PCR testing for FMR1-related disorders at the Children s Hospital at Westmead: past and present. Dr Karen Wong

Transcription

1 PCR testing for FMR1-related disorders at the Children s Hospital at Westmead: past and present Dr Karen Wong

2 FMR1-related disorders Fragile X syndrome Fragile X-associated tremor/ataxia (FXTAS) Fragile X-associated Primary Ovarian Insufficiency (FXPOI) Majority are caused by an expansion of CGG repeats at 5 -untranslated region of the first exon of the FMR1 gene.

3 FMR1-related disorders Normal individual 5-44 CGG repeats. Intermediate CGG repeats Premutation CGG repeats (FXTAS & FXPOI) Full mutation >200 CGG repeats (FRAXA) Lab (since 2000) Determine no. of CGG repeats by amplifying CGG repeats region in FMR1 using PCR ~800 patients Fragile X testing/year

4 Fragile X testing (2000 mid 2012) Batch of patients DNA set up PCR - Fu primers and Roche Expand kit Visualise on CE (Qiaxcel) AGRF for fragment separation (AB 3730) Each time perform a batch, 9 controls are included in PCR along with patients DNA.

5 Fragile X testing (2000 mid 2012) Traces from AGRF Linear regression to generate standard curve to determine no. CGG repeats based on 9 controls

6 Fragile X testing (2000 mid 2012) Anonymous patients DNA with Known CGG repeats (19-91) , ,30 29, ,30 31, P1 P2 P3 Standard curve generated based on fragment sizes and known CGG repeats of 9 controls.

7 Fragile X testing (2000 mid 2012) Patient s CGG repeats interpolated based on fragment size. 31 We use own controls as our fragments are GC rich; run differently to internal controls.

8 In house method Limitations Region GC rich, Taq only amplify fragments <100 CGG repeats. Cannot detect fragments >100 repeats. Cannot distinguish homozygote females and females heterozygote for a large PM/FM. Taq only amplify <50 repeats nicely repeats, MU increases. Cannot accurately size fragments >50 repeats.

9 CGG repeats <50 CGG repeats >50

10 In house method Limitations Region GC rich, Taq only amplify fragments <100 CGG repeats. Cannot detect fragments >100 repeats. Cannot distinguish homozygote females and females heterozygote for a large PM/FM Taq only amplify <50 repeats nicely repeats, MU increases. Cannot accurately size fragments >50 repeats. Cannot exclude mosaicism for a larger fragment in males or females showed up normal.

11 In house method Limitations Overcome limitations by sending: 1. Males and females CGG repeats >50 2. Homozygote females 3. Males that cannot amplify To another facility (SEALS) that performed Southern Blots/Amplidex to determine if there was a larger fragment.

12 Fragile X testing (mid 2012-now) Asuragen Amplidex PCR kit Amplify >200 CGG repeats (GC rich polymerase). Same as in-house except all controls in 1 tube F+R primers to generate fragment of a set size. In addition, contains 3 rd primer (CGGx5) binds along CGG repeats resulting in series of smaller fragments.

13 CGGCGGCGGCGGCGG FMR1-R GCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCGGCGGCGGCGGCGG FMR1-F

14 CGGCGGCGGCGGCGG FMR1-R GCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCGGCGGCGGCGGCGG FMR1-F

15 CGGCGGCGGCGGCGG CGGCGGCGGCGGCGG CGGCGGCGGCGGCGG CGGCGGCGGCGGCGG FMR1-R GCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCGGCGGCGGCGGCGG FMR1-F Generate a fragment of set sizes and a series of smaller fragments

16 Amplidex Control tube with 6 controls

17 Fragile X testing (mid 2012-now) Asuragen Amplidex PCR kit Able to resolve zygosity between females patients.

18 Fragile X testing (mid 2012-now) Asuragen Amplidex PCR kit Able to resolve zygosity between females patients.

19 Fragile X testing (mid 2012-now) Asuragen Amplidex PCR kit Able to resolve zygosity between females patients.

20 Fragile X testing (mid 2012-now) Asuragen Amplidex PCR kit Relatively expensive compared to in house method $7000/100 rxns Estimated cost (DNA extraction, reagents, labour, analysis) In House - $30/patient Amplidex - $90/patient Australian Medicare rebate on Fragile X testing ~$85 Not cost effective to use Amplidex for all patients!

21 Fragile X testing (mid 2012-now) Overcome this: We use new work scheme of: 1) In house method for all males 2) Amplidex for all females, males with CGG repeats >50, and males that don t amplify Able to Detect and size CGG repeats >50 Resolve zygosity in female patients ~$/patient more cost effective than $85 Participate in the full QAP scheme for Fragile X

22 Fragile X testing (mid 2012-now) Limitations of new work scheme 1) Small volume of GC rich polymerase (Amplidex)/rxn (0.05uL/rxn), cannot start a batch until min 10 patients. 2) Possibility of missing mosaicism for a larger fragment in males with a normal sized allele.

23 Open discussion Our interest How common is presence of mosaicism in males? How common is the presence of point mutations in FMR1? Is the use of the Amplidex method equivalent to the Southern Blot? -? Medicare rebate

24 Acknowledgement Julianne Jackson Gemma Jenkins Katrina Fisk Patricia Amiatu Tiffany Lai Lingli Zhang Bruce Bennetts CHW Molecular Genetics Laboratory CHW