The patient is an 11-year-old boy with a cognitive and behavioral profile compatible with, but not specific to, fragile X syndrome (). The patient has mild mental retardation with verbal greater than nonverbal skills [Differential Ability Scales (SS): Composite = 56, Verbal = 70, Nonverbal = 62, Spatial = 54] mild language delay [Clinical Evaluation of Language Fundamentals-4 (SS): Composite = 70, Language Content = 88, Working Memory =60; Receptive One Word Picture Vocabulary Test = 86, Expressive One Word Picture Vocabulary Test =95], and borderline-mildly impaired adaptive skills [Vineland Adaptive Behavior Scales (SS): Composite = 73, Communication = 66, Socialization = 84, Daily Living Skills = 79]. He also displays moderate to severe social avoidance, compatible with social anxiety, and ADHD-like symptoms by the Conner’s Rating Scales (inattention, hyperactivity, impulsivity) and by measures of attention and executive functioning (Wide Range Assessment of Memory and Learning-Second Edition, Delis Kaplan Executive Function System) that have responded well to treatment with selective serotonin reuptake inhibitors (SSRIs) and methylphenidate, respectively. Overall, the patient is physically a well-developed boy with mild hypotonia and no major anomalies. He has some joint laxity and some minor facial characteristics of fragile X (e.g., prominent jaw and thickening of nasal bridge), but not all of the manifestations usually seen in this disorder (e.g., normal size ears with incomplete cartilage). In summary, this patient has a more subtle presentation of fragile X syndrome than is typically seen. The patient had either inconclusive or normal results from standard clinical laboratory testing for fragile X and was referred to our laboratory for further analysis of the FMR1 gene.
Photograph of the patient with mosaic deletion of FMR1. He shows some (e.g., prominent jaw, thickening of nasal bridge) but not all (e.g., normal size ears) of the distinctive facial features of fragile X syndrome.
Clinical Fragile X Testing
Blood from the patient was sent for standard fragile X syndrome testing at two different clinical laboratories. Testing consisted of assessment of CGG repeat length by PCR and FMR1 DNA methylation by Southern analysis. After inconclusive results at both clinical laboratories, which in one instance reported weak signal for normal range CGG repeats, a sample was submitted to our laboratory for FMR1 gene sequencing. During sequencing, it was observed several of the exons, notably at the 5′ end of the gene and the promoter of FMR1, did not amplify efficiently, though enough product was able to be generated for sequence analysis. These and the previous results suggested that a mosaic deletion may be present. CGG repeat analysis by PCR and Southern analysis, specifically looking for evidence of a deletion, was then performed. CGG repeat analysis consists of PCR amplification using fluorescently labeled amplification primers flanking the CGG repeat, followed by separation of the products by capillary electrophoresis. The patient had a CGG repeat length of 23 CGGs, well within the normal range. However, compared to another normal male sample with 23 CGGs, the peak area was approximately 10-fold less in the patient’s DNA sample (). In the Southern blot, measurement of the 2.7 kb band intensity by phosphoimager analysis indicated there was ~10-fold reduction in intensity relative to an unaffected male tested in parallel (). Together, the PCR and Southern analyses indicate the patient has a mosaic deletion involving FMR1 in ~90% of his lymphocytes with 10% of his lymphocytes carrying an intact FMR1 gene. Sequencing of the FMR1 gene did not identify any mutations. Testing of the patient’s mother identified a 23 and a 30 CGG repeat allele with a normal female pattern on the Southern blot, indicating she does not carry a premutation allele. The sample was then submitted to the research laboratory for further evaluation.
Fig. 2 Clinical laboratory findings by standard fragile X testing. A: PCR amplification of the FMR1 CGG repeat of DNA isolated from the patient and a normal male with 23 CGG repeats. B: Southern analysis of DNA isolated from this patient with atypical fragile (more ...)
High-Density X Chromosome Microarray Analysis
Array comparative genomic hybridization (aCGH) was performed using a high-density microarray (P/ N: B3754001-00-01, Design name: HG18_CHRX_FT) from NimbleGen Systems to further characterize the deletion (). The array consists of 385,000 oligo probes ranging from 50 to 75 nucleotides applied to a glass slide using photomediated synthesis chemistry. The probes tile along the forward strand of the X chromosome at an average intermarker distance of 340 bp after repeat masking. Sample preparation and hybridization were performed in accordance with the manufacturer’s instructions. In brief, 2 μg of genomic DNA from the patient and a male reference sample were sonicated to generate 500–2,000 bp fragments. After fragmentation, the two samples were labeled with Cy3 and Cy5, respectively, during whole genome amplification using random 9 mers labeled with Cy3 or Cy5. Fifteen micrograms each of these labeled amplification products were combined and then hybridized to the microarray for 16 hr at 42°C. After hybridization, the arrays were washed and scanned at a 5 μm resolution using an Axon 4000B scanner.
Fig. 3 High-density X chromosome array analysis of the patient and sequencing of the junction fragment. A: View of entire X chromosome. Arrow indicates deleted region. B: View of X chromosome from coordinates 145,400,000 to 147,800,000. The top track graphs (more ...)
PCR Amplification and Sequencing of the Breakpoint
NimbleScan and Signal Map analysis software from NimbleGen were used to analyze the signal ratios on the array. A contiguous region spanning over 1 Mb encompassing FMR1 and FMR1NB showed a depressed signal (−0.33) relative to flanking sequence (0.001) on the X chromosome (). To confirm the deleted sequence, primers were designed to flank the potential break points. At the 5′ end of the deletion, a primer in the forward direction was designed to sequence just upstream of the first probe (CHRXFS146047722) having depressed signal (−0.231). Likewise, a primer in the reverse orientation was designed to sequence downstream of the last probe (CHRXFS147055822) showing depressed signal (−0.483). Using these primers, a 3.5 kb amplicon was generated with the patient’s DNA, but not with control DNA. This amplicon was gel eluted and sequenced in both directions.
PCR was performed using the LA Taq enzyme kit from Takara Bio, Inc. (Otsu, Shiga, Japan). Fifty nanograms of genomic template was used. The primer sequence for the upstream or 5′ end was AGGCTAATATCCTGGACGAAC (Hg18, ChrX starts at 146047123) and the downstream or 3′ end was TGAAAAACTGGAAGAAATCCAA (Hg18, ChrX starts at 147063517). Twenty-five microliters reaction volumes were made such that the final primer concentration was 0.2 μM, final dNTP concentration was 0.25 mM, buffer plus magnesium, and 1.5 U of LA Taq were used. The cycling conditions were 94°C for 4 min, 35 cycles of 94°C for 20 sec and 60°C for 8 min, with a final extension of 72°C for 5 min. The 3.5 kb band was purified from an agarose gel using Qiagen’s Gel Elution Kit (Cat. No. 28704) (Valencia, CA). The PCR product was bidirectionally sequenced using standard dideoxy chain termination methods from Applied Biosystems, Inc. (Foster City, CA) ().
Fig. 4 Sequence of the junction fragment (bottom portion of panel) and corresponding breakpoints in the Ensembl genome browser (top portion of the panel). The proximal break is 146,047,696 and the distal breakpoint is 147,061,090 resulting in the deletion of (more ...)