A Pseudo-Full Mutation Identified in Fragile X Assay Reveals a Novel Base Change Abolishing an EcoRI Restriction Site
2008; Elsevier BV; Volume: 10; Issue: 5 Linguagem: Inglês
10.2353/jmoldx.2008.080059
ISSN1943-7811
AutoresShujian Liang, Harold N. Bass, Hanlin Gao, Caroline Astbury, Mehdi R. Jamehdor, Yong Qu,
Tópico(s)Genomic variations and chromosomal abnormalities
ResumoDiagnostic testing for the fragile X syndrome is designed to detect the most common mutation, a CGG expansion in the 5′-untranslated region of the fragile X mental retardation (FMRI) gene. PCR can determine the number of CGG repeats less than 100, whereas Southern analysis can detect large premutations, full mutations, and their methylation status. Bands larger than 5.8 kb observed via Southern analysis are usually considered a methylated full mutation, causing fragile X syndrome in males and varied clinical presentations in females. We observed a 10.9-kb band on a Southern blot assay from an autistic girl with language delay. Further investigation identified a novel G-to-A transition at an EcoRI cleavage site, upstream of the CGG repeat region of the FMRI gene. This base change abolished the EcoRI restriction site, resulting in a 10.9-kb pseudo-full mutation. This G-to-A base change has not been previously reported and was not identified in a subsequent analysis of 105 male and 30 female patient samples. The clear 10.9-kb band detected on a Southern blot assay for fragile X syndrome mimics a large, methylated full mutation, which could result in a misdiagnosis without the benefit of family studies and further testing. Diagnostic testing for the fragile X syndrome is designed to detect the most common mutation, a CGG expansion in the 5′-untranslated region of the fragile X mental retardation (FMRI) gene. PCR can determine the number of CGG repeats less than 100, whereas Southern analysis can detect large premutations, full mutations, and their methylation status. Bands larger than 5.8 kb observed via Southern analysis are usually considered a methylated full mutation, causing fragile X syndrome in males and varied clinical presentations in females. We observed a 10.9-kb band on a Southern blot assay from an autistic girl with language delay. Further investigation identified a novel G-to-A transition at an EcoRI cleavage site, upstream of the CGG repeat region of the FMRI gene. This base change abolished the EcoRI restriction site, resulting in a 10.9-kb pseudo-full mutation. This G-to-A base change has not been previously reported and was not identified in a subsequent analysis of 105 male and 30 female patient samples. The clear 10.9-kb band detected on a Southern blot assay for fragile X syndrome mimics a large, methylated full mutation, which could result in a misdiagnosis without the benefit of family studies and further testing. The fragile X syndrome is the most common inherited disorder associated with mental retardation, with an estimated prevalence of 1 in 4000 in males and 1 in 8000 in females. The syndrome in males is characterized by typical facial features including large ears, speech delay, autistic behavior, macroorchidism, and mental retardation. Females with a full mutation exhibit a wide spectrum of phenotypes, ranging from no detectable learning or behavioral deficits to mild learning disabilities to effects as severe as those in a male fragile X patient with a full mutation.1Sherman S Pletcher BA Driscoll DA Fragile X syndrome: diagnostic and carrier testing.Genet Med. 2005; 7: 584-587Crossref PubMed Scopus (223) Google Scholar Although mutations, such as deletions and point mutations, have been identified in fragile X patients, more than 99% of cases have been associated with an expansion of a segment of CGG repeats in the 5′-untranslated region (UTR) of the fragile X mental retardation (FMR1) gene. According to the Technical Standards and Guidelines for Fragile X Testing from the ACMG (http://www.acmg.net/Pages/ACMG-Activities/stds-2002/fx.htm, last accessed April 3, 2008), there are four allelic forms of the CGG repeat lengths: normal (5 to 44 repeats), intermediate (gray zone, 45 to 54 repeats), premutation (55 to 200 repeats), and full mutation (>200 to 230 repeats). Full mutations with a large CGG expansion in this region are associated with inhibition of transcription of the FMR1 gene, causing deficiency or absence of the fragile X mental retardation protein. Therefore, standard laboratory techniques for fragile X syndrome diagnosis are designed to quantitate the number of CGG repeats in the 5′-UTR of the FMR1 gene. These techniques include a PCR, which detects normal and premutation alleles ( 200 CGG repeats) alleles and their methylation status. In the Southern blot assay for fragile X syndrome, a normal male usually shows a 2.8-kb band, and a normal female, 2.8- and 5.2-kb bands. In females, a 2.8-kb band represents the active, unmethylated X chromosome, whereas a 5.2-kb band represents the inactive, methylated X chromosome. Methylated full mutations are not detectable by PCR and are often shown as bands larger than 5.8 kb on a Southern blot. Here, we report a 10.9-kb band identified with a Southern blot assay for fragile X syndrome from an autistic girl with language delay. However, this large band does not represent a methylated full mutation and is not associated with an expanded CGG repeat in the 5′-UTR in the FMR1 gene.Materials and MethodsCase ReportsA 7-year-old girl was evaluated for autistic features and language delay that became evident by age 2 years. The child was the product of a full-term pregnancy to a healthy 20-year-old primigravida and her 22-year-old partner. The mother was of Mexican ancestry, and the father, Salvadoran. There was no parental consanguinity and no first- or second-degree relatives of the proband with congenital anomalies or developmental delay. Both parents are physically and cognitively normal. The child had no siblings. Delivery took place by primary cesarean section for fetal heart rate decelerations. Birth weight was 3.2 kg, and length, 53 cm. The neonatal course was uneventful.By age 2 years, delays in socialization and in expressive and receptive language led to a diagnosis of classical autism. The child has remained in good general health, with no seizures, hospitalizations, regression of milestones, or other serious health problems.The child weighed 39 kg (98th percentile), was 132 cm tall (80th percentile), and had a 52.5-cm head circumference (75th percentile). Ear length of 6 cm was between the 50th and 75th percentiles. The patient exhibited no dysmorphism, and she was physically normal.High-resolution chromosome analysis showed a normal female karyotype (46, XX). Molecular cytogenetic analysis using an LSI Prader-Willi/Angelman region probe (GABRB3; Abbott Laboratories, Des Plaines, IL) showed no deletion or duplication in 10 metaphase and 40 interphase cells. This analysis excluded the possibility that her autistic feature was due to a 15q duplication.2Bundey S Hardy C Vickers S Kilpatrick MW Corbett JA Duplication of the 15q11-13 region in a patient with autism, epilepsy and ataxia.Dev Med Child Neurol. 1994; 36: 736-742Crossref PubMed Scopus (127) Google ScholarStandard Molecular Diagnostic Testing for Fragile XMolecular diagnostic testing was performed using both PCR and Southern blot analysis to determine the CGG repeat size at the 5′-UTR of the FMR1 gene and its methylation status. Blood was drawn into EDTA tubes from the index patient, parents, and normal controls. DNA was isolated with the Puregene DNA isolation procedure (Qiagen, Valencia, CA). The primer set for PCR was from Fu et al,3Fu YH Kuhl DPA Pizzuti A Pieretti M Sutcliffe JS Richards S Verkerk AJMH Holden JJA Fenwick Jr, RG 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.Cell. 1991; 67: 1047-1058Abstract Full Text PDF PubMed Scopus (1747) Google Scholar with a forward primer (5′-GACGGAGGCGCCGCTGCCAGG-3′) and a reverse primer (5′-GTGGGCTGCGGGCGCTCGAGG-3′) bracketing the CGG repeat region. Genomic DNA was amplified using the FailSafe PCR System (FSP995J premix) from Epicenter Biotechnologies (Madison, WI) according to the manufacturer's instructions. The reaction was first denatured for 10 minutes at 94°C, followed by 30 cycles of 20 seconds at 98°C and 3 minutes at 72°C. A final extension of 10 minutes was performed at 72°C. The PCR products were fractionated on a 2% agarose gel and detected with ethidium bromide using UV illumination (Quantity One; Bio-Rad, Hercules, CA). For the standard Southern analysis, 6 μg of genomic DNA was double-digested with NruI (a methylation-sensitive enzyme, cutting only unmethylated, active X chromosome) and EcoRI (100 U of each enzyme at 37°C for 6 hours), electrophoresed on a 0.7% agarose gel, transferred to a nylon membrane, and hybridized with digoxiginin-labeled probe pFXa1NHE (Chemicon International, Temecula, CA). This probe hybridizes to the region from a NheI site to an EcoRI site (GenBank accession no. L29074.1; Figure 1). After hybridization, the gel was visualized by immuno-chemiluminescence using a Sure Blot Chem Hybridization and Detection kit (Chemicon International) according to the manufacturer's instructions.Determination of the Origin of the 10.9-kb BandA 10.9-kb band was detected in the proband but not in her parents on the standard Southern blot assay as described above. Therefore, the following investigation was pursued to determine the origin of the 10.9-kb band.Modified Southern Blot AnalysisSix micrograms of genomic DNA from the patient, parents, and controls was digested with PstI only, EcoRI only, and EcoRI/NruI (100 U of the enzyme at 37°C for 6 hours). Figure 1 shows the restriction map surrounding the FMR1 gene and the predicted fragments after the digestions. The digested products were subjected to Southern blot analysis as described above.PCR Analysis of the EcoRI SitePCR primers were designed to bracket the region of the EcoRI site at the 11,114 position 5′ of the FMR1 gene (GenBank accession no. L29074.1; Figure 1). These PCR primers are: special forward primer (SF), 5′-TAGGCTTGAGCAACGAACTG-3′, and special reverse primer (SR), 5′-TGTGTCCCTGGCACATTAAA-3′, resulting in a 300-bp PCR product (from nucleotide position 10,993 to 11,292). The PCR reaction was performed using 200 ng of genomic DNA under the following conditions: 1 cycle at 95°C for 10 minutes, 35 cycles at 96°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute, with a final extension of 72°C for 10 minutes. PCR products were digested with 20 U of EcoRI, incubated at 37°C for 2 hours, separated on a 2.5% agarose gel, and visualized with ethidium bromide using UV illumination as described above. This test has been performed on DNA collected from 105 male and 30 female patient samples to determine the allele frequency.Sequence AnalysisThe PCR primers mentioned above (SF and SR) were also used for sequencing. The PCR products were purified using a DNA Clean & Concentrator kit (Zymo Research, Orange, CA) and sent to the DNA sequencing laboratory (City of Hope National Medical Center and Beckman Research Institute, Duarte, CA). The sequencing was done by capillary electrophoresis using Big Dye fluorescent sequencing reagents and a Hitachi AB model 3730 capillary DNA analyzer. The data were collected with AB DNA Sequencing Collection software V 3.0 and analyzed by Analysis software V 5.3.1.ResultsStandard Molecular Testing ResultsThe blood specimen from the index patient was subjected to fragile X testing due to her autistic features and language delay. Standard PCR analysis revealed two normal alleles with 23 of 32 CGG repeats from the proband, 18 of 23 CGG repeats from the mother, and a normal allele with 32 CGG repeats from the father (data not shown). Standard Southern blot analysis (EcoRI/NruI double digestion) showed that the index patient had a 10.9-kb band on the Southern blot, in addition to the normal 2.8- and 5.2-kb bands (Figure 2, lane 2). However, there were normal 2.8- and 5.2-kb bands for the mother (Figure 2, lane 5) and a normal 2.8-kb band for the father (Figure 2, lane 9).Figure 2Standard Southern blot of the family. Lanes 2, 5, and 9 are from the index patient (IP), mother (M), and father (F), respectively. Lanes 10 and 11 are the normal (NC) and abnormal controls (AC; Coriell GM07537A). Other lanes represent results from the same batch run from other patients. MW, the Lambda/HindIII MW marker (Chemicon International).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Modified Southern AnalysesSouthern blot using PstI digestion only was performed to search for or rule out the presence of a CGG expansion in the proband (Figure 3A). The index patient (Figure 3A, lane 2), her mother (Figure 3A, lane 3), and her father (Figure 3A, lane 4) all showed the predicted, normal-sized bands approximately 1.1 kb in size, same as the normal control (Figure 3A, lane 5). Figure 3A, lane 6, is an abnormal control (Coriell GM07537A) with a full mutation band (approximately 2.1 kb in size, with 336 CGG repeats). No extra band was observed in the index family, ruling out the possibility that the 10.9-kb band observed on the standard Southern blot assay was due to the large CGG expansion at the 5′-UTR of the FMR1 gene. Because the size of 10.9-kb could result from an abolished EcoRI cutting site at the 11,114 position (see Figure 1d), a Southern blot after EcoRI digestion with and without NruI was analyzed for comparison (Figure 3B). In a standard Southern blot assay for fragile X syndrome with EcoRI/NruI double digestion, a normal female should have 2.8- and 5.2-kb bands (Figure 3B, lane 2), and a normal male, a 2.8-kb band (Figure 3B, lane 10). With EcoRI digestion only, both normal female and male should have a single 5.2-kb band (Figure 3B, lanes 3 and 11). As shown in Figure 3B, the proband's mother had the same banding pattern (Figure 3B, lanes 4 and 5) as a normal female (Figure 3B, lanes 2 and 3), and the father had a normal 2.8-kb band after EcoRI/NruI double digestion (Figure 3B, lane 8). However, when his DNA was digested with EcoRI only, the father showed one 10.9-kb band (Figure 3B, lane 9) instead of a normal 5.2-kb band, indicating that the EcoRI cutting site at the 11,114 position is absent. Thus, the proband inherited this 10.9-kb band from her father and the 5.2-kb band from her mother (Figure 3B, lane 7, with EcoRI digestion only). As a result, it is not surprising to observe the 10.9-kb band in the standard Southern analysis (EcoRI/NruI double digestion) from the index patient (Figure 3B, lane 6), when some of her father's X chromosome is fully methylated (inactivated), abolishing the NruI site.Figure 3Determination of the origin of the 10.9-kb fragment from the index patient. A: Southern blot using PstI digestion only showing bands of 0.99, 1.03, and 1.08 kb (the largest band approximately 1.1 kb in size). An abnormal control (AC, lane 6) shows a full mutation (Coriell GM07537A), with an additional 2.1-kb band (336 CGG repeats). No CGG expansion was noted in the family study (lanes 2 to 4) or in a normal control (NC, lane 5). MW, the Lambda/HindIII MW marker (Chemicon International). B: Southern analysis of the family digested with EcoRI, with (+) and without (−) NruI. MW, molecular marker as in A; NFC, normal female control; M, mother; IP, index patient; F, father; NMC, normal male control; AC, abnormal control as in A. Note that father and the index patient both showed the 10.9-kb fragment (lanes 6, 7, and 9). C: PCR products with (+) and without (−) EcoRI digestion. MR, 100-bp Molecular Ruler (Bio-Rad); NC, normal control; M, mother; IP, index patient; F, father.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PCR Analysis of the EcoRI SiteA PCR analysis was designed to bracket the 11,114 EcoRI site. In a normal individual, the PCR product should be 300 bp in size without EcoRI digestion and should show a 122/178-bp banding pattern after digestion. Figure 3C reveals that the proband's mother had a 300-bp band without EcoRI digestion (Figure 3C, lane 4) and a 122/178-bp banding pattern after digestion (Figure 3C, lane 5), as observed in a normal control (Figure 3C, lane 2 and 3). The father had only a 300-bp band with and without EcoRI digestion (Figure 3C, lane 8 and 9), indicating the abolished EcoRI cutting site. The index patient had one 300-bp band without EcoRI digestion (Figure 3C, lane 6). After digestion, three bands of 122, 178, and 300 bp were observed, confirming her heterozygous status (Figure 3C, lane 7) at the 11,114 EcoR1 restriction site. The same PCR/restriction digestion test was performed on 165 alleles (105 male and 30 female patient samples from our laboratory). All showed the same digestion pattern as the normal control (Figure 3C, lanes 2 and 3; data not shown).Sequence AnalysisSequence analysis surrounding the 11,114 EcoRI cutting site (GenBank accession no. L29074.1; Figure 1) from both directions confirmed a G→A transition at position 11,114 in the father, changing GAATTC to AAATTC and eliminating the EcoRI restriction site (Figure 4a). The index patient inherited the G→A base change from her father and a normal allele from her mother, showing both G and A on DNA sequencing (Figure 4b). The mother had a G at position 11,114, as did the normal control (Figure 4c). No other sequence variation was detected in the 300-bp region bracketing the 11,114 EcoRI cutting site in the index patient, parents, and normal control (data not shown).Figure 4Confirmation of a base change at EcoRI cutting site by sequencing. a: Arrow points to the position 11,114, where a G→A transition occurred in the father. b: The index patient is heterozygous, with both G and A. c: Mother has a normal G at the same position.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionWe report here a 10.9-kb pseudo-full mutation band observed on a Southern blot analysis for fragile X testing from an autistic girl with language delay. In clinical laboratory practice, a real full mutation band for fragile X syndrome usually accompanies with variable size or methylation mosaic bands or with a smear pattern. However, the 10.9-kb band detected in the index patient is a very clean band (Figure 2, lane 2). Therefore, a parental study was requested. This report demonstrated that this 10.9-kb band observed in the index patient was inherited from her father and was the result of a G→A transition upstream of the CGG repeats in the 5′-UTR of the FMR1 gene, abolishing the 11,114 EcoR1 restriction site. The G→A base change has not been identified from 165 alleles (105 males and 30 females), suggesting that this is not a common polymorphism. The father, who carried the transition on his X chromosome, was physically and cognitively normal, indicating that this base change may not be clinically significant. Therefore, the final report for the index patient was interpreted as “the 10.9-kb band was not the result of a large CGG expansion in the FMR1 gene.” Interestingly, a pseudo-deletion case resulting from an A→G transition at position 14,744, which created an EcoRI cutting site downstream of the 5′-UTR of the FMR1 gene, was reported in 2000.4Daly TM Rafii A Martin RA Zehnbauer BA Novel polymorphism in the FMR1 gene resulting in a “pseudodeletion” of FMR1 in a commonly used fragile X assay.J Mol Diagn. 2000; 2: 128-131Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar The authors showed that this rare base change was a novel polymorphism that does not cause an FMR1 gene deletion.Molecular diagnostic testing for fragile X syndrome has been available since 1991.3Fu YH Kuhl DPA Pizzuti A Pieretti M Sutcliffe JS Richards S Verkerk AJMH Holden JJA Fenwick Jr, RG 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.Cell. 1991; 67: 1047-1058Abstract Full Text PDF PubMed Scopus (1747) Google Scholar The basic techniques used are PCR and Southern analysis. Usually, PCR is used to determine the number of CGG repeats less than 100, whereas Southern analysis is used to detect large premutations (100 to 200 CGG repeats), full mutations (>200 CGG repeats), and their methylation status. These tests are reported with more than 99% sensitivity, and positive results are 100% specific (http://www.acmg.net/Pages/ACMG-Activities/stds-2002/fx.htm, last accessed April 3, 2008). In practice, some laboratories would screen CGG repeats using PCR analysis to exclude males with a normal CGG repeat or females showing two normal alleles.5Biancalana V Beldjord C Taillandier A Szpiro-Tapia S Cusin V Gerson F Philippe C Mandel JL The French National Working Group on Fragile X syndrome Five years of molecular diagnosis of fragile X syndrome (1997–2001): a collaborative study reporting 95% of the activity in France.Am J Med Genet. 2004; 129A: 218-224Crossref PubMed Scopus (35) Google Scholar To detect a larger repeat expansion, the Southern blot assay is usually used only if no PCR products are detected in a male patient and if females show only one band on PCR analysis. In this report, the index patient and her mother both showed two normal alleles (23 of 32 and 18 of 23 CGG repeats, respectively) and the father had a normal 32 CGG repeat. Therefore, the laboratories that use PCR as the first screening method would not have run a Southern analysis in this case. On the other hand, if this 10.9-kb fragment were identified for the proband without additional family studies, it could have been misinterpreted as a methylated full mutation. Furthermore, if the father's X chromosome is preferentially activated (without methylation) during his daughter's embryological development, the 10.9-kb fragment may not be observed (or in a trace amount) on a standard Southern assay with EcoRI/NruI double digestion. These may be some of the reasons that this G→A base change has not been reported after 17 years of routine fragile X testing.The FMR1 gene is highly conserved at the sequence and amino acid levels, as evidenced by its presence in the human, mouse, Caenorhabditis elegans, Xenopus laevis, Drosophila melanogaster, and chicken,6Crawford DC Acuna JM Sherman SL FMR1 and the fragile X syndrome: Human genome epidemiology review.Genet Med. 2001; 3: 359-371Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar but the base G at the position 11,114, upstream of the 5′-UTR of the FMR1 gene, seems not well conserved. The conservation score is approximately 10% of the maximal, based on the PhastCons package.7Siepel A Haussler D Phylogenetic hidden Markov models.in: Nielsen R Statistical Methods in Molecular Evolution. Springer, New York2005: 325-351Crossref Google Scholar As shown in Figure 5 (derived from University of California Santa Cruz Genome Bioinformatics, http://genome.ucsc.edu/cgi-bin/hgTracks, accessed April 10, 2008), this base is conserved in the chimpanzee, Rhesus monkey, bushbaby monkey, Guinea pig, dog, cat, cow, elephant, and tenrec but not in the rabbit. Treeshrew, mouse, rat, shrew, hedgehog, horse, and armadillo have one or more unalignable bases in the gap region, due to either an excessive evolutionary distance between the species or an independent insertion/deletion in the aligned region among these species. This may explain why the father who carries this G→A change is clinically normal.Figure 5DNA sequence alignment around the 11,114 EcoRI restriction site with 18 species showing conservation status of the base G (derived from University of California Santa Cruz Genome Bioinformatics, http://genome.ucsc.edu/cgi-bin/hgTracks, last accessed April 10, 2008).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although the father has not presented any measurable symptoms, the significance of the 10.9-kb band will not be clear until additional individuals with this G→A base change have been identified and clinically evaluated. Further studies of the FMR1 gene and its expression in this family may help to resolve the issue. Nevertheless, whenever a clean 10.9-kb band is observed on a Southern blot analysis in a fragile X assay, family studies and further testing should be undertaken for appropriate clinical interpretation. The fragile X syndrome is the most common inherited disorder associated with mental retardation, with an estimated prevalence of 1 in 4000 in males and 1 in 8000 in females. The syndrome in males is characterized by typical facial features including large ears, speech delay, autistic behavior, macroorchidism, and mental retardation. Females with a full mutation exhibit a wide spectrum of phenotypes, ranging from no detectable learning or behavioral deficits to mild learning disabilities to effects as severe as those in a male fragile X patient with a full mutation.1Sherman S Pletcher BA Driscoll DA Fragile X syndrome: diagnostic and carrier testing.Genet Med. 2005; 7: 584-587Crossref PubMed Scopus (223) Google Scholar Although mutations, such as deletions and point mutations, have been identified in fragile X patients, more than 99% of cases have been associated with an expansion of a segment of CGG repeats in the 5′-untranslated region (UTR) of the fragile X mental retardation (FMR1) gene. According to the Technical Standards and Guidelines for Fragile X Testing from the ACMG (http://www.acmg.net/Pages/ACMG-Activities/stds-2002/fx.htm, last accessed April 3, 2008), there are four allelic forms of the CGG repeat lengths: normal (5 to 44 repeats), intermediate (gray zone, 45 to 54 repeats), premutation (55 to 200 repeats), and full mutation (>200 to 230 repeats). Full mutations with a large CGG expansion in this region are associated with inhibition of transcription of the FMR1 gene, causing deficiency or absence of the fragile X mental retardation protein. Therefore, standard laboratory techniques for fragile X syndrome diagnosis are designed to quantitate the number of CGG repeats in the 5′-UTR of the FMR1 gene. These techniques include a PCR, which detects normal and premutation alleles ( 200 CGG repeats) alleles and their methylation status. In the Southern blot assay for fragile X syndrome, a normal male usually shows a 2.8-kb band, and a normal female, 2.8- and 5.2-kb bands. In females, a 2.8-kb band represents the active, unmethylated X chromosome, whereas a 5.2-kb band represents the inactive, methylated X chromosome. Methylated full mutations are not detectable by PCR and are often shown as bands larger than 5.8 kb on a Southern blot. Here, we report a 10.9-kb band identified with a Southern blot assay for fragile X syndrome from an autistic girl with language delay. However, this large band does not represent a methylated full mutation and is not associated with an expanded CGG repeat in the 5′-UTR in the FMR1 gene. Materials and MethodsCase ReportsA 7-year-old girl was evaluated for autistic features and language delay that became evident by age 2 years. The child was the product of a full-term pregnancy to a healthy 20-year-old primigravida and her 22-year-old partner. The mother was of Mexican ancestry, and the father, Salvadoran. There was no parental consanguinity and no first- or second-degree relatives of the proband with congenital anomalies or developmental delay. Both parents are physically and cognitively normal. The child had no siblings. Delivery took place by primary cesarean section for fetal heart rate decelerations. Birth weight was 3.2 kg, and length, 53 cm. The neonatal course was uneventful.By age 2 years, delays in socialization and in expressive and receptive language led to a diagnosis of classical autism. The child has remained in good general health, with no seizures, hospitalizations, regression of milestones, or other serious health problems.The child weighed 39 kg (98th percentile), was 132 cm tall (80th percentile), and had a 52.5-cm head circumference (75th percentile). Ear length of 6 cm was between the 50th and 75th percentiles. The patient exhibited no dysmorphism, and she was physically normal.High-resolution chromosome analysis showed a normal female karyotype (46, XX). Molecular cytogenetic analysis using an LSI Prader-Willi/Angelman region probe (GABRB3; Abbott Laboratories, Des Plaines, IL) showed no deletion or duplication in 10 metaphase and 40 interphase cells. This analysis excluded the possibility that her autistic feature was due to a 15q duplication.2Bundey S Hardy C Vickers S Kilpatrick MW Corbett JA Duplication of the 15q11-13 region in a patient with autism, epilepsy and ataxia.Dev Med Child Neurol. 1994; 36: 736-742Crossref PubMed Scopus (127) Google ScholarStandard Molecular Diagnostic Testing for Fragile XMolecular diagnostic testing was performed using both PCR and Southern blot analysis to determine the CGG repeat size at the 5′-UTR of the FMR1 gene and its methylation status. Blood was drawn into EDTA tubes from the index patient, parents, and normal controls. DNA was isolated with the Puregene DNA isolation procedure (Qiagen, Valencia, CA). The primer set for PCR was from Fu et al,3Fu YH Kuhl DPA Pizzuti A Pieretti M Sutcliffe JS Richards S Verkerk AJMH Holden JJA Fenwick Jr, RG Warren ST Oostra BA
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