Contents lists available at SciVerse ScienceDirect
Gene
j ourna l homepage: www.e lsev ie r .com/ locate /gene
Short Communication
Mosaicism for FMR1 gene full mutation and intermediate allele in afemale foetus: A postzygotic retraction event
Susana Isabel Ferreira a, Luís Miguel Pires a, José Ferrão a, Joaquim Sá b,Armando Serra c, Isabel Marques Carreira a,d,e,⁎a Laboratório de Citogenética e Genómica, Faculdade de Medicina da Universidade de Coimbra, Coimbra, Portugalb Serviço de Genética Médica, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugalc Serviço de Genética e Biologia Forenses, Instituto Nacional de Medicina Legal e Ciências Forenses, I.P. Delegação do Centro, Coimbra, Portugald CIMAGO — Centro de Investigação em Meio Ambiente, Genética e Oncobiologia, Faculdade de Medicina da Universidade de Coimbra, Coimbra, Portugale CENCIFOR — Centro Ciências Forenses, Instituto Nacional de Medicina Legal e Ciências Forenses, I.P., Coimbra, Portugal
Abbreviations: 5′UTR, 5′unstranslated region; Array-CHybridization; FMR1, Fragile X Mental Retardation 1Retardation Protein; IQ, Intelligence Quotient; PCR,STR, Short Tandem Repeat; TP PCR, Triplet Repeat Prim⁎ Corresponding author at: Laboratório de Citogenét
Medicina da Universidade de Coimbra, Pólo Ciências d2, Azinhaga de Santa Comba, 3000-354 Coimbra, Portufax: +351 239 480 035.
Fragile X syndrome is caused by the expansion of an unstable CGG repeat in the 5′UTR of FMR1 gene. The occur-rence of mosaicism is not uncommon, especially in male patients, whereas in females it is not so often reported.Herewe report a female foetus thatwas subject to prenatal diagnosis, because of hermother being a premutationcarrier. The foetus was identified as being amosaic for an intermediate allele and a full mutation of FMR1 gene, inthe presence of a normal allele. Themosaic statuswas confirmed in three different tissues of the foetus – amnioticfluid, skin biopsy and blood – the last two obtained after pregnancy termination. Karyotype analysis andX-chromosome STR markers analysis do not support the mosaicism as inheritance of both maternal alleles. Oli-gonucleotide array-CGH excluded an imbalance that could contain the primer binding site with a different repeatsize. The obtained results give compelling evidence for a postzygotic expansionmechanismwhere the foetusmo-saic pattern originated from expansion of the mother's premutation into a full mutation and consequent regres-sion to an intermediate allele in a proportion of cells. These events occurred in early embryogenesis before thecommitment of cells into the different tissues, as the three tested tissues of the foetus have the samemosaic pat-tern. The couple has a son with Fragile X mental retardation syndrome and choose to terminate this pregnancyafter genetic counselling.
Fragile X syndrome is themost common form of X-linkedmental re-tardation. It is caused by the expansion of an unstable CGG repeat in the5′untranslated region of the Fragile Xmental retardation 1 gene (FMR1)(Maddalena et al., 2001; Pandey et al., 2004). The polymorphic CGG re-peat region is classified into 4 groups according to repeat length(Maddalena et al., 2001). Normal alleles have a number of CGG repeatsthat ranges from ~5 to ~44, often interrupted by AGG triplets that con-fer stability to the alleles (Maddalena et al., 2001). The second class ofalleles is named intermediate, with CGG repeats ranging from ~45 to~54 (Maddalena et al., 2001). Intermediate alleles can be occasionally
GH, array-ComparativeGenomicgene; FMRP, Fragile X MentalPolymerase Chain Reaction;ed PCR.ica e Genómica, Faculdade dea Saúde, Sub-Unidade 1 - Pisogal. Tel.: +351 239 480 026;
ira).
rights reserved.
unstable upon transmission and potential precursors of premutationsin future generations (Rifé et al., 2004). Premutation alleles rangefrom ~55 to ~200 CGG repeats, being highly unstable alleles only duringmaternal transmission to the next generation (Warren and Jin, 2000).When premutations expand they originate fullmutation alleles, typical-ly havingmore than 200 CGG repeats and beingmethylated, resulting ingene silencing and absence of Fragile X mental retardation protein(FMRP) that causes the phenotype (Rifé et al., 2004).
The occurrence of mosaicism of FMR1 gene is not uncommon, espe-cially in male patients, with mosaics for full mutation/premutation, fullmutation/normal allele and full mutation/deletion reported (GarcíaArocena et al., 2000; Nolin et al., 1994; Orrico et al., 1998; Schmuckerand Seidel, 1999). However, in females, mosaicism is not so oftenreported. Fan et al. (2005) reported a female mosaic for an FMR1 fullmutation and a deletion allele and Hantash et al. (2010) reported twofemale mosaics for premutation/full mutation alleles. More recently,Sharony et al. (2012) reported 25 females with three peaks for FMR1gene analysis, all within the normal to intermediate range, and onlyone allele within the premutation range. They suggested that themosa-icism could be explained by triple X syndrome, low level mosaicism ofsex chromosomes or by partial duplication of the X-chromosome
422 S.I. Ferreira et al. / Gene 527 (2013) 421–425
containing the fragile site with a different fragment size (Sharony et al.,2012). Here we report a female foetus with a 46,XX normal karyotype,that was subject to prenatal diagnosis, due to the premutation carrierstatus of the pregnant mother. The prenatal diagnosis identified mosa-icism for intermediate allele and a full mutation of FMR1 gene, in thepresence of a normal allele.
2. Materials and methods
Genomic DNA was extracted from peripheral and foetal bloodlymphocytes using Jetquick blood and cell culture DNA Midi Spin kit(Genomed, Löhne, Germany). DNA concentration and purity wereevaluated using a NanoDrop1000 Spectrophotometer (Thermo Scien-tific, Waltham, USA).
An amniocentesiswas performed at 18 weeks of gestation, three daysafter the diagnosis of Fragile X mental retardation syndrome in thecouple's 22 months old son. The amniocytes were cultured in Ham'sF-10 medium (Gibco®, Life Technologies, Paisley, UK) supplementedwith 10% foetal bovine serum (Gibco®), and when confluence wasachieved the cells were scrapped and foetal DNA was extracted usingthe High Pure PCR Template Preparation Kit (Roche Applied Science,Indianapolis, USA) according to the manufacturer's instructions. DNAfrom skin biopsy of the expelled foetus was extracted using the sameprotocol.
FMR1 gene CGG repeat number was determined by conventionalPCR using primers C and F described by Fu et al. (1991) and by TripletRepeat Primed PCR (TP PCR) using Asuragen AmplideX® FMR1 PCRKit (Asuragen, Austin, USA). Conventional PCR was performed as de-scribed by Amos Wilson et al. (2008) using an Applied Biosystems ABI2720 Thermal Cycler (Applied Biosystems, California, USA). Sampleswere analysed by capillary electrophoresis on anABI Prism 3130Genet-ic Analyzer (Applied Biosystems) andGeneMapper software version 4.1(Applied Biosystems) was used to determine the alleles dimension bycomparison with the size standard ROX 500 (Applied Biosystems).
TP PCR was performed according to the manufacturer's instruc-tions using an Applied Biosystems ABI Veriti Thermal Cycler (AppliedBiosystems). Samples were analysed by capillary electrophoresis onan ABI Prism 3130 Genetic Analyzer (Applied Biosystems) andGeneMapper software version 4.1 (Applied Biosystems) was used todetermine the alleles dimension by comparison with the size stan-dard ROX1000 (Asuragen).
X-chromosome STR markers analysis was performed on the foetusand in both progenitors using the Investigator Argus X-12 kit (Qiagen,Hilden, Germany). PCR amplification for 12 X-STR loci was performedaccording to the manufacturer's instructions. For genetic typing,
Fig. 1. Pedigree of the studied family with CGG repeat numb
samples were analysed on an ABI Prism 3500 Genetic Analyzeralong with GeneMapper ID-X 1.2 software (Applied Biosystems) bycomparison with BTO size standard 550 (Qiagen).
Oligonucleotide array-CGH analysis was performed on the foetus'DNA with an Agilent SurePrint G3 Human Genome microarray 180K(Agilent Technologies, Santa Clara, CA, USA) to exclude a genomic im-balance in FMR1 gene region (Ferreira et al., 2012).
3. Results
The study initiated by testing for Fragile X Syndrome a 22 month oldboy (III-1 — Fig. 1) with possible developmental delay. He is the firstchild of healthy non-related parents. At the time of the first evaluation,the mother (II-2) has a 16 weeks pregnancy. After conventional PCRanalysis of FMR1 gene in the proband, as no amplificationwas obtained,the genomic DNA was analysed by TP PCR. The result revealed the pro-band to be a full mutation carrier, with more than 200 CGG repeats(Fig. 2). Blood from the mother was requested, to determine theproband's full mutation origin, and after TP PCR analysis themother re-vealed to be a premutation carrier. Themother had a normal allele with37 CGG repeats interrupted by 3 AGG repeats, and a premutation with111/118 CGG repeats without AGG interruptions (Fig. 2). As there wasa pregnancy in course, prenatal diagnosis by amniocentesis wasperformed at 18 weeks gestation. The foetus (III-2) was in risk ofbeing a premutation or full mutation carrier, due to the mother's geno-type. When TP PCR was performed on DNA obtained from amnioticfluid three alleles were obtained: a normal allele with 29 CGG repeats,an intermediate allele with 48 CGG repeats and a full mutation withmore than 200 CGG repeats (Fig. 2). The foetus revealed to be a normalfemale by karyotype analysis [46,XX] and array-CGH analysis revealedno genomic imbalances at the X-chromosome. X-chromosome STRmarkers analysis performed on DNA obtained from amniotic fluidand in both progenitors revealed the foetus to have inherited the pa-ternal allele and only one maternal allele (Fig. 3). With these resultsand after genetic counselling, the couple choose to terminate thepregnancy.
TP PCR analysis was performed on the father's (II-3) blood, whichrevealed to have a normal allele with 29 CGG repeats and 2 AGG in-terruptions (Fig. 2). Foetal blood and skin biopsy samples from thefoetus were collected after pregnancy termination, DNA extractedand CGG repeat number were determined. The results were coinci-dent with the ones obtained from the amniotic fluid sample.
The grandmother and one aunt of the child, respectively relativesI-1 and II-1, were studied. The grandmother revealed to be carrier of a30 CGG repeat normal allele and a 92 CGG repeat premutation with 1
er result for FMR1 gene depicted above each individual.
Fig. 2. TP PCR results for family members I-1, II-2, II-3, III-1and III-2. AGG interruptions are marked with a horizontal trace.
423S.I. Ferreira et al. / Gene 527 (2013) 421–425
AGG interruption at the 5′ end of the gene, which was transmitted toher daughter (II-2) with slight expansion (Fig. 2). The aunt (II-1) hadtwo normal alleles, having inherited the mother's normal allele. Fa-miliar studies will proceed on the maternal side in order to identifyrelatives in risk of being premutation carriers.
Fig. 3. X-chromosome STR analysis results. The first electropherogram corresponds to the X-the third one corresponds to the X-chromosome profile of the father's DNA. Maternal inherleles with the male symbol (♂). The foetus is homozygous for three alleles – represented witthe obtained profile it is possible to conclude that the foetus has only inherited one of the
4. Discussion
There is a significantly higher prevalence of FMR1 gene mosaicismreported in males compared to females. Here we report a female foe-tus mosaic for FMR1 gene full mutation and an intermediate allele, in
chromosome profile of the foetus DNA, the one in the middle to the mother's DNA andited alleles are signed with the female symbol (♀) above and the paternal inherited al-h the two symbols♀♂ – that were inherited from both the father and the mother. Withmother's X chromosomes.
424 S.I. Ferreira et al. / Gene 527 (2013) 421–425
the presence of a normal allele. This normal allele with 29 CGG re-peats and 2 AGG triplets was inherited from the male partner, andtherefore must be present in all cells. The pregnant woman carrieda normal allele with 37 CGG repeats interrupted by 3 AGG triplets,and a premutation with no AGG interruptions. Alleles within normalrange and interrupted by AGG triplets are stable and have neverbeen found to expand when transmitted to the descendents (Nolinet al., 2013). Nolin et al. (2003, 2011) concluded in two different stud-ies that for premutations within the 100–200 CGG range, 97–98% ex-panded to full mutation. The remaining contracted either to smallerpremutations or to intermediate alleles (Nolin et al., 2003, 2011).More recently, Yrigollen et al. (2012) demonstrated that the predictedrisk of expansion of a maternal premutation with more than 110 CGGwith 0 AGG interruptions is of 100%. Considering these data, the foetusfull mutation allele must have arisen by expansion of the mother'spremutation. The most intriguing result is the origin of the foetusintermediate allele with 48 CGG repeats. Karyotype analysis andX-chromosome STRmarkers analysis do not support themosaicismas in-heritance of both maternal alleles. Indeed, X-chromosome STR markersanalysis results revealed that the foetus has inherited only oneX-chromosome from themother. Oligonucleotide array-CGH analysis ex-cluded an imbalance at FMR1 gene region that could contain the primerbinding sitewith a different size. Paternal transmissions of normal and in-termediate size alleles are less stable than maternal transmission, withthe length of the 3′ end representing a risk factor for this repeat instability(Sullivan et al., 2002). When the length of the 3′ end was b24 CGG, theobserved mutation rate in paternal alleles was 0% (Sullivan et al., 2002).In the reported case, the allele of the male partner has 29 CGG repeatsinterrupted by 2 AGG (Sullivan et al., 2002). According to this, the pater-nal allele is supposed to be very stable, and not expected to expand.
Studies in full mutation males have revealed that their spermcarries only premutation alleles (Reyniers et al., 1993). This has ledto the assumption that expansion of premutation to full mutation oc-curs somatically during early embryogenesis (Reyniers et al., 1993).
Taking this data into consideration, knowing that the foetusinherited only one X maternal chromosome and that normal allelesare stable, themost probablemechanism for the presence of mosaicismis postzygotic regression of the full mutation allele into an intermediateallele with 48 CGG repeats. It seems more likely that the initial eventwas the expansion of the maternal premutation into a full mutation,that then, due to instability, contracted to the smaller intermediate al-lele in a proportion of cells. We cannot disregard that the intermediateallele could have resulted from direct contraction of the premutation,but this presumes that two events would have to occur at an initialstage: contraction of the maternal premutation into the intermediateallele in some cells and expansion to full mutation in the remainingcells. The observation that, in general, patients show aminor proportionof the contracted allele supports the generation of mosaicism by apostzygotic retraction event of a full mutation (Fan et al., 2005;Moutou et al., 1997; Schmucker and Seidel, 1999). The overall resultsare compatible with the female foetus being a mosaic, in which onecell line has the normal 29 CGG allele and the full mutation, and theother cell line the normal 29 CGG allele and the 48 CGG intermediateallele.
We propose that the foetus mosaic pattern originated from expan-sion of the mother's premutation into a full mutation and consequentregression to an intermediate allele in a proportion of cells. Theseevents occurred before the divergence of the different tissues, as thethree tested tissues of the foetus – amniotic fluid, skin and blood –
have the same mosaic pattern.We do not know the distribution of the mosaic in brain cells, but as
skin and nervous system tissue develop from the ectoderm (Dobkin etal., 1996), and given the agreement of the mosaic status between thedifferent studied tissues, it is likely that the brain would also have thesame mosaic pattern. The impact of the full mutation mosaicism inthe female's phenotype would be conditioned by the X-inactivation
pattern, and there would be an increased risk of intellectual disability.Women with a full mutation could have less severe disease thanmales. A 23-year-old woman with the full Fragile X mutation who hadno dysmorphic features and above-average intelligence combinedwith significant impairments due to anxiety and learning disabilityhas been described (de Vries et al., 1996). The authors concluded thatwomen with Fragile X syndrome can present primarily with learningand emotional problems. There is a significant association betweenthe proportion of normal FMR1 alleles on the active X chromosomeand IQ, known since de Vries et al. (1996) and Angkustsiri et al.(2008). This is valid for X-chromosome inactivation and should bevalid for full mutation mosaicism at the relevant neuron tissue, butthis quantification is not possible in the clinical practice. The couple'sdecision of terminating the pregnancywas accepted by themedical eth-ical commission from the hospital, according to the national law.
In conclusion, this case provides further evidence for the occur-rence of a postzygotic retraction event of a full mutation, derivedfrom the expansion of a maternal premutation, into an intermediateallele. It also demonstrates the accuracy of TP PCR as a reliable tech-nique for mosaicism detection and prenatal diagnosis of FMR1 genealleles, with a fast turnaround time.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors wish to thank the family.
References
Amos Wilson, J., et al., 2008. Fragile Xperts Working Group of the Association for Mo-lecular Pathology Clinical Practice Committee. Consensus characterization of 16FMR1 reference materials: a consortium study. J. Mol. Diagn. 10, 2–12.
Angkustsiri, K., Wirojanan, J., Deprey, L.J., Gane, L.W., Hagerman, R.J., 2008. Fragile Xsyndrome with anxiety disorder and exceptional verbal intelligence. Am. J. Med.Genet. A 146, 376–379.
de Vries, B.B., et al., 1996. Mental status of females with an FMR1 gene full mutation.Am. J. Hum. Genet. 58, 1025–1032.
Dobkin, C.S., et al., 1996. Tissue differences in Fragile X mosaics: mosaicism in bloodcells may differ greatly from skin. Am. J. Med. Genet. 64, 296–301.
Fan, H., Booker, J.K., McCandless, S.E., Shashi, V., Fleming, A., Farber, R.A., 2005. Mosaicismfor an FMR1 gene deletion in a Fragile X female. Am. J. Med. Genet. A 136, 214–217.
Ferreira, S.I., Matoso, E., Venâncio, M., Saraiva, J., Melo, J.B., Carreira, I.M., 2012. Critical re-gion in 2q31.2q32.3 deletion syndrome: report of two phenotypically distinct pa-tients, one with an additional deletion in Alagille syndrome region. Mol. Cytogenet.5, 25.
Fu, Y.H., et al., 1991. Variation of the CGG repeat at the Fragile X site results in geneticinstability: resolution of the Sherman paradox. Cell 67, 1047–1058.
García Arocena, D., de Diego, Y., Oostra, B.A.,Willemsen, R.,Mirta Rodriguez,M., 2000. A Frag-ile X case with an amplification/deletion mosaic pattern. Hum. Genet. 106, 366–369.
Hantash, F.M., et al., 2010. Qualitative assessment of FMR1 (CGG)n triplet repeat statusin normal, intermediate, premutation, full mutation, and mosaic carriers in bothsexes: implications for Fragile X syndrome carrier and newborn screening. Genet.Med. 12, 162–173.
Maddalena, A., et al., 2001. Technical standards and guidelines for Fragile X: the first of aseries of disease-specific supplements to the Standards and Guidelines for Clinical Ge-netics Laboratories of the American College of Medical Genetics. Quality AssuranceSubcommittee of the Laboratory Practice Committee. Genet. Med. 3, 200–205.
Moutou, C., Vincent, M.C., Biancalana, V., Mandel, J.L., 1997. Transition frompremutation to full mutation in Fragile X syndrome is likely to be prezygotic.Hum. Mol. Genet. 6, 971–979.
Nolin, S.L., Glicksman, A., Houck Jr., G.E., Brown, W.T., Dobkin, C.S., 1994. Mosaicism inFragile X affected males. Am. J. Med. Genet. 51, 509–512.
Nolin, S.L., et al., 2003. Expansion of the Fragile X CGG repeat in females withpremutation or intermediate alleles. Am. J. Hum. Genet. 72, 454–464.
Nolin, S.L., et al., 2011. Fragile X analysis of 1112 prenatal samples from 1991 to 2010.Prenat. Diagn. 31, 925–931.
Nolin, S.L., et al., 2013. Fragile X AGG analysis provides new risk predictions for 45–69repeat alleles. Am. J. Med. Genet. A 161, 771–778.
Orrico, A., Galli, L., Dotti, M.T., Plewnia, K., Censini, S., Federico, A., 1998. Mosaicism forfull mutation and normal-sized allele of the FMR1 gene: a new case. Am. J. Med.Genet. 78, 341–344.
Pandey, U.B., Phadke, S.R., Mittal, B., 2004. Molecular diagnosis and genetic counselingfor Fragile X mental retardation. Neurol. India 52, 36–42.
Reyniers, E., et al., 1993. The full mutation in the FMR-1 gene of male Fragile X patientsis absent in their sperm. Nat. Genet. 4, 143–146.
Rifé, M., et al., 2004. Analysis of CGG variation through 642 meioses in Fragile X fami-lies. Mol. Hum. Reprod. 10, 773–776.
Schmucker, B., Seidel, J., 1999. Mosaicism for a full mutation and a normal size allele intwo Fragile X males. Am. J. Med. Genet. 84, 221–225.
Sharony, R., et al., 2012. Three peaks in the polymerase chain reaction Fragile X analy-sis. J. Med. Screen. 19, 112–115.
Sullivan, A.K., Crawford, D.C., Scott, E.H., Leslie, M.L., Sherman, S.L., 2002. Pater-nally transmitted FMR1 alleles are less stable than maternally transmitted al-leles in the common and intermediate size range. Am. J. Hum. Genet. 70,1532–1544.
Warren, S.T., Jin, P., 2000. Understanding the molecular basis of Fragile X syndrome.Hum. Mol. Genet. 9, 901–908.
Yrigollen, C.M., et al., 2012. AGG interruptions within the maternal FMR1 gene reducethe risk of offspring with Fragile X syndrome. Genet. Med. 14, 729–736.
![Retraction - Hindawi Publishing ?· MalariaResearchandTreatment majorcomplications[ ].ehaematologicalabnormalities…](https://static.documents.pub/doc/80x56/5b4f45237f8b9a2a6e8bf093/retraction-hindawi-publishing-malariaresearchandtreatment-majorcomplications.jpg)
