Detection of Cryptic Fragile X Full Mutation Alleles by Southern Blot In

G C A T T A C G G C A T genes

Case Report Detection of Cryptic Fragile X Full Mutation Alleles by Southern Blot in a Female and Her Foetal DNA via Chorionic Villus Sampling, Complicated by Mosaicism for 45,X0/46,XX/47,XXX

Alison Pandelache 1, David Francis 1, Ralph Oertel 1, Rebecca Dickson 2, Rani Sachdev 3, Ling Ling 4, Dinusha Gamage 4 and David E. Godler 4,5,*

1 Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC 3052, Australia; [email protected] (A.P.); [email protected] (D.F.); [email protected] (R.O.) 2 Maternal Fetal Medicine Department, Royal Hospital for Women, Randwick, NSW 2031, Australia; [email protected] 3 Centre for Clinical Genetics, Sydney Children’s Hospital, Randwick, NSW 2031, Australia; [email protected] 4 Diagnosis and Development, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC 3052, Australia; [email protected] (L.L.); [email protected] (D.G.) 5 Faculty of Medicine, Dentistry and Health Sciences, Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia * Correspondence: [email protected]; Tel.: +61-3-8341-6496 Citation: Pandelache, A.; Francis, D.; Oertel, R.; Dickson, R.; Sachdev, R.; Abstract: We describe a female with a 72 CGG FMR1 premutation (PM) (CGG 55–199) and family Ling, L.; Gamage, D.; Godler, D.E. history of fragile X syndrome (FXS), referred for prenatal testing. The proband had a high risk of Detection of Cryptic Fragile X Full having an affected pregnancy with a full mutation allele (FM) (CGG > 200), that causes FXS through Mutation Alleles by Southern Blot in hypermethylation of the FMR1 promoter. The CGG sizing analysis in this study used AmplideX a Female and Her Foetal DNA via triplet repeat primed polymerase chain reaction (TP-PCR) and long-range methylation sensitive PCR Chorionic Villus Sampling, Complicated by Mosaicism for (mPCR). These methods detected a 73 CGG PM allele in the proband’s blood, and a 164 CGG PM 45,X0/46,XX/47,XXX. Genes 2021, 12, allele in her male cultured chorionic villus sample (CVS). In contrast, the Southern blot analysis 798. https://doi.org/10.3390/ showed mosaicism for: (i) a PM (71 CGG) and an FM (285–768 CGG) in the proband’s blood, and (ii) a genes12060798 PM (165 CGG) and an FM (408–625 CGG) in the male CVS. The FMR1 methylation analysis, using an EpiTYPER system in the proband, showed levels in the range observed for mosaic Turner syndrome. Academic Editor: Giovanni Neri This was conﬁrmed by molecular and cytogenetic karyotyping, identifying 45,X0/46,XX/47,XXX lines. In conclusion, this case highlights the importance of Southern blot in pre- and postnatal testing Received: 8 April 2021 for presence of an FM, which was not detected using AmplideX TP-PCR or mPCR in the proband Accepted: 18 May 2021 and her CVS. Published: 24 May 2021

Keywords: fragile; X syndrome; FMR1; mosaicism; CGG; cultured chorionic villus; sample; southern Publisher’s Note: MDPI stays neutral blot; full mutation; 45,X0/46,XX/47,XXX with regard to jurisdictional claims in published maps and institutional afﬁl- iations. 1. Introduction Fragile X syndrome (FXS) is the most common single gene disorder associated with inherited disability and autism, affecting 1:4000 males and 1:5000–8000 females [1]. It is Copyright: © 2021 by the authors. usually caused by the cytosine-guanine-guanine (CGG) expansion ≥200 repeats referred to Licensee MDPI, Basel, Switzerland. as a full mutation (FM) in the 50UTR end of the FMR1 gene. This FM expansion usually This article is an open access article distributed under the terms and leads to an abnormal methylation of the FMR1 promoter that encompasses the CpG island 0 conditions of the Creative Commons located in the 5 of the CGG repeat, as well as FMR1 exon 1 and a portion of the FMR1 0 Attribution (CC BY) license (https:// intron 1, both located in the 3 of the CGG expansion [2]. Methylation of this locus is also creativecommons.org/licenses/by/ affected by X-inactivation in females [3] and has been associated with silencing of FMR1 4.0/). transcription and loss of fragile X mental retardation protein (FMRP), which is essential

Genes 2021, 12, 798. https://doi.org/10.3390/genes12060798 https://www.mdpi.com/journal/genes Genes 2021, 12, 798 2 of 9

for normal neurodevelopment [4,5]. The levels of methylation at this locus have also been associated with intellectual functioning in both males [6] and females [7,8] affected with FXS. Moreover, abnormal methylation of the FMR1 promoter is not restricted to FM alleles, with changes in DNA methylation associated with X-inactivation skewing in individuals affected with different types of sex chromosome aneuploidy [3], and in a proportion of males with idiopathic developmental delay with normal size (NS) (CGG < 45) or intermediate size (CGG 45–54) alleles [9]. The FMR1 alleles with CGG sizes between 55 and 199 repeats, termed premutation (PM), are considerably more common than FM, found in approximately one in 300 females and approximately one in 800 males, in the general population [10]. These alleles usually have a completely unmethylated FMR1 promoter. The PM alleles in females have also been associated with an increased risk of having a child affected with FXS, with this risk increases proportionally with an increase in the CGG size of the maternal PM allele [11]. Adenine-guanine-guanine (AGG) interruptions (found at the 50 end of the CGG expansion) have also been suggested to modify the chance of expansion [12]. This information is used to counsel couples requesting prenatal diagnosis of FXS [13,14]. Typically, prenatal testing involves sex determination by karyotype and ﬂuorescence in situ hybridization (FISH), and CGG sizing using routine polymerase chain reaction (PCR) and Southern blot analysis [15]. Triplet repeat primed PCR (TP-PCR) and long-range PCR, using commercial kits for FMR1 CGG screening and linkage techniques, have also been used in these settings [16]. In contrast, FMR1 methylation analysis on proband and/or foetal DNA is not typically performed as part of prenatal testing for FXS, despite being suggested to be informative in mosaic cases in these settings [17,18].

2. Clinical Report 2.1. Clinical History and Consent A twenty-six-year-old female with a family history of FXS was referred for prenatal testing due to a family history of FXS. Her initial results found a normal size allele (<44 CGGs) of 30 CGG repeats and a 73 CGG PM allele detected by AmplideX triplet repeat primed PCR (TP-PCR) sizing after cascade carrier testing. The proband’s maternal cousin’s three children were previously found to be carrying PM and FM alleles. The proband’s mother was identified to have a 37 CGG and 59 CGG PM as part of the cascade testing by AmplideX TP-PCR. The proband did not have any history of developmental delay and was neurotypical. The proband’s height was 152 cm on the 3rd centile and she reported that she had a “hole in the heart” at birth and a unilateral left solitary kidney after removal of her right polycystic kidney in childhood. She also reported being treated for hypothyroidism and oligomenorrhea and had difficulty conceiving. Initial prenatal testing involved TP-PCR on fetal DNA via chorionic villus sampling (CVS) at 12 weeks plus 2 days gestation, and identified a single 156 CGG PM with an atypical stutter pattern up to 170 CGG. This raised concern that the allele represented a biased PCR result with a possibility of mosaicism for FM alleles. The case was then reflexed for Southern blot analysis [19]. Verbal consent was obtained from the proband when her samples were submitted for the follow-up clinical testing at the Victorian Clinical Genetics Services for her anonymized clinical information and fragile X testing results to be used in this study. For these reasons, additional ethical approval was not required.

2.2. Sample Processing Maternal DNA was extracted from ethylenediaminetetraacetic acid (EDTA) preserved blood and DNA was extracted from a CVS from a local laboratory. Further proband testing of established phytohaemagglutinin (PHA) stimulated peripheral blood lymphocyte cultures was performed, with a harvest of metaphase chromosomes using a standard laboratory procedure, as previously described [20]. The Giemsa-banded analysis of 60 cells was examined to produce a conventional karyotype analysis. The molecular karyotype analysis was also performed, as previously described [18]. Genes 2021, 12, x FOR PEER REVIEW 3 of 10

lymphocyte cultures was performed, with a harvest of metaphase chromosomes using a standard laboratory procedure, as previously described [20]. The Giemsa-banded analysis of 60 cells was examined to produce a conventional karyotype analysis. The molecular karyotype analysis was also performed, as previously described [18].

2.3. FMR1 CGG Sizing Routine FMR1 testing involved first-line PCR-based assessment of CGG repeat size using a validated PCR assay, with the upper limit of detection of 170 CGGs in males and , , 798 3 of 9 Genes 2021 12 157 CGGs in females [15]. All DNA samples that showed a CGG size in the PM range, or failed to show a PCR product, were referred for second-line confirmatory testing by

Southern2.3. blotting, FMR1 CGG using Sizing a 520-bp probe segment (Pfxa3) of the 1-kb Pst 1 fragment and ps8 control,Routine as previouslyFMR1 testing described involved first-line [19]. PCR-basedTwo different assessment triplet of CGG primed repeat sizelong-range PCR commercialusing akits validated were PCR also assay, used with for the CGG upper limitsizing, of detection i.e., AmplideX of 170 CGGs TP in males-PCR and and AmplideX mPCR 157(Asuragen, CGGs in females Austin, [15 ].TX, All USA), DNA samples performed that showed as previously a CGG size by in the[21,22] PM range,, and on the same or failed to show a PCR product, were referred for second-line confirmatory testing by DNA samplesSouthern blotting,as those using analyzed a 520-bp by probe Southern segment blot. (Pfxa3) The of the AmplideX 1-kb Pst 1 fragmentTP-PCR and has a precision of ±2 repeatps8 control, error, as either previously classified described as [19 low]. Two (NS different-54 CGG) triplet or primed as increased long-range risk PCR (55–200 CGG or >200commercial CGG) determined kits were also using used for Gene CGG sizing,Mapper i.e., AmplideX5.0 (Thermo TP-PCR Fisher and AmplideX Scientific, Waltham, mPCR (Asuragen, Austin, TX, USA), performed as previously by [21,22], and on the same MA, USADNA), samplesas previously as those analyzed by [21] by. Southern blot. The AmplideX TP-PCR has a precision Confirmatoryof ±2 repeat error, testing either classified utilized as low a combination (NS-54 CGG) or as of increased standard risk (55–200 CGG CGGsizing PCR [15], AmplideXor >200 based CGG) analysis, determined and using Southern Gene Mapper blot, 5.0 (Thermoto exclude Fisher the Scientific, presence Waltham, of FM MA, alleles in the CVS sample.USA), as CGG previously sizing by [21using]. AmplideX TP-PCR for the proband identified a 30 and a Confirmatory testing utilized a combination of standard CGG sizing PCR [15], Am- 72 CGGplideX PM basedallele analysis, (II:2), andand Southern a 30 CGG blot, to and exclude a 164 the presenceCGG PM of FM allele alleles in in the the CVSmale CVS (III:3) (Figuresample. 1). The CGG standard sizing using CGG AmplideX sizing TP-PCR PCR for[15] the detected proband identified a 30 CGG a 30 and and a 72 a CGG74 CGG allele in the probandPM allele (II:2) (II:2), whilst and a 30 it CGG had and failed a 164 amplification CGG PM allele in for the the male CVS CVS (III:3)sample (Figure (III:3).1). The standard CGG sizing PCR [15] detected a 30 CGG and a 74 CGG allele in the proband (II:2) whilst it had failed amplification for the CVS sample (III:3).

0 100 200 300 400 500 600 700 800 900 1000 A. B. 10,000 8000 II:2 I:1 I:2 [37 / 59 CGGs] 30 6000

4000 (FU)

2000 72

0 Units

0 100 200 300 400 500 600 700 800 900 1000 2000

II:1 [30 CGGs] II:2 [30 / 72-75 CGGs] 1600 III:3

1200 Fluoresce 600

400 30 164 0 III:1 M/C 6/40 III:2 [30 / 30 CGGs] III:3 CVS [30 / 164-171 CGGs] Fragment size (bp).

100 300 500 700 900 1100 1300 1500 C. 100 300 500 700 900 1100 1300 1500 D. 3600 Control digestion 20,000 Control digestion III:3 II:2 2800 16,000

12,000 (FU) 2000 (FU) 8000 1200 4000

400 Units

Units 0 0

100 300 500 700 900 1100 1300 1500 100 300 500 700 900 1100 1300 1500 HPAII digestion 3600 HPAII digestion 20,000 II:2 III:3 16,000 2800

12,000 2000

Fluoresce Fluoresce 8000 1200 4000 400 0 0 Fragment size (bp). Fragment size (bp).

Figure 1. (A) Pedigree of the studied family with CGG sizing determined using an AmplideX TP-PCR. Males are represented Figure 1. (A) Pedigree of the studied family with CGG sizing determined using an AmplideX TP-PCR. Males are by squares, females by circles and triangle sex unknown. A black outline indicates examined and half shaded indicates represented bycarrier. squares, A black females arrow indicates by circles proband. and III:1 triangle Miscarriage sex atunknown. 6 of 40 weeks; A (bBlack) AmplideX outline TP-PCR indicates CGG sizing, examined with no FMand half shaded indicates carrier.alleles A detectedblack arrow in either indicates III:3 male CVS proband. or II:2 proband; III:1 Miscarriage (C) II:2 and (D) III:3at 6 AmplideX of 40 weeks; mPCR, ( withB) A themplideX upper panel TP in-PCR blue CGG sizing, with no FM allelesrepresenting detected CGG in sizing either based III:3 on control male digestionsCVS or II:2 and p theroband; lower panel (C) in II:2 green and representing (D) III:3 relativeAmplideX methylation mPCR, based with the upper panel in blue onrepresentingHpaII methylation CGG sensitive sizing restriction based enzyme-based on control digestion digestions followed and by the long-range lower PCR. panel No FMin allelesgreen are representin detected g relative methylation basedin either on III:3 HpaII male CVSmethylation or II:2 female. sensitive Skewed methylationrestriction for enzyme the 75 CGG-based PM allele digestion is suggested followed by 80% by methylation. long-range PCR. No Note: numbers on each panel next to speciﬁc peaks indicate CGG sizes. FM alleles are detected in either III:3 male CVS or II:2 female. Skewed methylation for the 75 CGG PM allele is suggested by 80% methylation. Note: numbers on each panel next to specific peaks indicate CGG sizes.

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The 30 CGG allele in the male CVS is likely a result of maternal cell contamination, The 30 CGG allele in the male CVS is likely a result of maternal cell contamination, as it is the same size as the normal size allele in the mother (Figure 1). Interestingly, its as it is the same size as the normal size allele in the mother (Figure1). Interestingly, its methylation is different in CVS (32%) as compared with the mother’s normal size allele methylation is different in CVS (32%) as compared with the mother’s normal size allele (18%) (Figure 1C,D). One potential explanation could be that, by chance, the maternal cell (18%) (Figure1C,D). One potential explanation could be that, by chance, the maternal cell contamination involved a greater proportion of cells that had the normal size allele on the contamination involved a greater proportion of cells that had the normal size allele on the inactive X chromosome as compared with maternal blood. Another explanation may be inactive X chromosome as compared with maternal blood. Another explanation may be related to technicaltechnical limitslimits ofof thethe mPCR mPCR assay assay used used for for quantitative quantitative methylation methylation analysis analysis in inasmall a small proportion proportion of of cells cells associated associated with with maternal maternal contamination. contamination. Importantly,Importantly, no FM alleles were detected by AmplideX TP-PCRTP-PCR in both the proband and the CVS. In contrast, FM allele smears were detected in both the proband and male CVS using Southern blot ( (FigureFigure2 2B).B). OnOn thethe basisbasis ofof thethe SouthernSouthern blotblot results,results, thethe probandproband chose to terminate terminate the the pregnancy. pregnancy. Unfortunately, tissues from the terminated foetus were not available to confirm conﬁrm these findings. ﬁndings.

A. B. 1 2 3 4

I:1 I:2 [37 / 59 CGGs] 768 to 625 285 to 408 II:1 [30 CGGs] II:2 [30 / 74 / 285-768 CGGs]

159 C. 82 72

III:3 CVS 30 III:1 M/C 6/40 III:2 [30 / 30 CGGs] [159 / 408-625 CGGs] III:3 II:2

Figure 2. (A) Pedigree of the studied family with CGG sizing determined using Southern blot and Figure 2. (A) Pedigree of the studied family with CGG sizing determined using Southern blot and standard CGG sizing PCR. Males are represented by squares, females by circles and triangle sex standard CGG sizing PCR. Males are represented by squares, females by circles and triangle sex unknown. The The black black outline outline indicates indicates examined, examined, half half shaded shaded indicates indicates carrier, carrier, and andfull shaded full shaded indicatesindicates affected affected with with FXS. FXS. The b blacklack arrows arrows indicate indicate proband proband and and a diagonal line indi indicatescates being being deceased. III:1 Miscarriage at 6 of 40 weeks (B)) Southern blotblot LaneLane 11 (male(male PMPM control),control), Lane Lane 22 (III:3 (maleIII:3 male PM/FM PM/FM CVS), CVS Lane), Lane 3 (II:2 3 PM/FM(II:2 PM/FM female), female Lane), Lane 4 (male 4 (male normal normal CGG CGG size size control). control).

2.4. FMR1 FMR1 Methylation Analysis Analysis The FMR1 methylation testi testingng was performed for both CVS (III:3) and proband proband (II:2) usingusing the the EpiTYPER EpiTYPER system system targeting targeting 12 12 CpG CpG sites sites within within the the fragile X related epigenetic element 2 (FREE2) (located at the FMR1 exon 1/intron 1 1 boundary) boundary) [23] [23],, and and the the AmplideX AmplideX mPCR commercial kit targeting two restriction sites on either side of the CGG repeat mPCR commercial kit targeting twoHpaII HpaII restriction sites on either side of the CGG (one within the CpG island and the other within FMR1 exon 1) [21]. For the CVS at 12 repeat (one within the CpG island and the other within FMR1 exon 1) [21]. For the CVS at weeks of gestations, these analyses were performed after DNA methylation had been 12 weeks of gestations, these analyses were performed after DNA methylation had been establishment (reported to occur at 11 weeks of gestation for the FMR1 promoter in a establishment (reported to occur at 11 weeks of gestation for the FMR1 promoter in a human foetus) [24]. AmplideX mPCR showed that, in the proband HpaII sites, both the 30 human foetus) [24]. AmplideX mPCR showed that, in the proband HpaII sites, both the and 171 CGG alleles were approximately 30% methylated. The proband’s X-inactivation 30 and 171 CGG alleles were approximately 30% methylated. The proband’s X- skewing was observed with the 74 CGG allele being 80% methylated, whilst the 30 CGG inactivation skewing was observed with the 74 CGG allele being 80% methylated, whilst allele was only 18% methylated. The AmplideX mPCR assay, however, did not detect an the 30 CGG allele was only 18% methylated. The AmplideX mPCR assay, however, did FM allele in either the proband (11:2) or CVS DNA (III:3) (Figure1C). not detect an FM allele in either the proband (11:2) or CVS DNA (III:3) (Figure 1C). The EpiTYPER methylation results in the CVS sample across 12 FREE2 CpGsites approachedThe EpiTYPER 0%; while methylation for the proband results FREE2 in the methylation CVS sample approached across 12 10%. FREE2 This CpG wassites an approachedunexpected ﬁnding0%; while for for the the female proband proband, FREE2 as thismethylation was signiﬁcantly approached below 10%. the This methylation was an unexpectedlevels typically finding observed for for the the female same CpG proband, sites in as females this was with significantly NS, PM, and below FM alleles the

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(Table1). These methylation levels were, however, within the range described in females with mosaic Turner syndrome (24).

Table 1. Comparisons of the CpG10-12 FREE2 methylation for the male CVS and proband blood with the sex chromosome aneuploidy, typically developing female and male reference cohorts from earlier studies [3,17,25].

Group Tissue N CGG Size CMA d Meth. % MAX % MIN % 46,XY Blood 14 <40 2 (±4) 4 0 controls a 46,XX b Blood 35 <40 27 (±10) 38 16 control 47,XXX c Blood 8 N/A 47,XXX 43 (±8) 47 38 45,Xo c Blood 11 <40 45,X 1 (±3) 4 1 159, (III:3) CVS 1 46,XY 3 408–625 30, 72, 45,X/46,XX/ (II:2) Blood 1 8 285–768 47,XXX Note: Methylation % for reference samples is expressed as mean (±2 standard deviations). a Convenience sample of consenting typically developing males. b De-identiﬁed sample of females recruited in a population FXS carrier screening study. c De-identiﬁed sample taken as part of fragile X cascade testing and routine molecular microarray testing/karyotyping as part of previous studies. d Chromosomal microarray-based molecular karyotyping and standard karyotyping for III:3.

2.5. Follow-Up Conventional Cytogenetic and Molecular Karyotype Analysis for II:2 A conventional and molecular karyotype was requested by the referring doctor (Table1, Figures3 and4). A conventional cytogenetic karyotype analysis was performed using Giemsa/Trypsin/Leishman stain banding (G-banding) to produce visible staining of condensed metaphase chromosomes from venous blood. G-banding identified three different karyotypes in the proband including mosaicism for X0/XX/XXX cell lines, a female karyotype with sex chromosome mosaicism. Among the 60 cells examined, 38 cells (approximately 63%) showed a 45,X0 cell line, 19 cells (approximately 32%) showed a 47,XXX cell line, and the remaining three cells (approximately 5%) showed a normal female (46,XX) karyotype (Figure3). The proband’s short stature, history of congenital heart disease, renal anomaly, hypothyroidism, and oligomenorrhea are clinically consistent with a 45,X0 phenotype. The molecular karyotype analysis of the whole genome was performed using chromosomal microarray (Figure4). Single nucleotide polymorphisms (SPN) duogenotype comparisons were performed for the whole genome between the proband mother and foetus and showed sharing of one allele (identity by state 1), in over 97,600 informative probes used. This confirmed a parent-child relationship. Comparisons from chromosome 1 are shown in Figure4 as a representation. The SNP duo genotype comparison between the mother’s 45,X cell line and the foetus’s abnormal X chromosomes were manually modified and the process of deduction confirmed that the abnormal X was found in the maternal 45,X0 cell line showing identity by state equaling zero. The father’s DNA sample was not available, and thus was not used in these analyses. Considering that the previously reported methylation of FREE2 for XXX females was ~43%, for X0 was 0%, and for XX was ~27% (Table1), by combining these figures with proportions of each cell line observed in the blood of the female, from karyotyping, FREE2 methylation would equate to approximately 15% ((XXX: 43% methylation times 0.32) + (XX: 27% M times 0.05) + (X0: 0% methylation times 0.63)). This is consistent with the molecular karyotype analysis (Figure4) and FREE2 methylation results observed for the proband (Table1), providing a likely explanation for why FREE2 was hypomethylated in this female. Genes 2021, 12, x FOR PEER REVIEW 6 of 10

Genes 2021, 12, 798 proband (Table 1), providing a likely explanation for why FREE2 was hypomethylated6 of 9 in this female.

A. B.

(45,X 63% of cells) (46,XX 5% of cells) C. D.

Mitotic non-disjunction Mitotic disjunction of X chromosomes of X chromosomes

47,XXX 45,X 46,XX [19/60 cells] [38/60 cells] [3/60 cells] (47,XXX 32% of cells)

FigureFigure 3. 3. GG banded chromosomechromosome analysis analysis of II:2of II:2 proband proband blood blood and conventionaland conventional karyotypes karyotypes identify mosaic cell lines. ( ) 45,X0 was found in 38/60 karyotypes analysed; ( ) 47,XXX was found in 19/60 GenesGenes 20212021, 12,,12 x ,FOR 0 PEER REVIEW identify mosaic cellA lines. (A) 45,X0 was found in 38/60 karyotypesB analysed; (B) 47,XXX was7 of 7 10 of 10

foundkaryotypes in 19/60 analysed; karyotypes (C) 46,XX analysed; was found (C) 46,XX in 3/60 was karyotypes found in analysed. 3/60 karyotypes (D) Diagram analysed. of post ( zygoticD) Diagramnondisjunction of post and zygotic generation nondisjunction of the mosaic and cell generation line 45,X[38]/47,XXX[19]1/46,XX[3]3. of the mosaic cell line 45,X[38]/47,XXX[19]1/46,XX[3]3.

A. 41,992 7992 1 A_Cultured cell CVS 24,672 8184 17,091 0 B_Proband Mother 24,691 8098 17,176 0

FigureB. 4. 1Cont. 21,984 8 0 6437 A_Cultured cell CVS B 15,473 0 A 12,956 0 B_Proband Mother B 15,353 8 A 13,068 0

FigureFigure 4. Linkage 4. Linkage analysis analysis using using chromosomal chromosomal microarray. microarray. (A) (SingleA) Single nucleotide nucleotide polymorphisms polymorphisms (SNP)(SNP) duo duo image image for forchromosome chromosome 1 showing 1 showing identity identity by state by state(sharing (sharing one allele) one allele) for the for whole the whole chromosome.chromosome. Whole Whole genome genome comparison comparison confirmed conﬁrmed child child-parent-parent relationship. relationship. Note: Note: Chromosome Chromosome 1 1 SNPduoSNPduo Output Output A_Culture A_Culture CVS CVS–B_Proband–B_Proband Mother Mother Average Average Identity Identity by by State State (IBS):1.84; (IBS):1.84; (B ()B ) SNP SNP duo for chromosome X between the foetus and the X0 cell line showing IBS0, that is sharing duo for chromosome X between the foetus and the X0 cell line showing IBS0, that is sharing no no alleles for most of the chromosome including the FMR1 gene. Note: Chromosome X SNPduo alleles for most of the chromosome including the FMR1 gene. Note: Chromosome X SNPduo Output Output A_Cultured CVS–B_Proband Mother Average IBS:1.1.547. A_Cultured CVS–B_Proband Mother Average IBS:1.1.547.

3. Conclusions3. Conclusions ThisThis study study describes describes a female a femaleprobandproband ref referrederred for for prenatal prenatal testing testing due due to toa family a family historyhistory of ofFXS FXS with with 45,X0/46,XX/47,XXX 45,X0/46,XX/47,XXX cell cellline lines,s, and and mosaic mosaicismism for forPM PM and and FM FM alleles alleles.. AlthoughAlthough previous previous studies studies have have reported reported 45,X0/46,XX 45,X0/46,XX mosaicism mosaicism in inFM FM females females [26] [26 and] and havehave suggested suggested that that it itmay may be be much much more more common thanthan expectedexpected in in females females with with a FMa FM [27 ], [27]until, until now, now, these these cases cases have have not not been been described described in prenatalin prenatal settings. settings. Moreover, Moreover, it has it has been beensuggested suggested that that the the presence presence of an of FMan FM on anon X an chromosome X chromosome may may predispose predispose it to lossit to duringloss duringmitosis, mitosis, and that and this that may this occur may due occur to structural due to structural changes of changes the metaphase of the metaphase chromosome chromosomewith an FM with [26– 28an]. FM [26,27,28]. MosaicismMosaicism for for 45,X0/46,XX 45,X0/46,XX /47,XXX /47,XXX in the in the proband proband described described in inthis this report report has has implicationsimplications for for future future genetic genetic counselling counselling regarding regarding reproductive reproductive options options and and disease disease risksrisks observ observeded in inmosaic mosaic Turner Turner syndrome. syndrome. It is It important is important to tonote note that that it was it was initially initially

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A. 41,992 7992 1 A_Cultured cell CVS 24,672 8184 17,091 0 B_Proband Mother 24,691 8098 17,176 0

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B. 1 21,984 8 0 6437 A_Cultured cell CVS B 15,473 0 A 12,956 0 B_Proband Mother B 15,353 8 A 13,068 0

FigureFigureFigure 4. Linkage 4. Linkage analysis analysis analysis using using using chromosomal chromosomal chromosomal microarray. microarray. microarray. (A (A) ) (Single SingleA) Single nucleotide nucleotide nucleotide polymorphisms polymorphisms (SNP)(SNP)(SNP) duo duo image image for for forchromosome chromosome chromosome 1 showing 1 1 showing showing identity identity identity by by state by state state(sharing (sharing (sharing one one allele) one allele) allele) for for the the for whole whole the whole chromosome.chromosome.chromosome. Whole Whole Whole genome genome genome comparison comparison comparison confirmed confirmed confirmed child-parentchild child-parent-parent relationship. relationship. relationship. Note: Note: Note: Chromosome Chromosome Chromosome 1 1 1 SNPduoSNPduoSNPduo Output Output A_Culture A_Culture A_Culture CVS CVS–B_Proband CVS–B_Proband–B_Proband MotherMother Mother Average Average Average Identity Identity Identity by by State by State State (IBS):1.84; (IBS):1.84; (IBS):1.84; (B) ( SNPB ()B ) SNP SNP duo for chromosome X between the foetus and the X0 cell line showing IBS0, that is sharing duoduo for chromosome X X between between the the foetus foetus and and the the X0 X0 cell cell line line showing showing IBS0, IBS0, that that is sharing is sharing no no no alleles forfor mostmost of of the the chromosome chromosome including including the theFMR1 FMRgene.1 gene. Note: Note: Chromosome Chromosome X SNPduo X SNPduo Output alleles for most of the chromosome including the FMR1 gene. Note: Chromosome X SNPduo Output OutputA_Cultured A_Cultured CVS–B_Proband CVS–B_Proband Mother Mother Average Average IBS:1.1.547. IBS:1.1.547. A_Cultured CVS–B_Proband Mother Average IBS:1.1.547. 3. Conclusions 3. Conclusions3. Conclusions This study describes a femaleproband referred for prenatal testing due to a family ThisThis study study describes describes a female a femaleprobandproband ref referrederred for for prenatal prenatal testing testing due due to toa family a family historyhistory of ofFXS FXS with with 45,X0/46,XX/47,XXX 45,X0/46,XX/47,XXX cell cell line lines,s, and and mosaic mosaicismism forfor PMPM and and FM FM alleles. alleles. historyAlthough of previousFXS with studies 45,X0/46,XX/47,XXX have reported 45,X0/46,XX cell lines, and mosaicism mosaicism in FM for females PM and [26 FM] and alleles. Although previous studies have reported 45,X0/46,XX mosaicism in FM females [26] and Althoughhave suggested previous that it studies may be have much reported more common 45,X0/46,XX than expected mosaicism in females in FM with females a FM [ [2726],] and havehaveuntil suggested suggested now, these that that cases it itmay have may be notbe much much been more described more common in prenatal thanthan expectedexpected settings. Moreover,in in females females it with with has a been FMa FM [27 ], [27]untilsuggested, until now, now, that these these the cases presence cases have have of not an not FMbeen been on described an described X chromosome in prenatalin prenatal may settings. predispose settings. Moreover, itMoreover, to loss during it has it has been beensuggestedmitosis, suggested and that that that the this the presence may presence occur of an of due FMan to FM on structural anon X an chromosome X changes chromosome of may the metaphasemay predispose predispose chromosome it to lossit to duringloss duringmitosis,with anmitosis, FM and [26 that and–28 this]. that may this occur may due occur to structural due to structural changes of changes the metaphase of the metaphase chromosome chromosomewithMosaicism an FM with [26– for28an]. FM 45,X0/46,XX [26,27,28] /47,XXX. in the proband described in this report has implicationsMosaicismMosaicism for for future for 45,X0/46,XX 45,X0/46,XX genetic counselling /47,XXX /47,XXX in regarding the in the proband proband reproductive described described options in inthis and this report disease report has has implicationsimplicationsrisks observed for for future in future mosaic genetic genetic Turner counselling counselling syndrome. regarding It regarding is important reproductive reproductive to note thatoptions options it was and andinitially disease disease risksrisksflagged observ observed ined this in study inmosaic mosaic through Turner Turner skewed syndrome. syndrome. X-inactivation It is It important is important patterns to using tonote note FREE2 that that it methylation was it was initially initially testing, not typically performed in prenatal settings. This mosaicism was subsequently confirmed by microarray and karyotype analyses performed to identify the origins of this skewed X-inactivation. Conversely, mosaicism for PM and FM alleles found in the

proband’s blood, and her male CVS have implications for both counselling related to FXS, as well as for future diagnostic testing. Speciﬁcally, for the male prenatal case, CVS and the proband mother FM alleles were missed by two independent commercial assays, AmplideX PT-PCR and AmplideX mPCR that are often used for prenatal and diagnostic testing for FXS [16]. This FM allele was detected in both samples using Southern blot analysis. Such a discordance may be explained by either or both primer sites for AmplideX TP-PCR and AmplideX mPCR assays being lost in this mosaic female and her CVS. Although the exact mechanism for this is unknown, this explanation is consistent with similar observations previously reported in a mosaic FXS male in post-natal settings, where one of the primer sites was lost due to a microdeletion proximal to the CGG expansion [29]. Altogether, these cases highlight the importance of Southern blot testing in prenatal and postnatal diagnostic testing for FXS. Genes 2021, 12, 798 8 of 9

Author Contributions: R.D. and R.S. clinically assessed all participants in this study and were involved in the follow-up, including genetic counselling; A.P., D.F., R.O., L.L., D.G. and D.E.G. performed the molecular analyses and were involved in providing results interpretation related to the clinical observations; A.P. and D.E.G. prepared the drafts of the paper. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Victorian Government’s Operational Infrastructure Support Program, NHMRC (no. 1,049,299 and no. 1,103,389 (D.E.G).); the Royal Children’s Hospital Foundation (D.E.G.); the Medical Research Future Fund (MRF1141334 (D.E.G.)); and the the Financial Markets Foundation for Children (no. 2017–361 (D.E.G.)). Informed Consent Statement: Verbal consent was obtained from the proband when her samples were submitted for the follow-up clinical testing at the Victorian Clinical Genetics Services for her anonymized clinical information and fragile X testing results to be used in this study. For these reasons additional ethical approval was not required. Data Availability Statement: The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Acknowledgments: The authors would like to thank all the study participants for being involved in the study. We would also like to thank the various departments at Victorian Clinical Genetic Services, Cytogenetics laboratory for performing conventional karyotyping; the Cyto-molecular team for performing standard PCR, Southern blot, and molecular karyotype analysis; and the RGCS laboratory for performing confirmatory testing of FXS participants using the AmplideX FMR1 TP-PCR screening assay. We would also like to thank Solange Aliaga from the Diagnosis and Development Laboratory (MCRI) for performing AmplideX mPCR and EpiTYPER based testing. Conflicts of Interest: David E. Godler is named as an inventor on patent applications (PCT/AU2010/ 000169 and PCT/AU2014/00004) related to the technology described in this article. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

FXS Fragile X Syndrome FMR1 Fragile X mental retardation 1 FMRP Fragile X mental retardation protein CGG Cytosine-guanine-guanine AGG Adenine-guanine-guanine PCR Polymerase chain reaction CVS Chorionic villus sampling FISH Fluorescence in situ hybridization PM Premutation FM Full mutation SNP Single nucleotide polymorphism TP-PCR Triplet repeat primed PCR

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