You are here

Article Text

Article menu

Download PDFPDF

Letters to JMG
FMRP expression studies in blood and hair roots in a fragile X family with methylation mosaics
  1. B B A de Vries1,
  2. L-A Severijnen2,
  3. A Jacobs1,
  4. R Olmer2,
  5. D J J Halley2,
  6. B A Oostra2,
  7. R Willemsen2
  1. 1Department of Human Genetics, University Medical Centre Nijmegen, The Netherlands
  2. 2Department of Clinical Genetics, Erasmus University Rotterdam, The Netherlands
  1. Correspondence to:
 Dr B B A de Vries, Department of Clinical Genetics, University Medical Centre Nijmegen, P O Box 9101, 6500 HB Nijmegen, The Netherlands; 
 b.devries{at}antrg.umcn.nl

https://doi.org/10.1136/jmg.40.7.535

Statistics from Altmetric.com

    Request Permissions

    If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

    The fragile X syndrome is a common cause of familial mental retardation with an estimated prevalence of 1/4000-1/6000 for males in western countries.1–3 This X linked disorder is characterised by mental retardation with additional features like a long face with large protruding ears, macro-orchidism, and eye gaze avoidance.4–6 The causative mutation is an amplification of a trinucleotide (CGG) repeat in the 5′ UTR of the FMR1 gene. Normal people have between six and 54 CGG repeats, carriers of the premutation have between 55 and 200, and affected subjects have more than 200 CGG repeats in their FMR1 gene, the so called full mutation.7,8 The latter expansion is accompanied by hypermethylation of the repeat and its upstream region resulting in a shutdown of transcription and absence of the FMRP.9–11

    In fragile X patients, two special subclasses of mosaicism can be distinguished on the basis of size and methylation pattern: (1) subjects with a premutation in a proportion of their cells in addition to a full mutation, often referred to as “size mosaics”; this pattern can be observed in 20–40% of male patients12,13; (2) subjects with intercellular variations in the methylation status of a full mutation, “methylation mosaics”.14 In a large multicentre study, “methylation mosaicism” was observed in 3% of the males with a full mutation.12

    Key points

    • In fragile X patients two special subclasses of mosaicism can be distinguished on the basis of size and/or methylation pattern: patients with full mutation and premutation, called “size mosaics”, and patients with intercellular variations of the methylation status, called “methylation mosaics”.

    • Within a known fragile X family, three brothers with methylation mosaic patterns were studied using the FMRP antibody test on both blood smears and hair roots. The index patient aged 10 years (case 1) was diagnosed at the age of 5 years; he was mildly retarded and had some clinical fragile X features. DNA analysis showed a full mutation (200–250 repeats) with 86% unmethylated mutations. His 5 year old brother (case 2) had a mutation of 177 repeats that was unmethylated in 67% of cells. His (early) development was considered normal and he lacked additional fragile X features. The third brother (case 3) had a mutation of 183–187 repeats that was unmethylated in 86% of cells. His development was normal at the age of 3 years and he did not have any significant fragile X features.

    • In cases 1, 2, and 3, FMRP expression in blood (in duplicate) was 20% and 10%, 22% and 10%, and 7% and 2%, respectively, and 67%, 85% and 88% of their hair roots expressed FMRP.

    • These results suggest that FMRP expression in hair roots gives a better reflection of the mental development than FMRP expression in leucocytes, which is consistent with the common embryonic origin of hair roots and neuronal cells.

    In 1995, Willemsen et al15 developed an FMRP antibody test for detecting the presence or absence of FMRP in lymphocytes and later in hair roots.16 This test allowed for screening for the fragile X syndrome among mentally retarded males17 and in addition made quantification of the number of FMRP expressing cells in patients possible. Using this technique in blood smears, Tassone et al18 were able to find correlations between FMRP expression and IQ in males with size mosaicism and methylation mosaicism. Very recently, a highly significant correlation has been found between FMRP expression in hair roots and cognitive functioning in females carrying a full mutation (R Willemsen, in press).

    Here, a study of FMRP expression in blood and hair roots is reported in three male sibs with a varying degree of methylation mosaicism.

    PATIENTS AND METHODS

    The three brothers who are the subjects of this report are from a fragile X family known to the Department of Clinical Genetics, Rotterdam. The family was ascertained through a son of the mother’s sister who had the classical clinical presentation of the fragile X syndrome confirmed by a fully methylated full mutation of the FMR1 gene.

    DNA analysis

    Genomic DNA was isolated19 from blood leucocytes digested with HindIII and the methylation sensitive enzyme EagI and hybridised with probe pP2 according to standard protocols.20

    Sizing of the Southern blot and densitometry were done using a Kodak Electrophoresis Documentation and Analysis System 120.

    Protein analysis

    Blood smears were made from one drop of blood within two hours after collection. Slides were air dried. Hairs were plucked from different locations on the scalp and analysed within 24 hours. The FMRP was visualised by using monoclonal antibodies 1A1 against FMRP.21 Further immunoincubations were performed according to procedures described previously15,22 (http://www.eur.nl/FGG/CH1/frax/). A total of 100 leucocytes were analysed per patient and scoring for FMRP expression was performed by two people independently.

    Determination of IQ levels

    The Wechsler Intelligence Scale for Children-Revised (WISC-R), the McCarthy Scales of Children’s Abilities (MSCA), the Wechsler Preschool and Primary Scale of Intelligence (WIPPSI-R), and the Peabody Picture Vocabulary Test-Revised (PPVT-R) were used to test the intellectual abilities by one examiner (AJ) who was not informed about the genetic status of the children tested. The WISC-R is suitable for children from the age of 6 years whereas the MSCA, the WIPPSI-R, and PPVT-R should be used for younger children. The verbal, performance, and full scale IQ scores were calculated.

    RESULTS

    Case reports

    Case 1

    This boy was born after a normal pregnancy and delivery with a birth weight of 3250 g. In his first year of life he had frequent ENT related problems which disappeared after tonsillectomy and the insertion of grommets. His early development was somewhat slow but within the normal range: he sat at 8 months, walked unaided at 18 months, and spoke his first words at 1 year. At the age of 2 years hyperactive behaviour was noted which disappeared at 4 years of age. However, at that age he appeared to be unable to attend normal school. For further evaluation he was referred to our centre and because of the family history (the mother’s sister had two sons with the fragile X syndrome) DNA analysis of the FMR1 gene was performed.

    At the age of 5 years he had normal physical measurements, height 1.17 m (70th centile) and head circumference 52.5 cm (75th centile). He had a long, narrow face with a normal chin, normally shaped and sized ears, periorbital fullness, and normal teeth (fig 1). His testes were mildly enlarged for his age (4 ml/4 ml). He had hyperextensible finger joints and relatively broad and short halluces. His behaviour was normal with normal eye contact.

    Figure 1
    Figure 1

    (A) Case 1 at the age of 5 years, (B) case 2 at the age of 5 years, and (C) case 3 at the age of 3 years.

    Psychological testing at the age of 10 years, using the WISC-R, showed a full scale IQ score of 55 with a verbal IQ score of 56 and a performance IQ score of 61.

    Case 2

    This boy is the 4 year younger brother of case 1. He was also born after an uneventful pregnancy and delivery with a normal birth weight. His early development was normal: he sat at 9 months, stood at 10 months, and walked unaided at 14 months. Because of the diagnosis in his older brother, he was tested for the fragile X syndrome at the age of 1 year 3 months.

    At the age of 5 years he had normal physical measurements, height 110 m (25th centile) and head circumference 51.8 cm (60th centile). He had a normal face, except for a broad forehead which was observed in the father as well, some periorbital fullness, and normal ears (fig 1). His genitals were normal. His behaviour was normal with normal eye contact.

    Psychological testing at the age of 5 years showed a full scale IQ score of 81 with the MSCA and 75 with the WIPPSI-R.

    Case 3

    This boy is the 6 years younger brother of case 1. He was born after a normal pregnancy and delivery with a birth weight of 3750 g. His development was normal: he walked at 14 months and spoke normal sentences at 3 years.

    At the age of 3 years he had normal physical measurements, height 1.00 m (50th centile) and head circumference 51.5 cm (70th centile). He had no dysmorphic facial features, except for a broad forehead which was observed in the father as well; he had normal sized and shaped ears. His genitals were normal. He had some hyperextensibilty of MCP V. His behaviour was normal with normal eye contact. Psychological testing at the age of 3 years 8 months showed a full scale IQ score of 91 with the MSCA and 97 with the PPVT-R.

    Molecular findings

    In case 1, a full mutation was found in his leucocytes using Southern blot analysis: a 14% methylated 560 bp larger than normal band (~217 repeats) and a 86% unmethylated 670 bp larger than normal band (~253 repeats) (fig 2).

    Figure 2
    Figure 2

    Analysis with probe pP2 of HindIII/EagI digested DNA (leucocytes) of the mother, cases 1, 2, and 3, and a normal sib.

    Protein analysis in blood smears (in duplicate) showed 10% and 20% of the leucocytes and 67% of the hair roots (10/15) expressing FMRP.

    In case 2, a mutation that was smaller than in case 1 was found in leucocytes using Southern blot analysis: a 33% methylated 440 bp larger than normal band (~177 repeats) and a 67% unmethylated 440 bp larger than normal band (~177 repeats).

    Protein analysis in blood smears (in duplicate) showed 10% and 22% of the leucocytes and 85% of the hair roots (17/20) expressing FMRP.

    In case 3, a mutation similar in size to that of case 2 was also found in leucocytes using Southern blot analysis: a 14% methylated 470 bp larger than normal band (~187 repeats) and an 86% unmethylated 460 bp larger than normal band (~183 repeats) (fig 2).

    Protein analysis in blood smears (in duplicate) showed 2% and 7% of the leucocytes and 88% of the hair roots (23/26) expressing FMRP.

    The test results are summarised in table 1.

    View this table:
    Table 1

    Summary test results in the three brothers

    DISCUSSION

    According to the definition of mosaicism, all fragile X males are mosaic as they have different amplified repeat sequences in the FMR1 gene in different cells. However, two special subclasses can be distinguished; 20–40% of fragile X males have a premutation in a proportion of cells and the full mutation in the remaining (majority) of cells. In general, the proportion of cells with a premutation is lower than <30% and various studies have shown that the cognitive functioning of these fragile X patients is not significantly better than the males with a full mutation, suggesting that the number of FMRP expressing cells with a premutation is insufficient.12,23 However, high functioning males with a size mosaic pattern have been described24–26 and Tassone et al18 detected a correlation between FMRP expression in blood smears and IQ in mosaic males.

    For the second group of mosaic patients, the so called “methylation mosaics” the situation is different. These patients have a full mutation in all cells but in a proportion of cells the full mutation is unmethylated. The cells with an unmethylated full mutation are able to produce FMRP and can therefore function normally. Depending on their proportion, they are able to compensate for the loss of functioning of the cells with a methylated full mutation. Various reports on patients with methylation mosaicism suggest that a proportion of cells with an unmethylated full mutation of at least 40% of normal is likely to be required for normal cognitive functioning.24,27–36 This is supported by actual FMRP studies in blood smears of patients with methylation mosaicism that showed that all mosaic patients with a normal IQ had FMRP in ⩾50% of lymphocytes.18,32 This situation is reminiscent of the situation in females with a full mutation where cognitive function is related to the X inactivation pattern.12,14,37–39

    FMRP expression studies in blood smears of methylation mosaic males have been reported. Smeets et al31 reported normal protein expression in cell lines of two normal functioning adults with an unmethylated full mutation. De Vries et al32 reported three cousins with 75%, 40%, and 10% cells expressing FMRP who had an unmethylated full mutation in 90%, 35%, and 10% of the cells, respectively. The latter two were both retarded whereas the adult males with 75% FMRP expressing cells had a normal IQ. Tassone et al18 found a correlation between IQ and FMRP expression in blood smears in 13 males with a partially methylated full mutation. They also found three non-retarded mosaic males with expression of FMRP in ⩾50% of lymphocytes.18 The findings of normal FMRP expression in partially unmethylated full mutations were in contrast with the report of Feng et al,40 who found markedly diminished FMRP production in fibroblast clones from transcripts with more than 200 repeats. These conflicting findings raise the question of whether unmethylated full mutations have normal or diminished FMRP expression, what is the relation to cognitive functioning, and what is the correct tissue to study. Interestingly, Tassone et al41 reported a six-fold increase of FMR1 mRNA levels in methylation mosaic males suggesting the existence of a compensatory response to impeded FMRP production.

    In the oldest of the three reported brothers, the size of the (un)methylated alleles are all in the full mutation range whereas the other two brothers have (un)methylated alleles in the high premutation range; thus the latter two do have partially methylated premutation sized alleles which is quite rare.

    The proportion of cells expressing FMRP in a blood smear ascertained by the FMRP antibody test did not correspond very well with the proportion of unmethylated FMR1 alleles (pre- or full mutation sized) as ascertained by DNA blotting analysis. It suggests that in leucocytes the translation might also be hampered in the large unmethylated premutation sized alleles. It also shows that accurate prediction of mental functioning in males with an intercellular variation of the methylation status through FMRP studies in blood smears is, like DNA analysis, less valid. However, FMRP expression in hair roots did reflect the cognitive functioning in the three brothers. Both brothers with normal IQs (81 and 91) had a high proportion of FMRP expressing hair roots (85% and 90%, respectively). This is consistent with the common embryonic origin, ectoderm, of hair roots and neuronal cells whereas blood is of mesodermal origin. Of course a larger number of males with (un)methylated full mutations need to be tested to assess the validity of the relationship between FMRP expression in hair roots and mental functioning.

    Acknowledgments

    We thank the patients and their family for their kind support.

    REFERENCES

    1. ↵
      Turner G, Webb T, Wake S, Robinson H. Prevalence of fragile X syndrome. Am J Med Genet1996;64:196–7
      OpenUrlCrossRefPubMedWeb of Science
    2. Murray A, Youings S, Dennis N, Latsky L, Linehan P, McKechnie N, Macpherson J, Pound M, Jacobs P. Population screening at the FRAXA and FRAXE loci: molecular analyses of boys with learning difficulties and their mothers. Hum Mol Genet1996;5:727–35
      OpenUrlAbstract/FREE Full Text
    3. ↵
      De Vries BB, van den Ouweland AM, Mohkamsing S, Duivenvoorden HJ, Mol E, Gelsema K, van Rijn M, Halley DJ, Sandkuijl LA, Oostra BA, Tibben A, Niermeijer MF. Screening and diagnosis for the fragile X syndrome among the mentally retarded: an epidemiological and psychological survey. Collaborative Fragile X Study Group. Am J Hum Genet1997;61:660–7.
      OpenUrlPubMedWeb of Science
    4. ↵
      Fryns JP. X-linked mental retardation and the fragile X syndrome: a clinical approach. In: Davies KE, ed. The fragile X syndrome. Oxford: Oxford University Press, 1989:1–39.
    5. Hagerman RJ. Physical and behavioural phenotype. In: Hagerman RJ, Cronister A, eds. Fragile-X syndrome: diagnosis, treatment and research. Baltimore: The Johns Hopkins University Press, 1996:3–87.
    6. ↵
      De Vries BBA, Halley DJJ, Oostra BA, Niermeijer MF. The fragile X syndrome. J Med Genet1998;35:579–89.
      OpenUrlAbstract/FREE Full Text
    7. ↵
      Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A, Reiner O, Richards S, Victoria MF, Zhang FP, Eussen BE, van Ommen GJB, Blonden LAJ, Riggins GJ, Chastain JL, Kunst CB, Galjaard H, Caskey CT, Nelson DL, Oostra BA, Warren ST. Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell1991;65:905–14.
      OpenUrlCrossRefPubMedWeb of Science
    8. ↵
      Fu YH, Kuhl DP, Pizzuti A, Pieretti M, Sutcliffe JS, Richards S, Verkerk AJ, Holden JJ, Fenwick R Jr, Warren ST, Oostra BA, Nelson DL, Caskey CT. Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell1991;67:1047–58.
      OpenUrlCrossRefPubMedWeb of Science
    9. ↵
      Pieretti M, Zhang FP, Fu YH, Warren ST, Oostra BA, Caskey CT, Nelson DL. Absence of expression of the FMR-1 gene in fragile X syndrome. Cell1991;66:817–22.
      OpenUrlCrossRefPubMedWeb of Science
    10. Sutcliffe JS, Nelson DL, Zhang F, Pieretti M, Caskey CT, Saxe D, Warren ST. DNA methylation represses FMR-1 transcription in fragile X syndrome. Hum Mol Genet1992;1:397–400.
      OpenUrlAbstract/FREE Full Text
    11. ↵
      Verheij C, Bakker CE, de Graaff E, Keulemans J, Willemsen R, Verkerk AJ, Galjaard H, Reuser AJ, Hoogeveen AT, Oostra BA. Characterization and localization of the FMR-1 gene product associated with fragile X syndrome. Nature1993;363:722–4.
      OpenUrlCrossRefPubMedWeb of Science
    12. ↵
      Rousseau F, Heitz D, Tarleton J, MacPherson J, Malmgren H, Dahl N, Barnicoat A, Mathew C, Mornet E, Tejada I, Maddalena A, Spiegel R, Schinzel A, Marcos JAG, Schoderet DF, Schaap T, Maccioni L, Russo S, Jacobs PA, Schwartz C, Mandel JL. A multicenter study on genotype-phenotype correlations in the fragile X syndrome, using direct diagnosis with probe StB12.3: the first 2,253 cases. Am J Hum Genet1994;55:225–37.
      OpenUrlPubMedWeb of Science
    13. ↵
      Nolin SL, Glicksman A, Houck G Jr, Brown WT, Dobkin CS. Mosaicism in fragile X affected males. Am J Med Genet1994;51:509–12.
      OpenUrlCrossRefPubMedWeb of Science
    14. ↵
      Rousseau F, Heitz D, Biancalana V, Blumenfeld S, Kretz C, Boue J, Tommerup N, Van Der Hagen C, DeLozier-Blanchet C, Croquette MF, Gilgenkrantz, S., Jalbert P, Voelckel MA, Oberle I, Mandel JL. Direct diagnosis by DNA analysis of the fragile X syndrome of mental retardation. N Engl J Med1991;325:1673–81.
      OpenUrlCrossRefPubMedWeb of Science
    15. ↵
      Willemsen R, Mohkamsing S, de Vries B, Devys D, van den Ouweland A, Mandel JL, Galjaard H, Oostra B. Rapid antibody test for fragile X syndrome. Lancet1995;345:1147–8.
      OpenUrlCrossRefPubMedWeb of Science
    16. ↵
      Willemsen R, Anar B, De Vries BBA, Willems PJ, Galjaard H, Oostra BA. Non-invasive screening for the fragile X syndrome using hair root analysis. Am J Hum Genet1998;63:224.
    17. ↵
      De Vries BBA, Mohkamsing S, Van den Ouweland AMW, Halley DJJ, Niermeijer MF, Oostra BA, Willemsen R. Screening with the FMR1 protein test among mentally retarded males. Hum Genet1998;103:520–2.
      OpenUrlCrossRefPubMed
    18. ↵
      Tassone F, Hagerman RJ, Ikle DN, Dyer PN, Lampe M, Willemsen R, Oostra BA, Taylor AK. FMRP expression as a potential prognostic indicator in fragile X syndrome. Am J Med Genet1999;84:250–61.
      OpenUrlCrossRefPubMedWeb of Science
    19. ↵
      Miller S, Dykes D, Polesky H. A simple salting out procedure for extracting DNA from nucleated cells. Nucleic Acids Res1988;16:1215.
      OpenUrlFREE Full Text
    20. ↵
      Oostra BA, Jacky PB, Brown WT, Rousseau F. Guidelines for the diagnosis of fragile X syndrome. J Med Genet1993;30:410–13.
      OpenUrlAbstract/FREE Full Text
    21. ↵
      Devys D, Lutz Y, Rouyer N, Bellocq JP, Mandel JL. The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation. Nat Genet1993;4:335–40.
      OpenUrlCrossRefPubMedWeb of Science
    22. ↵
      Willemsen R, Anar B, De Diego Otero Y, De Vries BBA, Hilhorst-Hofstee Y, Smits A, Van Looveren E, Willems PJ, Galjaard H, Oostra BA. Noninvasive test for the fragile X syndrome, using hair root analysis. Am J Hum Genet1999;65:98–103.
      OpenUrlCrossRefPubMed
    23. ↵
      De Vries BB, Wiegers AM, de Graaff E, Verkerk AJ, Van Hemel JO, Halley DJ, Fryns JP, Curfs LM, Niermeijer MF, Oostra BA. Mental status and fragile X expression in relation to FMR-1 gene mutation. Eur J Hum Genet1993;1:72–9.
      OpenUrlPubMedWeb of Science
    24. ↵
      Hagerman RJ, Hull CE, Safanda JF, Carpenter I, Staley LW, O’Connor RA, Seydel C, Mazzocco MM, Snow K, Thibodeau SN, Kuhl D, Nelson DL, Caskey CT, Taylor A. High functioning fragile X males: demonstration of an unmethylated fully expanded FMR-1 mutation associated with protein expression. Am J Med Genet1994;51:298–308.
      OpenUrlCrossRefPubMedWeb of Science
    25. Merenstein SA, Sobesky WE, Taylor AK, Riddle JE, Tran HX, Hagerman RJ. Molecular-clinical correlations in males with an expanded FMR1 mutation. Am J Med Genet1996;64:388–94.
      OpenUrlCrossRefPubMedWeb of Science
    26. ↵
      Cohen IL, Nolin SL, Sudhalter V, Ding XH, Dobkin CS, Brown WT. Mosaicism for the FMR1 gene influences adaptive skill development in fragile X-affected males. Am J Med Genet1996;64:365–9.
      OpenUrlCrossRefPubMed
    27. ↵
      Loesch DZ, Huggins R, Hay DA, Gedeon AK, Mulley JC, Sutherland GR. Genotype-phenotype relationships in fragile X syndrome: a family study. Am J Hum Genet1993;53:1064–73.
      OpenUrlPubMedWeb of Science
    28. McConkie-Rosell A, Lachiewicz AM, Spiridigliozzi GA, Tarleton J, Schoenwald S, Phelan MC, Goonewardena P, Ding X, Brown WT. Evidence that methylation of the FMR-I locus is responsible for variable phenotypic expression of the fragile X syndrome. Am J Hum Genet1993;53:800–9.
      OpenUrlPubMed
    29. Merenstein SA, Shyu V, Sobesky WE, Staley L, Berry-Kravis E, Nelson DL, Lugenbeel KA, Taylor AK, Pennington BF, Hagerman RJ. Fragile X syndrome in a normal IQ male with learning and emotional problems. J Am Acad Child Adolesc Psychiatry1994;33:1316–21.
      OpenUrlPubMedWeb of Science
    30. Rousseau F, Robb LJ, Rouillard P, Der Kaloustian VM. No mental retardation in a man with 40% abnormal methylation at the FMR-1 locus and transmission of sperm cell mutations as premutations. Hum Mol Genet1994;3:927–30.
      OpenUrlAbstract/FREE Full Text
    31. ↵
      Smeets HJ, Smits AP, Verheij CE, Theelen JP, Willemsen R, van de Burgt I, Hoogeveen AT, Oosterwijk JC, Oostra BA. Normal phenotype in two brothers with a full FMR1 mutation. Hum Mol Genet1995;4:2103–8.
      OpenUrlAbstract/FREE Full Text
    32. ↵
      De Vries BB, Jansen CA, Duits AA, Verheij C, Willemsen R, Van Hemel JO, Van den Ouweland AM, Niermeijer MF, Oostra BA, Halley DJ. Variable FMR1 gene methylation of large expansions leads to variable phenotype in three males from one fragile X family. J Med Genet1996;33:1007–10.
      OpenUrlAbstract/FREE Full Text
    33. Lachiewics AM, Spiridigliozzi GA, McConkie-Rosell A, Burgess D, Feng Y, Warren ST, Tarleton J. A fragile X male with a broad smear on Southern blot analysis representing 100–500 CGG repeats and no methylation at the Eag1 site of the FMR1 gene. Am J Med Genet1996;64:278–82.
      OpenUrlCrossRefPubMed
    34. Wang Z, Taylor AK, Bridge JA. FMR1 fully expanded mutation with minimal methylation in a high functioning fragile X male. J Med Genet1996;33:376–8.
      OpenUrlAbstract/FREE Full Text
    35. Wohrle D, Salat U, Glasser D, Mucke J, Meisel-Stosiek M, Schindler D, Vogel W, Steinbach P. Unusual mutations in high functioning fragile X males: apparent instability of expanded unmethylated CGG repeats. J Med Genet1998;35:103–11.
      OpenUrlAbstract/FREE Full Text
    36. ↵
      Taylor AK, Tassone F, Dyer PN, Hersch SM, Harris JB, Greenough WT, Hagerman RJ. Tissue heterogeneity of the FMR1 mutation in a high-functioning male with fragile X syndrome. Am J Med Genet1999;84:233–9.
      OpenUrlCrossRefPubMedWeb of Science
    37. ↵
      Taylor AK, Safanda JF, Fall MZ, Quince C, Lang KA, Hull CE, Carpenter I, Staley LW, Hagerman RJ. Molecular predictors of cognitive involvement in female carriers of fragile X syndrome. JAMA1994;271:507–14.
      OpenUrlCrossRefPubMedWeb of Science
    38. Reiss AL, Freund LS, Baumgardner TL, Abrams MT, Denckla MB. Contribution of the FMR1 gene mutation to human intellectual dysfunction. Nat Genet1995;11:331–4.
      OpenUrlCrossRefPubMedWeb of Science
    39. ↵
      De Vries BB, Wiegers AM, Smits AP, Mohkamsing S, Duivenvoorden HJ, Fryns JP, Curfs LM, Halley DJ, Oostra BA, van den Ouweland AM, Niermeijer MF. Mental status of females with an FMR1 gene full mutation. Am J Hum Genet1996;58:1025–32.
      OpenUrlPubMedWeb of Science
    40. ↵
      Feng Y, Zhang F, Lokey LK, Chastain JL, Lakkis L, Eberhart D, Warren ST. Translational suppression by trinucleotide repeat expansion at FMR1. Science1995;268:731–4.
      OpenUrlAbstract/FREE Full Text
    41. ↵
      Tassone F, Hagerman RJ, Loesch DZ, Lachiewicz A, Taylor AK, Hagerman PJ. Fragile X males with unmethylated, full mutation trinucleotide repeat expansions have elevated levels of FMR1 messenger RNA. Am J Med Genet2000;94:232–6.
      OpenUrlCrossRefPubMedWeb of Science