Screening for Fragile X Syndrome (Murray et al.) - NIHR Journals ...

More documents

Recommendations

Info

Screening and diagnostic testsand, hence, optimise detection, a radioactive probewhich is compatible with restriction enzyme choiceis recommended (Rousseau et al, 1992).A simplified profile of the DNA banding patternproduced from a double digest is shown in Figure 4.Normal males have a single band whereas, infemales, a second band is seen representing themethylated, inactivated X chromosome. A fourbandedpattern is produced by a female with aPM, since she will have the mutated and normal Xchromosome in both the inactive and active state.In males, a methylated band is seen only for an FM.Southern blotting has several disadvantages inthe screening context. First, it cannot be usedto determine accurately the repeat size, which isimportant for small PMs. Second, there is a longlaboratory turn-round time of up to 10 days, largelybecause of the radioactive detection of fragments;using a phospho-imaging detection system (such asthat produced by Molecular Synthetics Ltd) couldreduce turn-round time to 1 day. Third, the test isexpensive at about £50–75 per test.DNA amplification by polymerasechain reactionA rapid and relatively cheap method of assessingthe repeat size is to amplify part of the DNA.The most common technique is the polymerasechain reaction (PCR), in which the enzyme DNApolymerase is used to process and copy a specifiedsequence. The PCR product can be detected byradioactive (Fu et al, 1991; Pergolizzi et al, 1992;Erster et al, 1992) or other means (Brown et al,1993; El-Aleem et al, 1995; Nanba et al, 1995;Haddad et al, 1996; Wang et al, 1995a). Thus, themethod works on smaller quantities of less purifiedstarting material, either blood or mouthwash(Hagerman et al, 1994a), than Southern blotting.The turn-round time for PCR testing is approximately1 week and, for a high throughput, thecost may be reduced to £10 per sample.PCR is most suitable for detecting PMs and largenormal alleles as it enables improved size resolutionof small repeat sequences (Heitz et al, 1992;Macpherson et al, 1992a). Its use is limited for thedetection of large FMs, and it is unable to determinemethylation status. There is a tendency forlarge FMs to fail to amplify. This is less of a problemin males since absence of an expanded fragmentcan be taken to indicate that an FM may be presentand demonstration of a small repeat size is sufficientto exclude an FM. In females, however, theabsence of one of the two bands expected afterPCR analysis is ambiguous. A small single band isconsistent with(a) preferential amplification of the smaller oftwo alleles, one of which might be mutated(Brown et al, 1993; Erster et al, 1992; Pergolizziet al, 1992)(b) normal alleles homozygous for size(c) normal alleles differing by only one CGGrepeat, which are practically indistinguishable.Brown and colleagues (1993) have devised amethodology which avoids selective amplificationof the smaller allele in heterozygotes enablingthem to be differentiated from homozygotes.However, a single allele on PCR is still notfully informative.PCR and selectiveSouthern blottingSequential testing is a reasonable compromisebetween Southern blotting and PCR. The protocol5.2 kb2.8 kbNormalfemaleNormalmalePMfemalePMmaleFMfemaleFMmaleFM malemosaic30FIGURE 4 Example of test results using a double digest
Health Technology Assessment 1997; Vol. 1: No. 4is to perform PCR on all samples but when thereis a failure to amplify or there is a single band ina female to perform a Southern blot. Over onequarterof females are homozygous for repeat size(see Table 2) and, in our experience, a further 8%have alleles differing by one repeat. Thus, thisapproach requires a fair amount of Southernblotting, adding an extra week to the turn-roundtime and, thus, additional costs.Blotting PCR productsAlthough FMs are amplified inefficiently, theirlarge expansions become ideal hybridisationtargets for CGG containing oligonucleotides.Blotting methods for these PCR products havebeen described for fragile X syndrome. Becausethe initial reaction uses DNA amplification, onlysmall amounts of starting material are required.Non-radioactive blotting of amplified productsis feasible (Pergolizzi et al, 1992).PCR-based methylation assayThis method relies on the fact that the unmethylatedFMR-1 gene can be digested by certainenzymes, whereas the methylated gene is resistantto digestion. Thus, in males, where methylationoccurs with an FM but not a PM or normal allele,digestion with a methylation-sensitive enzymefollowed by amplification should only yieldproducts if there is an FM. Females with an FMare not detected by this method since they alwayshave one of their X chromosomes methylated(Wang et al, 1995a). There is also the risk ofincomplete digestion.Measurement of FMRPAn antibody test has been described to measureFMRP (Willemsen et al, 1995). This could be usedto detect an FM in males since those affected shouldproduce no protein. The test will not be useful infemales since protein is produced from the normalactive X chromosome even when the other chromosomehas an FM. Also, even for males the test islimited, because mosaics produce FMRP to a degree.PM alleles yield normal quantities of FMRP.Alternatives to standard PCRSeveral alternative methods to standard PCR havebeen described, although none are in routine use.Repeat expansion detectionFirst described by Schalling and colleagues (1992),repeat expansion detection (RED) uses a thermostableDNA ligase in the usual cycling reaction.Complementary oligonucleotides are used, whichanneal along the entire length of one strand ofthe repeat sequence. Adjacent oligonucleotidesare then joined by adding ligase. The ligatedproducts from one cycle continue to ligate toproducts generated from the original DNA in allsubsequent cycles, until the whole expansion hasbeen replicated. However, sensitivity is rather poorin comparison with PCR because the techniqueinvolves no amplification.Ligase chain reactionThe ligase chain reaction (LCR) does allow amplificationbut to date has generally been restrictedto the mapping of point mutations involving thegeneration of short stretches of DNA. LCR is lesssuited to the amplification of longer stretches ofDNA because of its reliance on complementaryself-annealing primers. It has an advantage overPCR in that it is a non-processive system andshould not show undue bias against the detectionof large expansions.HybridsA method combining LCR and PCR has beendescribed by both Abbott Laboratories Ltd andRoche Diagnostics Ltd for the detection of microorganisms,and this may adapt well to detectingCGG amplifications although it has not as yet beentried. A modified RED with the hybrid PCR–LCRdetection system is another possibility.Testing protocolsGiven the technical, practical and financialconstraints of the various laboratory techniquesavailable, it is possible to devise a reasonabletesting protocol for the main applications.ScreeningFor antenatal, pre-conceptual, cascade andneonatal screening, the first choice would bePCR followed by selective Southern blottingof ambiguous alleles. Since antenatal and preconceptualscreening concentrate on females,one-third of samples may also need Southernblotting, thus increasing the average cost oftesting to about £30 per sample. Nonetheless,this would offer a cheaper, faster and moreaccurate alternative to Southern blotting of allsamples. A more sophisticated protocol wouldinvolve the sequencing of grey zone alleles for31
Page 1 and 2: Health Technology Assessment 1997;
Page 5 and 6: Screening for fragile X syndromeJen
Page 7: Health Technology Assessment 1997;
Page 38 and 39: Screening and diagnosis26extended t
Page 50 and 51: Modelling allele dynamicsshown in T
Page 52 and 53: Modelling allele dynamicsTABLE 12 R
Page 54 and 55: Modelling allele dynamicsfrequency.
Page 56 and 57: Assessment of screening potentialac
Page 58 and 59: Human and financial costs of screen
Page 60 and 61: Human and financial costs of screen
Page 62 and 63: Recommendations5. A central registr

Screening for Fragile X Syndrome (Murray et al.) - NIHR Journals ...

Create successful ePaper yourself

Delete template?

Save as template?