AMERICAN COLLEGE OF MEDICAL GENETICS
Standards and Guidelines for Clinical Genetics Laboratories
2006 Edition.

 
Technical Standards and Guidelines for Fragile X Testing: A Revision to the Disease-Specific Supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics

Supercedes Technical Standards and Guidelines for Fragile X Testing: The First of a Series of Disease-Specific Supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics (Genetics in Medicine 2001;3(3):200.

Approved by the Board of Directors of the American College of Medical Genetics on October 2, 2000.

Reviewed and Revised: 2005 by the Fragile X Working Group of the Laboratory Quality Assurance Committee

Fragile X Molecular Working Group 2005 for the Quality Assurance Committee of the American College of Medical Genetics:

Elaine B. Spector, PhD
University of Colorado School of Medicine Department of Pediatrics

Kathryn Kronquist, PhD
University of Colorado School of Medicine Department of Pathology

Disclaimer

These standards and guidelines are designed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines does not necessarily ensure a successful medical outcome. These standards and guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical molecular geneticist should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. It may be prudent, however, to document in the laboratory record the rationale for any significant deviation from these standards and guidelines.

Preface

The ACMG Laboratory Quality Assurance (Lab QA) Committee has the mission of maintaining high technical standards for the performance and interpretation of genetic tests. In part, this is accomplished by the publication of the document "ACMG Standards and Guidelines for Clinical Genetics Laboratories," which was published in its second edition in 1999 and is now maintained online (see http://www.acmg.net/Pages/ACMG_Activities/stds-2002/stdsmenu-n.htm).This subcommittee also reviews the outcome of national proficiency testing in the genetics area and may choose to focus on specific diseases or methodologies in response to those results. Accordingly, the subcommittee selected fragile X syndrome to be the first topic in a new series of supplemental sections, recognizing that it is one of the most frequently ordered genetic tests and that it has many alternative methods with different strengths and weaknesses. This document follows the outline format of the general Standards and Guidelines. It is designed to be a checklist for genetic testing professionals who are already familiar with the disease and the methods of analysis.

FX 1

Introduction

Disease-specific statements are intended to augment the current general ACMG Standards and Guidelines for Clinical Genetics Laboratories. Individual laboratories are responsible for meeting the CLIA/CAP quality assurance standards with respect to appropriate sample documentation, assay validation, general proficiency and quality control measures.

FX 2

Background On Fragile X Syndrome

FX 2.1

Gene Symbol/Chromosome Locus

FMR1 is the gene symbol recognized by the HUGO nomenclature committee (www.gene.ucl.ac.uk/nomenclature/). The gene has also been referred to as FXA in the past. The chromosome locus is Xq27.3.

FX 2.2

OMIM Number: 309550

FX 2.3

Brief clinical description

The features of fragile X syndrome include specific cognitive deficits and certain characteristic, but nonspecific physical features and behaviors. Most individuals with the premutation do not show fragile X syndrome-related features; however, some with high repeat sizes (>100 repeats) have been identified with learning difficulties, emotional problems, or even mental retardation.1 Females with premutations are at high risk for premature ovarian failure. 2-6 Older males with premutations have been reported to have the Fragile X Tremor Ataxia syndrome (FXTAS). FXTAS is a late onset, progressive development of intention tremor and ataxia often accompanied by progressive cognitive and behavioral difficulties including memory loss, anxiety, reclusive behavior, deficits of executive function and dementia.7-11 Older females with premutations may also exhibit movement disorders, although this is more rare.12 Penetrance is age related in both premutation-associated disorders; however, accurate age-specific estimates are not available at this time. For more information on these disorders, see the online Gene Clinics profile at www.geneclinics.org and the National Fragile X Foundation at www.nfxf.org.

FX 2.4

Mode of inheritance

Inheritance of the FMR1 mutation is X-linked, although the pattern of fragile X syndrome is complicated due to the characteristics of the unstable repeat sequence mutation. In typical fragile X families, the mutation is a multi-step expansion occurring over one or more generations in a region of CGG repeats in the 5' untranslated region of the gene. Small expansions (premutations) are not generally associated with cognitive deficits in males and females. Large expansions (full mutations) are penetrant in all males and many females. With extremely rare exceptions, the parent of origin of the expansion to the full mutation is female.

FX 2.5

Gene Description/Normal Gene Product

The gene product is FMRP, fragile X mental retardation protein, a widely expressed RNA-binding protein. The fragile X syndrome is caused by a loss of the fragile X mental retardation protein (FMRP). FMRP is a selective RNA binding protein that can form a messenger ribonucleoprotein complex that can associate with polysomes.13 FMRP has been shown to behave in vitro as an inhibitor of protein translation.14 At the neuroanatomic level the fragile X brain differs from normal brain due to the presence of unusually long and thin dendritic spines in the cortical regions.15,16 The dendritic spines are the location where excitatory synaptic transmission occurs. FMRP appears to be associated with polyribosomes within dendritic spines of "wild-type" neurons.17 From these data, it has been proposed that FMRP is a translation suppressor that regulates protein synthesis locally in dendrites in response to synaptic stimulation signals.18 In the fragile X brain, translation of certain mess ages may be exaggerated because the normal inhibition provided by FMRP is absent.19

Studies of FMR1 mRNA expression provide evidence that expansion in the premutation range perturbs gene expression and may have pathophysiological consequences, particularly those related to FXTAS and ovarian failure (section 2.3). Reductions in the amount of FMRP have been found in both lymphocytes and transformed lymphocytes of premutation carriers.20,21 Using a highly sensitive fluorescent assay Kenneson et al.21 demonstrated a decrement in FMRP in individuals with expansions only slightly larger than the upper edge of the normal range. The reduction in FMRP is associated with an increase in FMR1 Mrna1,20-22 in individuals with premutations. Expansion of the CGG repeats into the premutation range can shift transcription of FMR1 mRNA from the usual, downstream-most start site to upstream sites. The utilization of alternative start sites may be correlated with increased FMR1 mRNA transcription levels.23

FX 2.6

Mutational mechanism/abnormal gene product

Fragile X syndrome is caused by the deficiency or absence of FMRP. Theoretically, this can occur through any type of deletion or inactivating mutation, but in well over 99% of cases there is an expansion of a segment of CGG repeats in the 5' UT region of FMR1. Large CGG expansions in this region are associated with hypermethylation and inhibition of transcription.

FX 2.7

Listing of Mutations

Mutations at locations other than the CGG repeat have been described. A listing can be found in the Human Gene Mutation Database at www.uwcm.ac.uk/uwcm/mg/hgmd0.html. Guidelines for detecting these relatively rare mutations are beyond the scope of this document.

FX 2.8

Prevalence and Ethnic Association of Common Mutations

All major ethnic groups appear to be susceptible to expansion of the FMR1 CGG region.24,25

FX 2.8.1

Full Mutations: Crawford et al. provided an extensive review of the literature and indicated a prevalence of fragile X syndrome ranges from 1/3,717 to 1/8,198 in Caucasians males in the general population.24 The female prevalence rate is presumed to be approximately one-half of the male rate. In another study carried out over 4 years in metropolitan Atlanta, Crawford et al.26 determined the prevalence of the fragile X syndrome to be 1/ 2,545 African-American males and 1/3,717 Caucasian males. However, the prevalence estimate for Caucasian males, determined from this and from other studies, fell within the 95% confidence interval for African-American males. The prevalence of the fragile X mutation in an Afro-Caribbean population in the French West Indies was similar (1/2,539) to that in the African-American population in Atlanta.27 Falik et al. have suggested that the Tunisian Jewish population is the only other ethnic group to have a higher prevalence of the fragile X syndrome than the Caucasian population.28 However, these studies were not been supported by the data of Tolodano-Alheder et al.25 Further studies are required to determine if the frequency of the fragile X syndrome differs in ethnic populations.

FX 2.8.2

Premutations: Previous screens for the prevalence of pre-mutations in Canada studied 10,624 unselected women and estimated the carrier frequency to be 1/ 259 women. The range of CGG repeats was 55 to 101.29 Later studies revealed a prevalence of premutation alleles in males of 1/760.30

A study of 9,459 women in Israel found 1/152 with alleles >54 repeats.31 In the individuals with no family history of the fragile X syndrome 1/166 women were determined to have premutations with a CGG repeat range of 55 to 101. This estimate of the premutation carrier frequency is approximately two-fold higher than that reported in the studies performed in Canada.

Toledano-Alhader et al.25 obtained similar values when studying 14,334 preconceptual or pregnant women in Israel – namely 1/113 women with >54 CGG repeats. This study excluded women with a family history of mental retardation. In addition, they found that the premutation carriers were well distributed among all the Jewish ethnic groups in contrast to a previous study.28

A third study of approximately 2,000 mothers and their newborns in the general Italian population found a premutation carrier frequency of 1/109 females and 1/225 newborn males (56-70 CGG repeats).32 A fragile X screen of 10,000 newborn males in Taiwan showed a prevalence of 1/1,674,33 Thus the carrier frequencies vary widely among populations and may be higher than those determined in the French-Canadian population.

FX 2.9

Special Testing Considerations

FX 2.9.1

Sensitivity and specificity: CGG-repeat expansion full mutations account for >99% of cases of fragile X syndrome. Therefore, tests that effectively detect and measure the CGG repeat region of the FMR1 gene are >99% sensitive. Positive results are 100% specific. There are no known forms of FMRP deficiency that do not map to the FMR1 gene. Fragile X syndrome should not be confused with the unrelated mental retardation syndrome associated with the FXE locus.

FX 2.9.2

Diagnostic versus predictive testing: Molecular testing for expanded FMR-1 alleles is used for confirmative diagnosis and carrier detection. Positive results are considered diagnostic rather than predictive, inasmuch as penetrance of fragile X syndrome is virtually 100% in males and the age of onset is not variable.

FX 2.9.3

Prenatal testing: This test can be used for prenatal diagnosis in both amniotic fluid cells and chorionic villus samples (CVS). Laboratories offering CVS testing must be aware of this tissue’s unique properties:

(a) Methylation associated with lyonization is usually not present and methylation associated with full mutations may or may not be present.34 In the past, the hypomethylated status of this locus in this tissue had been thought of as a limitation or possible source of confusion. To the contrary, because it is unwarranted to use methylation status or X-inactivation for phenotypic prediction of a full mutation, the possible hypomethylation of this tissue is no disadvantage, provided that the tissue-specific basis of the hypomethylation is understood.34,35 It is an acceptable option to omit methylation analysis entirely when testing CVS specimens. In the minor fraction of CVS cases with a result that is ambiguous between a large premutation and a small full mutation by size criteria alone, a follow-up amniocentesis may be required;

(b) The degree of somatic variation in a full mutation "smear" has a wider range of possibilities than is typically seen in blood specimens, from very limited to extraordinarily diffuse;

(c) Mosaicism between trophoblasts and somatic cells is theoretically possible. For this reason, when CVS results indicate a premutation, follow-up amniocentesis has been suggested to rule out mosaicism for a full mutation. However, there is no known occurrence of this type of mosaicism.

FX 3

Guidelines

FX 3.1

Definition of Normal and Mutation Categories

There are four allelic forms of the gene: Normal, intermediate, premutation and affected. The associated number of CGGs for each can be defined based on our current information to date. It must, however, be recognized that the borders of each definition may change with increased empirical data and research.

FX 3.1.1

Normal Alleles: Normal alleles have a range of ~5 to ~44 repeats. The most common repeat length by far is 29 or 30 CGG repeats. Normal alleles have no meiotic or mitotic instability.

FX 3.1.1.1

In stable, normal alleles, the CGG region is interrupted by an AGG triplet after every 9 or 10 CGG repeats. The AGG triplets are thought to anchor the region during replication

and prevent strand slippage. Direct testing for the AGG triplets is not routinely performed.

FX 3.1.2

Intermediate (Gray Zone, Inconclusive, Borderline): The range from ~45 to ~54 repeats is intermediate (also referred to gray zone, inconclusive or borderline). Alleles in this range can be considered normal in the sense that such alleles have not been observed to expand to a full mutation in one generation. Moreover, at this point, there is no observed increased risk for the specific premutation-associated disorders, although data are limited. Alleles of this size may be associated with fragile X syndrome in future generations or in distant relatives. Minor increases and decreases in repeat number can occur when alleles of this size are passed on, but there is no measurable risk of a child with fragile X syndrome in the next generation. Alleles in this range can be referred to as premutations if they are confirmed by family studies to be traceable to a known full mutation or unambiguous premutation.

FX 3.1.3

Premutation: Premutation alleles range from ~55 to ~200 repeats. These alleles are long repeat tracks that are unstably transmitted from parent to child. They show no somatic variation. They are not hypermethylated and are not associated with fragile X syndrome. Women with alleles in this range are considered to be at risk for having affected children, although to date all known mothers of affected children have alleles of 59 repeats or higher.36-38 Female members of families with CGG repeats in this range can benefit from being monitored in future pregnancies.

FX 3.1.3.1

The upper limit of premutations is sometimes said to be ~230. In fact, both numbers (200 and 230) are rough estimates derived from Southern blots where large premutations were measured with increases of 0.5 to 0.6 kb, implying roughly 170 to 200 more triplet repeats than normal.

FX3.1.4

Full mutations: Full mutations have over 200 to 230 repeats, typically several hundred to several thousand repeats. There is usually broad somatic variation within each patient. Hypermethylation is typically present on most or all copies. The appearance of full mutations may vary in CVS as compared to blood and amniocytes. Methylation may or may not be present and the degree of somatic heterogeneity ranges from distinctly limited to extraordinarily diffuse.

FX 3.1.5

Mosaicism: Mosaicism due to de novo somatic mutations does not occur at the FMR1 CGG-repeat region but size mosaics and methylation mosaics have been observed.39-42 When mosaicism is present, tissue-specific differences can be seen. Individuals with size or methylation mosaicism may be higher functioning that individuals with fully methylated full mutations.

FX 3.1.5.1

Size mosaics: This term refers to subpopulations of full mutations and premutations. Occasionally there also may be minor subpopulations with near normal or normal length.

FX 3.1.5.2

Methylation mosaics: This term refers to full mutations with subpopulations that remain unmethylated.

FX 3.2

Methodological Considerations

All general guidelines for Southern blots and polymerase chain reaction (PCR) in the ACMG Standards and Guidelines for Clinical Genetics Laboratories apply (www.acmg.net). The following additional details are specific for fragile X. For this test, there are many valid methods with different strengths and weaknesses. Laboratories very often need to use more than one method because no single method can detect all types of mutations equally well or with equal precision. For instance, for carriers with pre/full mosaic pattern it would be questionable whether PCR alone would detect the full mutation leading to a different risk assessment for fragile X, FXTAS and POF.43 For this reason, the ACMG policy statement43 recommends that Southern blot analysis always be conducted, even if a premutation allele is identified by PCR.

FX 3.2.1

Southern blots

FX 3.2.1.1

Probe and restriction site combinations: Table 1 describes several single- and double-enzyme options that are commonly used and several probes that are available.44-47 Other restriction enzymes and probes can be used, if equivalence is demonstrated.

FX 3.2.1.2

In general, when using the StB 12.3 probe, small premutations are more easily detected when the normal fragment is small and/or electrophoretic migration is long, whereas large/diffuse full mutations are more easily detected when the normal fragment is large and/or electrophoretic migration is short.

FX 3.2.1.3

Controls should be included to confirm the proper choice and activity of restriction enzymes and probe. They should ideally represent the more difficult-to-recognize genotypes. To verify digestion and hybridization parameters, a normal control will suffice. However, in fragile X blots the abnormal controls are extremely important because they provide quality control on the resolution of small premutations and the detectability of diffuse smears. Cell lines possessing specific CGG repeat sizes may be obtained from the Corriel Institute for Medical Research (http://locus.umdnj.edu/ccr/).

FX 3.2.1.4

For female patients, it should be noted that the degree of separation between two differently sized normal alleles could appear identical with that between a normal and a premutation allele (e.g., 20 and 44 repeats vs. 35 and 59 repeats). A Southern blot with superior resolution and appropriate size standards or controls is required to distinguish between these possibilities. Alternatively, most PCR-based methods can provide the required resolution. Similar considerations apply to detection of premutation alleles in normal transmitting males.

FX 3.2.1.5

Because full mutations can be extremely diffuse and faint, signal: noise ratios must be very good. Laboratories are advised to be aware of the many different appearances of full mutations. Full mutations are not likely to be overlooked in males, inasmuch as the normal signal will be absent (or light, in size mosaics), but full mutations can be easily missed in females if the background is poor. Skewed X-inactivation may also present problems in the use of Southern blots in the detection of females with pre or full mutations.

FX 3.2.1.6

Migration distances should be interpreted using a standard ladder such as lambda Hind III fragments or a set of carefully chosen, independently tested human references.

FX 3.2.1.7

The following guidelines refer to methylation analysis using double digestion.

FX 3.2.1.7.1

In tissues other than CVS, methylation analysis reveals the degree of hypermethylation in full mutations and shows the distribution of X-inactivation in any female with two distinguishable alleles. Southern blot analysis with the addition of methylation sensitive enzyme digestion can:

(a) help discriminate between premutations and full mutations for the rare alleles that fall near the boundary (i.e., around 200 repeats); and

(b) detect rare individuals that are methylation mosaics.

FX 3.2.1.7.2

In tissues other than CVS, the results of routine methylation analysis and PCR

are sometimes confounded by an abnormal karyotype such as 45,X and 47,XXY. Individuals with testicular feminization (XY females) will have a male methylation pattern. Interpretation of results should be formulated using accurate karyotypic information.

FX 3.2.1.7.3

In tissues other than CVS, methylation analysis increases the difficulty of detecting females with small premutations who have highly skewed X-inactivation. Double digestion with a methyl-sensitive enzyme causes the signal from each allele in a female to be split into active and inactive bands, forming four bands in a carrier female. When X-inactivation is balanced in a carrier the two active bands are readily seen, although the two inactive bands may comigrate. However, if X-inactivation is heavily skewed, there will be only two visible bands. This is particularly challenging when the premutation is predominantly inactive because then it appears only in the upper region of the gel where resolution is considerably poorer. For an example of a carrier with extremely skewed X-inactivation, see Figure 1, lane 13. Lanes 3 and 4 show two females with oppositely skewed X-inactivation. The above data is true for the use of the StB 12.1 probe. Use of other probes such as PE 5.1 will yield an additional small control band.

FX 3.2.1.7.4

In CVS tissue, the FMR1 region usually does not have methylation associated with X-inactivation and it may or may not have hypermethylation associated with full mutations before 12.5 weeks.34 When testing CVS tissue, methylation analysis is optional. If done, it should never be used to predict the severity of a full mutation or the influence of X-inactivation. Incidentally, methylation analysis before 12.5 week’s gestation can serendipitously alert a laboratory to maternal cell contamination in chorionic villus specimens inasmuch as methylation associated with X-inactivation is usually not present at this locus in CVS tissue, a strong normal inactive band can be a sign of possible maternal cell contamination. Other explanations for such a band include X-inactivation in some fetal cells or incomplete digestion. Further investigation would be called for.

Table 1
Probe and restriction site combinations

Primary restriction sites    
and normal length
Optional internal
methyl-sensitive sites    
Probes and
references
EcoRI, 5.2 kb EagI, BssHI, NruI, etc StB12.3,44 E5.145
HindIII, 5.4 kb Ox1.946 BglII, 12 kb
PstI, 1.0 kb n/a pfxa3,47 Ox0.5546

 

Figure 1

Fig. 1: Southern blot using EcoRI and EagI digestion, probed with StB12.3, using extended electrophoresis to illustrate several subtle specimen types.

(1) Normal female.
(2) Full mutation male. Note the combination of a predominant band with a diffuse smear.
(3) Female with 28 and 52 repeats, with the smaller allele predominantly active.
(4) Female with 26 and 52 repeats, with the larger allele predominantly active.
(5) Female with 18 and ~80 repeats, with equal X-inactivation.
(6) Normal male.
(7) Normal male, underloaded and smiling due to DNA degradation. (The apparent line between lane 6 and 7 is a photographic artifact.)
(8) Normal female.
(9) Normal male.
(10) Normal male.
(11) Affected male, underloaded and very diffuse.
(12) Premutation male.
(13) Female with 20 and 70 repeats, with the smaller allele virtually exclusively active. The only evidence of abnormality is the slow migration of the "5.2 kb" band.
(14) Female with 27 and 42 repeats, with the larger allele somewhat more active.
(15–17) Unremarkable normal females and male.
 
Figure provided by Genetics & IVF Institute.

FX 3.2.2

PCR Methods

FX 3.2.2.1

Several sets of primers, PCR conditions and methods of separation and detection have been published.48-51 Other primers and methods can be used if equivalence is demonstrated. A particular region to be aware of in primer design is the deletional hotspot.52

FX3.2.2.2

All PCR reactions, for any locus, can theoretically fail to detect an allele if there is polymorphism at a primer binding site. There are no known polymorphisms that would affect any of the commonly used primers.

FX 3.2.2.3

Patient amplicon sizes should be determined using a standard ladder such as an M13 sequencing reaction or a set of carefully chosen, independently tested human references.

FX 3.2.2.4

Controls representing the genotypes to be distinguished should be run on each gel. The upper limit of allele size that can be successfully detected should be known and a control

corresponding to that size should be included in each run. Laboratories should confirm the size of their control DNA by sequencing or by exchange with another laboratory.

FX 3.2.2.5

Amplification of CG-rich regions is difficult and special conditions are required. The difficulty increases with increasing numbers of CGG repeats; therefore, many PCR strategies do not attempt to detect large alleles. In such a system, it is not possible to tell the difference between a female who is homozygous for a normal allele and one who has a large nonamplifiable second allele. Similarly, patients who are mosaics for premutations and full mutations will appear to have only premutations.

FX 3.2.2.6

When a PCR strategy is capable of detecting large alleles, amplification nevertheless may favor the smaller allele in any specimen with multiple alleles, i.e., females and mosaics.

Such methods should be validated with carrier females and mosaics, in addition to males. Because of disproportionate amplification, PCR is not reliable for determining the ratio of different species in a mosaic individual.

FX 3.2.2.7

In PCR amplification of samples from females and mosaics, heteroduplexes can form. If denaturing electrophoresis is used, conditions must be sufficiently denaturing to avoid heteroduplex artifact. If nondenaturing electrophoresis is used, steps must be taken to distinguish between heteroduplexes and true abnormal alleles.

FX 3.2.2.8

Basic PCR amplification is not affected by methylation. Although PCR tests specifically modified to detect methylation status have been described,53,54 the common PCR strategies that have been in use for many years are completely independent of methylation.

A genotype classification method using a methylation specific triple PCR method was described by Zhou et al.55 This method distinguishes all normal and premutation males and females and all full mutation males and females. This method may provide a suitable alternative for Southern blot and yields estimates of the allele sizes similar to other PCR based methods.

FX 3.2.2.9

When a PCR strategy is used to detect full mutations, the presence of a deletion hotspot in the CGG-repeat region should be noted.52 Primers located within the deletion hotspot may result in failure to detect the expanded allele. Primers located upstream of the deletion hotspot may result in apparent size mosaicism.

FX 3.3

Interpretations

FX 3.3.1

The following elements must be included in the report: In addition to the items described in the current general ACMG Standards and Guidelines for Clinical Genetics Laboratories, (www.acmg.net/Pages/ACMG_Activities/stds-2002/stdsmenu-n.htm/).

FX 3.3.1.1

State whether the method used was PCR, Southern blot, or both. If Southern blot, state the restriction enzymes and probes used. If PCR, state the method used for separation and detection.

FX 3.3.1.2

State the definitions used for normal, intermediate (gray-zone, borderline, inconclusive) premutation and full mutation.

FX 3.3.1.2.1

Note that it is not necessarily obvious that the borderline category refers to the border between normal and premutation and not to the border between premutation and full mutation. Similarly, note that the term instability, which is often used with regard to borderline alleles to describe minor intergenerational or mitotic changes, may unintentionally suggest a risk of having an affected child or personal late-onset symptoms.

FX 3.3.1.3

Classify the patient’s result using the defined categories. The term size mosaic should be used for alleles that have significant subpopulations in both the premutation and full mutation range.

FX 3.3.1.4

All positive results should state that genetic counseling is indicated and testing is appropriate for at-risk family members. In addition to first-degree female family members, male family members may be at risk for late onset development of symptoms of FXTAS.

FX 3.3.2

The following descriptive elements may appear, with caution:

FX 3.3.2.1

The size of the alleles may be reported. If so, the precision used in quoting the size must be supportable by the precision of the ladder used, the sharpness of the bands or peaks, degree of stutter, etc. It may be appropriate to state a range or use qualifying terms such as approximately. Descriptions such as "positive for an allele with 55–200 repeats" are ambiguous.

FX 3.3.2.2

Description of methylation may be provided. The two kinds of methylation must be clearly distinguished: methylation due to X-inactivation and hypermethylation of full mutations. The term methylation mosaic or incomplete methylation may be used if not all molecules in a full mutation are hypermethylated.

FX 3.3.3

The following helpful points on alternative diagnoses may be included:

FX 3.3.3.1

There are rare forms of FMRP deficiency not caused by CGG expansion, which may not be detected by this test.

FX 3.3.3.2

Mental retardation associated with other fragile X sites, in particular FXE, or other gene mutations will not be detected with this test.

FX 3.3.3.3

Routine chromosome analysis is recommended in the diagnostic workup of mental retardation.

FX 3.3.4

Comments on phenotype, if included, should be abstract rather than case-specific. The following concepts apply:

FX 3.3.4.1

All males with full mutations have fragile X syndrome to some degree. The severity cannot be predicted from the size of the full mutation, but if premutations are also present or if the majority of the full mutation molecules are unmethylated, the phenotype MAY be less severe.

FX 3.3.4.2

Females with full mutations exhibit a wide spectrum of phenotypes. They may be as severely affected as a male with an expanded fragile X allele (which is itself a range of phenotypes). Females with full mutations may also exhibit very mild learning disabilities or have no detectable deficits. The severity cannot be predicted from the size of the full mutation, nor can it be predicted from the pattern of X-inactivation.

FX 3.3.4.3

Individuals with premutations probably should no longer be interpreted as unaffected carriers. Women who carry a premutation are at risk for ovarian dysfunction and potentially infertility problems.3 Older men, and more rarely older women, with the premutation are at risk for FXTAS which can include: intention tremor and ataxia often accompanied by progressive cognitive and behavioral difficulties including memory loss, anxiety, reclusive behavior, deficits of executive function and dementia. If an individual referred because of mental retardation, autism, or learning disabilities is found to carry a premutation, no association can be stated at this point in time. It should be considered as a coincident finding unless FMRP deficiency or mosaicism for a full mutation can be found.

FX 3.3.4.4

Individuals with intermediate alleles should be interpreted as unaffected. Even more so than a premutation, an intermediate allele is considered a coincidence when found in a developmentally-delayed patient. FMRP deficiency or mosaicism for a full mutation can be investigated by methylation-sensitive Southern blot analysis, but with less likelihood of success because intermediate alleles are not uncommon in the general population.

FX 3.3.5

Comments on reproductive risk, if included, should be abstract rather than case-specific. The following concepts apply:

FX 3.3.5.1

All affected males and the overwhelming majority of affected females inherit their mutations from their mothers. The mothers carry either the premutations or full mutations.

FX 3.3.5.2

Women with full mutations have a theoretical 50% chance of passing on the full mutation with each pregnancy.

FX 3.3.5.3

Women with premutations have a theoretical 50% chance of passing on the fragile X mutation with each pregnancy. If it is passed on, the chance the allele will increase to a full mutation depends on its size in the mother. In the most recent collaborative study of Nolin et al.36, probabilities range from 3% for maternal alleles with CGG repeats between 55-59 (1/23 transmissions) to nearly 100% for maternal alleles with 90 CGGs and above. The smallest allele known to expand to the full mutation is 59 repeats. Laboratories should be familiar with Fu et al.48, Heitz et al.56, Kallinen et al.37 and Nolin et al.36,38 and any current publications on this topic.

FX 3.3.5.4

Men with premutations will almost always pass premutations to all of their daughters. An extremely rare phenomenon involves unaffected males with premutations who have had affected daughters, apparently by gonadal mosaicism for full mutations.57-59 The sons of men with premutations are not at risk for developing the fragile X syndrome or late onset FXTAS.

FX 3.3.5.5

Men and women alike with intermediate alleles are not known to expand to the full mutation in one generation, i.e., carriers of intermediate alleles do not have affected children in the next generation. Instability may be identified if the allele can be traced through the family to a known full mutation or unambiguous premutation. In the absence of such a connection, on a research basis it may be possible to show meiotic instability or a specific repeat sequence pattern (absence of AGG interruptions) that is at higher risk for instability. However at this point, sequencing is not clinically available for the repeat sequence. Also, more research is needed to determine the clinical value for predicting the risk to specific future generations, perhaps three or four before the expansion to the full mutation is observed.

FX 4

Alternative Testing Methods

FX 4.1

Cytogenetic analysis: Testing for the fragile site FXA at Xq27 is no longer an acceptable diagnostic method. Specificity and sensitivity are both insufficient.

FX 4.2

Protein analysis: Immunohistochemical staining for FMRP is a valid diagnostic method in lymphocytes.60 Williamsen et al.34 demonstrated that staining for the FMRP protein in chorionic villus samples could be used as an alternative prenatal diagnostic method for detection of full mutations in male fetuses. The situation is more complicated in female fetuses where some chorionic villi may be completely positive and others from the same sample may be completely negative for FMRP staining. The authors’ data sheds light on the timing of X-inactivation in chorionic villus cells of the female fetus. The diagnostic application of this method is not recommended at this time for the prenatal diagnosis of females carrying FMR1 full mutations.

FX 5

Policy Statements

FX 5.1

The American College of Medical Genetics issued a policy statement titled "Fragile X Syndrome: Diagnosis and Carrier Testing" in 1994 (Am J Med Genet 53:380 –381) which was updated in October of 2005 (Genet Med 7:584–587). This document is also available online at www.acmg.net/resources/policy-list.asp. These Standards and Guidelines are in general agreement with that statement.

FX 5.2

In 1995 the American College of Obstetrics and Gynecology issued a Committee Opinion, No. 161, on fragile X syndrome. These Standards and Guidelines are in general agreement with that opinion, with the exception of its recommendation for the use of amniocentesis for prenatal diagnosis. In experienced hands, chorionic villus samples are equally reliable and offer the advantage of first-trimester diagnosis to women who maybe at 50% risk for an affected pregnancy. However, it cannot be stressed enough that the unique properties of this tissue must be recognized.

FX 6

Acknowledgments

The authors would like to acknowledge the expert help of Dr. Stephanie L. Sherman.

This guideline is designed primarily as an educational resource for medical geneticists and other health care providers to help them provide quality medical genetic services. Adherence to this guideline does not necessarily ensure a successful medical outcome. This guideline should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the geneticist should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. It may be prudent, however, to document in the patient’s record the rationale for any significant deviation from this guideline.

FX 7

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