 1 in 8,000 females. Among 8,462 women in Israel who had
no family history of mental retardation, Pesso et al.(32)
identified one woman with the full mutation. In a separate study of
14,334 normal healthy women in Israel, Toledano-Alhadef et al.(33)
identified three women with a full mutation, resulting in a higher prevalence
(1 in 4,778 females) compared with Pesso et al.(32)
Neither study was population-based and both were biased toward testing
unaffected females. Thus, conclusions cannot be made about the prevalence
of the fragile X syndrome among females based on these studies. Large
population-based studies are needed to better understand the prevalence
of the full mutation and its expression among females. For this review, we defined premutations in females as either 61-200 repeats or 55-200 repeats. The former definition of premutations characterizes repeats that are always unstable and expand to the full mutation. The latter definition of premutations characterizes premutations of smaller size that can be found in families with the fragile X syndrome(24,36). The smallest premutation among families with the fragile X syndrome to expand to the full mutation in a single generation is 59 repeats (24,36). In the general population, however, these smaller premutations may or may not be unstable(37). Thus, the former estimate of premutations (Table 22-2) represent the lower limits of premutation carriers whereas the latter estimates of premutations possibly represent the upper limits of premutation carriers. Results from recent, large studies suggest that the lower limits of the prevalence of the premutation range from 1 in 231(33) to 1 in 468(32) females, and the upper limits range from 1 in 116(33)to 1 in 259(38) females (Table 2). Interactions The second reason to expect gene-gene interactions is that the normal function of FMRP is to regulate translation of other proteins. FMRP is an RNA-binding protein (2,45) that is capable of binding to itself as well as other proteins(46). In addition to its RNA-binding capabilities, FMRP has both a nuclear localization signal (NLS) and a nuclear export signal (NES)(47). The current model suggests that oligomerized FMRP, in conjunction with other proteins, shuttles specific mRNAs from the nucleus to the cytoplasm for translation. The identification of the specific mRNAs and their corresponding genes necessary for the complete understanding of the molecular consequences related to the fragile X syndrome phenotype(3). Laboratory tests The most popular and accepted method for DNA-based testing for the expanded CGG repeat is the Southern blot. Many different restriction enzymes can be used in combination to determine both expansion (EcoRI, PstI, BglII, HindIII, BclI) and methylation (SacII, BssHII, EagI, BstZI) status for an individual(7;48). Methylation status is particularly useful for distinguishing between borderline premutation and full mutation alleles (200-230 repeats)(36). Methylation sensitive enzymes can also describe the degree of methylation of the full mutation allele for both males and females as well as the X-inactivation pattern for females. However, neither of these measures can be used to predict the degree of mental retardation status for either sex(36). The main disadvantage of the Southern blot is that it requires a large amount of DNA and is laborious, both of which prevent the rapid and inexpensive screening of large populations. Because of these limitations, other diagnostic tests based on DNA and protein properties of the fragile X syndrome have been developed for fragile X screening. New DNA diagnostic tests have concentrated on the use of the polymerase chain reaction (PCR). Many different PCR protocols have been developed for the fragile X CGG repeat, with different degrees of amplification abilities and sizing accuracies. Regardless of the variations in protocol, compared with Southern blots, the PCR test is inexpensive, automated, and fast. Also, PCR can be performed on very small amounts of DNA, making collection of the samples relatively painless and convenient for the patients. The disadvantage of PCR is that the test results may not be straightforward for several reasons. The amplification of large repeats is difficult, especially in the presence of a second, smaller repeat. For many PCR protocols, the DNA fragment with the expanded repeat does not amplify. This is especially problematic for females and persons with repeat-size mosaicism who could appear to have a single, normal repeat size. To avoid these false negatives, many screening programs follow up by Southern blot any sample that fails to amplify by PCR and any female who appears to be homozygous. This strategy could potentially produce a false-negative result for persons who are normal/full mutation mosaic; however, few data exist to suggest that this occurs frequently. Most DNA-based methods can distinguish between premutations and full mutations. Because of ethical issues in identifying asymptomatic carriers, some proposed screening strategies are designed to identify only affected persons or those with the full mutation. Also, affected persons with point mutations and deletions that result in the loss of FMRP would not be routinely identified using the above methods that examine repeat size and potential sequencing of FMR1 is not routinely practiced for screening strategies or even for clinical diagnosis(49). For 99% of persons with the fragile X syndrome, affected status depends not only on the expansion of the repeat but on the subsequent lack of FMRP as well. The development of antibodies against FMRP has made screening possible on the basis of affected status only(45,50). In this protein-based assay, the percentage of FMRP detected in lymphocytes from blood smears is used to determine affected status(51-54). Typically, fewer than 40% of the lymphocytes from males with the fragile X syndrome have detectable amounts of FMRP(52). This protein-based test has recently been adapted for hair root(55;56) and prenatal(57;58) samples. Although promising, this technique cannot accurately identify affected females(42;54) and may not be appropriate for males who are normal/full or premutation/full mosaic. Potential contribution of genetic information
to improve health outcomes Although the medical and education communities recommend participation in programs and services after diagnosis of developmental delay or the fragile X syndrome, the effectiveness of early intervention services has not been yet demonstrated. This gap in research, however, is difficult to fill because it requires diagnosis with the fragile X syndrome soon after birth(63). In many countries, including the United States, the fragile X syndrome is commonly diagnosed through a referral for fragile X testing. A referral is indicated if a person has unexplained mental retardation, developmental delay, or autism, especially if physical or behavioral characteristics commonly associated with the fragile X syndrome are evident(64). Despite these recommendations, studies of school-aged children receiving special education in the Netherlands(19), the United States(28), and the United Kingdom(18) suggest that the current referral system can fail to identify toddlers and school-aged children who do not yet manifest the physical hallmarks of the syndrome(65;66). A second potential contribution resulting from the cloning of FMR1 could be the identification of families at risk of having a child with the fragile X syndrome. Based on the genetics of the fragile X syndrome, strategies for identifying at risk families usually focus on identifying female premutation and full mutation carriers. Ideally, women at risk would be identified before pregnancy, which would give these women the most options including egg donation, adoption, and prenatal diagnosis. Also, these women would be advised of their risk for premature ovarian failure (POF). Whereas the mean age of menopause is 51 years, women with POF experience the cessation of menses before age 40 years. Twenty-one percent (95% CI: 15%-27%) of premutation carriers develop POF compared with 1% of women in the general population67. Women identified as premutation carriers could be advised to plan their families earlier rather than later in their reproductive lives because of their risk for POF. Although knowledge of genotype status for the fragile X syndrome offers benefits to many families, this knowledge may also solicit poorly understood harms. One such harm is the impact carrier status may have on the psychology of an individual. Retrospective interviews of parents with children in whom the fragile X syndrome had been diagnosed indicate that many carrier parents feel guilty for passing the mutation to their children and are worried about the implications fragile X test results have on their families(68;69). Furthermore, a cross-sectional study of obligate carriers found that knowledge of carrier status was upsetting and caused a proportion of women to change their views about themselves(70). Finally, an important study by McConkie-Rosell and colleagues describes the emotions and attitudes of women without children with fragile X but who know before testing that they have a 50% chance of carrying the premutation allele. In this longitudinal study, white women over age 18 years who were at risk of carrying the premutation allele completed a structured interview and standardized measures both at the time of testing and 6 months after learning the results of the test(71). Results of the study suggest that the idea of being “at risk” before testing was upsetting and continued to be so only for the women found to be carriers(72). Surprisingly, non-carrier women surveyed after genetic testing viewed the fragile X syndrome as a more serious problem than they did the first time they were surveyed and than did carrier women(72). Results also suggest that women’s responses regarding feelings of self were not related to global self-concept but to the implications the positive carrier test would have on themselves and their families(71). These responses could be categorized into five areas of specific concerns: implications their positive test had for their children, reproductive options, possible expression of the fragile X syndrome phenotype in themselves, genetic identity, and regret of having not known sooner(71). Most non-carrier women and a small proportion of carrier women expressed relief or a positive emotion after learning carrier status(71;72). Although the only study of its kind in the literature, the results of the study may be limited to educated and married White women. Additional studies in this area are needed so that the impact of carrier testing can be fully understood in terms of race/ethnicity, education, economics, and marital status. Also, studies must be designed to explore the impact of carrier testing on individuals from the general population who have a very low prior risk of carrying the fragile X premutation or full mutation. A second potential harm associated with knowledge of genotype is related to insurance coverage. The focus of much concern is the use of genetic information that reveals susceptibility or carrier status of a presently healthy individual in determining health insurance coverage. In the United States, several states have enacted laws that prohibit the use of genetic information in pricing, issuing, or structuring of health insurance(73;74). At the federal level, the Health Insurance Portability and Accountability Act enacted in 1996 (HIPAA: Stat 1936, Pub L, No 110: 104-191) prohibits group health insurers from applying pre-existing condition exclusions to genetic conditions that are identified solely by genetic tests. Although fear of genetic discrimination has generated much attention, a recent survey of insurers, agents, and professionals in medical genetics (e.g., genetic counselors and clinicians) suggests that discrimination based solely on a genetic test is not common with regard to health insurance coverage(73;74). Consistent with these findings is one report about the effects of fragile X testing on health insurance coverage for 39 families living in Colorado(75). All families surveyed by Wingrove et al.(75) had a child affected with the fragile X syndrome. None of the families reported having their health insurance coverage cancelled as a result of genetic testing. Six families reported that carriers of the fragile X syndrome were declined for coverage; however, all six included a child affected with fragile X with their applications(75). Also, six families reported a member who refused testing because of fear of genetic discrimination(75). This result highlights the need to dispel the myths associated with genetic testing and health insurance that may otherwise discourage a person from seeking genetic testing. Future directions Beyond treatment of the symptoms of the syndrome, a new emphasis in research has been directed toward correcting the fragile X defect. Basic research suggests that reduced acetylation of histones H3 and H4 at the 5’ end of FMR1 leads to the condensation of chromatin and subsequent inhibition of transcription(26). The deacetylation is mediated through a methylcytosine binding protein, MeCP2, at the abnormally methylated CpG island observed in individuals with fragile X(79). Much interest lies in reactivating FMR1 by demethylating the CpG island. Although research groups have successfully reactivated FMR1 using 5 azadeoxycytidine (5 azadC), the chemical is too toxic for human use(26;80). Also, 5 azadC requires cell division, which makes it an unlikely candidate for neurons, the cells presumably most affected by the fragile X mutation. Chemical therapy of the primary defect is not yet a reality, but researchers remain hopeful that accumulating basic research and knowledge of the fragile X defect will translate into an effective treatment in the future. With regard to earlier diagnosis, as mentioned above, the referral system often used by physicians does not identify all young children with the fragile X syndrome. The current referral system relies, in part, on the classic physical features present in adults with the fragile X syndrome, which are not usually present in young children with fragile X(66). Often times the parents or guardians of young children with the fragile X syndrome will notice developmental delay or behavioral problems within the first few months of the children’s lives(81). In fact, an analysis of retrospective interviews of North Carolina mothers with children in whom fragile X was diagnosed reported that, on average, parents noted developmental delay at 9 months(81). Despite the early signs of developmental delay among this study group, the diagnosis of developmental delay averaged 24 months and the diagnosis of fragile X averaged 35 months(81). The lag time between the first signs of fragile X and a diagnosis could be related to the physician’s reluctance to test the child at such an early age. In the North Carolina study, 28 parents (68%) of affected children voiced frustration with their pediatricians or other health care providers for dismissing the parents’ concern for their children’s development(81). This frustration has also been documented in a similar survey of parents with children with fragile X in the United Kingdom(82). The importance of a prompt diagnosis for the fragile X syndrome cannot be understated. The benefits of an accurate diagnosis radiate beyond the person diagnosed because both immediate and extended family members will be affected by its consequences. The lag time between the birth of the first child with fragile X and a diagnosis must be short as possible to ensure that couples at risk have ample time to learn about the syndrome and to explore their reproductive options. As a result of this movement for a more timely diagnosis, researchers and parents have begun the discussion of general population screening programs for the fragile X syndrome. Population testing With regard to screening preconceptional or pregnant women in the United States, the policy for fragile X syndrome testing has not changed since a working group for the American College of Medical Genetics published their recommendations in 1994(64). That is, in the United States, the fragile X syndrome test is offered on a referral basis and not routinely to the general population. Examples of carrier screening offered to U.S. women with histories of fragile X or mental retardation(83;84) have been published in the literature. Only one program in the United States has reported screening reproductive-age women without a history of mental retardation. This program at the Genetics & IVF Institute in Fairfax, Virginia, reported offering fragile X carrier screening to women on a self-pay basis85;(86). Most women were referred to the clinic because of their advanced reproductive age. From December 1993 through June 1995, 3,345 women were offered testing, and 668 (21%) accepted. Most (69%) of these women did not have family histories of mental retardation. Among these women, three premutation carriers (60-199 repeats) were identified(85). Unlike the screening studies performed in the United States, investigators in Finland have implemented a large population-based carrier-screening program. The program implemented by the Kuopio University Hospital in Finland offered an FMR1 gene test free of charge to all pregnant women seeking prenatal care from July 1995 until December 199687. According to Ryynanen et al.(87), almost all pregnant women in Finland seek prenatal care and are registered in antenatal clinics during the 6th through 10th weeks of pregnancy because this registration is required for maternity allowance provided by the state. Among women without family histories of the fragile X syndrome, 1,477 (85%) elected genetic testing. Of these women, six were identified as premutation carriers (60-199 repeats) and all six women elected prenatal testing. The program has since screened an additional 1,358 women through December 1997. Six more women with the premutation allele were identified, and all six elected prenatal diagnosis(88). The program has also expanded to offer an FMR1 genetic test to pregnant women undergoing invasive prenatal testing because of advanced maternal age or history of trisomy pregnancy. In this expanded program, 241 (80%) of the 302 women offered the test consented, and one woman was identified as a premutation carrier(88). As in Finland, carrier testing is also widely employed and accepted in Israel. At least three groups have published results obtained from their large screening programs. Unlike the program in Finland, these programs offer the test on a self-pay basis and rely on either a self-referral or physician referral for testing. In one published report, the Rabin Medical Center screened 14,334 preconceptional or pregnant women who were self-referred and had no family histories of mental retardation between January 1992 and October 200033;(89). Women identified as carriers were offered prenatal testing free of charge as instructed by the Israeli Ministry of Health. A total of 204 women were identified as premutation carriers (51-200 repeats), and three women were identified as full mutation carriers(33). Of the pregnant women identified as premutation/full mutation carriers (n=173), only 14 women refused prenatal diagnosis(33). In a second report, the Genetic Institute of the Tel Aviv Sourasky Medical Center offered an FMR1 genetic test to 9,660 women during September 1994 through October 1998(37). A total of 38 premutation carriers (60-200 repeats) were identified, and all of these women consented to prenatal diagnosis(37). Finally, during January 1994 through March 1999, the Danek-Gertner Institute of Human Genetics screened 8,426 women of reproductive age who had no family histories of the fragile X syndrome or mental retardation(32). Among these women, 18 were identified as premutation carriers (61-199 repeats), and one was identified as a full mutation carrier (>200 repeats). Newborn screening for the fragile X syndrome has not yet been implemented in any country. Compared against the criteria published by the National Academy of Sciences (NAS)(90), the Institute of Medicine (IOM)(91), and Task Force on Genetic Testing(92), the fragile X syndrome meets at least two criteria essential for a successful program in newborn screening. Specifically, based on its morbidity and prevalence, the fragile X syndrome is an important public health problem. Also, approximately 99% of cases diagnosed thus far are caused by a single, inherited mutation, making the fragile X syndrome particularly amenable to DNA-based testing for an accurate diagnosis. Despite meeting these criteria, the fragile X syndrome does not meet several other key criteria for newborn screening. One crucial gap in research is the lack of a cure or effective treatment available for persons with the disorder, as mentioned previously. A second gap is the lack of knowledge of the potential harms associated with the diagnosis in an apparently healthy child. Many researchers and parents worry that a diagnosis at the newborn period would disrupt the parent-child bond. However, little evidence supports this, and many parents contend that a diagnosis would strengthen the parent-child bond through a greater understanding of the child’s special needs. Nevertheless, because the fragile X genetic test cannot predict the severity of mental retardation (especially regarding females with the full mutation), the effects of a diagnosis on the family’s perception of the child’s prognosis and future development are important to consider. Finally, a third gap is the lack of general consensus for the appropriate time to screen that would maximize the benefits of a diagnosis(93). Newborn screening will identify affected infants who are eligible for early intervention services. Newborn screening would also provide parents with information about their children’s future development and methods to optimize this development. However, identification of an affected person will also identify at risk families. Although these identified families could benefit from genetic counseling, newborn screening is neither ideal nor designed for identifying most at risk families for the fragile X syndrome in the general population(68;94). Also, many parents may want to know before pregnancy or birth about the fragile X syndrome. Indeed, a proportion of parents with fragile X children surveyed in the United Kingdom believed that a diagnosis based on newborn screening would be “too late.”(82) The debate concerning screening for the fragile X syndrome will no doubt continue. In the meantime, more information about the risk for expansion based on premutation size should be collected to better assess a women’s risk of conceiving a child with the fragile X syndrome. Also, the psychological impact genetic testing should be thoroughly explored because these results have implications that reach far beyond the fragile X syndrome in this new genetic age. Finally, effective treatments need to be developed and properly evaluated so that persons affected by the fragile X syndrome and their families can live life to the fullest potential.
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