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FRAX Case Study

FRAX Case Study. TWIN PEAKS. Fragile X. X-linked dominant disorder Most common inherited form of mental retardation Affecting 1 in 5000 males Expansion in the CGG repeat motif located in the 5’ UTR of the FMR1 gene. Patient KR 4 year old male Speech & Language delay, social difficulties

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FRAX Case Study

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  1. FRAX Case Study TWIN PEAKS

  2. Fragile X • X-linked dominant disorder • Most common inherited form of mental retardation • Affecting 1 in 5000 males • Expansion in the CGG repeat motif located in the 5’ UTR of the FMR1 gene

  3. Patient KR 4 year old male Speech & Language delay, social difficulties Karyotype, fragile X, 22q11 deletion Referred as a diagnosis case Cytogenetics - karyotype Molecular – FRX PCR Patient JH 12 year old male Learning difficulties Request for karyotyping and Fragile X Referred as a diagnosis case Cytogenetics - karyotype Molecular – FRX PCR

  4. Molecular Testing • PCR pre screen across the region of the FMR1 gene containing the CGG repeat units • PCR products were run on a Beckman Fragment Analyser • Repeat sizes of the alleles can be obtained • Southern Blot for detection of larger expansions.

  5. PCR FRX TRACE Patient 1 Patient 2 • Normal male with one X chromosome would be expected to show one corresponding peak • A normal female 2 X chromosomes would show 2 corresponding peaks (20-30% females are homozygous for repeat size, showing one peak) • Both Patients Displayed Two Distinct Peaks • 12 & 30 rpts • 30 & 33 rpts

  6. Cytogenetics Chromosome analysis of both patients revealed: • Patient 1 has a normal male karyotype of 47,XY in all cells tested • Patient 2 has the Klinefelter karyotype 47,XXY • Neither patient displayed a fragile site on the X chromosome

  7. Patient 2 • Southern Blot (Ln 6) • methylated and unmethylated X chromosome which is the typically pattern seen in unaffected females

  8. Patient 2 Report • Patient 2 was reported as not carrying a mutation in the Fragile X gene, however 2 normal sized alleles were detected indicating that he has Klinefelter syndrome • Klinefelter syndrome occurs at a frequency of 1 in 1000 males • Main clinical features are: infertility, hypogonadism and gynaecomastia • Klinefelter individuals can also have a tendency towards obesity and show a degree of learning difficulty • This diagnosis explains why this patient was referred with learning difficulties. • Patient was reported by cytogenetics

  9. Patient 1 Blood Spot PCR Test • For Patient 1 - Sample Mix Up • 2nd Extraction from Blood Spot.

  10. Patient 1 TRIS Multiplex • Sample Extracted on Autopure in batch of 8 • All Transfers are watched • All samples BS and DNA samples matched • Sample was sex matched • No sample contamination or sample mix up • Unlikely Sample mix up has occurred Blood Spot DNA

  11. Repeat Size Instability • Instability of the CGG repeat units through generations has been shown to be associated with repeat length and molecular structure of the repeats • It is thought the larger the repeat unit size the more unstable the allele is • Mosaicism involving premutation or full mutation size alleles has been described previously • Alleles in the normal repeat size range are thought to be stable (meiotically and mitotically) • This patient is an example of a normal sized allele that exhibits repeat length instability by the presence of the second normal length distinct band by PCR analysis

  12. Male FRX Double Peaks • This in not the first time this has been seen in our laboratory • A paper published in Canada in 2000, reported on nine developmentally delayed males with apparently unstable normal FMR1 alleles (Genetic Testing 2000 Vol.4, No.3, 235-239) • South West Thames Regional Genetics Service presented a poster at BSHG in 2001 on a six year old karotypically normal male with an apparently unstable normal FMR1 allele referred because of delayed speech • The Salisbury laboratory confirmed that they have seen a number of similar cases.

  13. Patient 1 Report • A normal fragile X report was issued on patient 1 stating that no mutation was detected in the FMR1 gene • The detection of two distinct bands was not mentioned as this may unnecessarily alarm the family

  14. Summary • Two patients appeared to have the same result (i.e. Klinefelter syndrome) but are in fact different • It is an example of how different testing procedures take results down a different path • Interaction between cytogenetic & molecular testing • Instability of the normal size FMR1 alleles is something to remember in the future when testing for fragile X syndrome • This patient is an example of a normal sized allele that exhibits repeat length instability by the presence of the second normal length distinct band by PCR analysis • It would be interesting to compare the size of the patients FMR1 allele to that of his mothers.

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