Original article

Use of two FISH probes provides a cost-effective, simple protocolto exclude an imprinting centre defect in routine laboratory testing

for suspected Prader–Willi and Angelman syndromeArabella Smith *, Lisa Robson, Luke St. Heaps

Department of Cytogenetics, Royal Alexandra Hospital for Children, Locked Bag 4001, Westmead, NSW 2145, Australia

Received 19 December 2001; accepted 3 October 2002

Abstract

From among the many suspected patients with Prader–Willi (PWS) or Angelman (AS) syndromes received for diagnosis in a routinegenetics laboratory, we present our protocol for the exclusion of a possible, rare imprinting centre (IC) defect. Deletion detection utilisingtwo FISH probes—SNRPN within the IC, and another probe outside the IC, on the same suspension remaining from the cytogenetic harvest,provides a simple, quick and cost-effective system for exclusion of an IC defect, for patients with an abnormal methylation analysis. © 2002Editions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Chromosome 15;SNRPN gene;UBE3A gene

1. Introduction

Prader–Willi (PWS) and Angelman (AS) syndromes arewell-described clinical dysmorphic syndromes[1,2], butdue to some overlap of features with both normal individu-als and those with other causes of intellectual handicap, adefinitive diagnosis nowadays involves DNA testing. Thecritical region for both syndromes is chromosome15(q11–13), a region known to be imprinted. PWS is causedby loss of paternal and AS by loss of maternal alleles withinthis region[3]. The genetic mechanisms leading to PWS andAS include deletion (70–75% of patients), uniparentaldisomy (UPD) (20–25% of PWS; 3–5% of AS) and animprinting centre (IC) defect (∼ 1% of PWS; 3–5% of AS)[3,4]. In addition,UBE3A mutation or other as yet unknownmechanisms account for∼ 20% of AS.

The PWS/AS chromosome region spans around 4.5 Mband large deletions of this size have two major proximalbreakpoints but a consistent single distal breakpoint[5,6].Class I deletions extend from D15S541 to D15S12 and classII from D15S543 to D15S12, taking out the IC. These

deletions are considered collectively as the common dele-tion; there is no phenotypic difference between them, theyare present in approximately equal proportions, they are thesame in both PWS and AS, they are the mechanism of PWSor AS in ∼ 70% of patients and there is no report ofrecurrence[6]. In comparison, microdeletions of the IC,within the SNRPN gene only, span from 6 to 250 kb DNA,with exon 1 deleted in PWS and the upstream bd exons ofSNRPN deleted in AS[7], and have a potentially high (50%)recurrence risk[8,9].

Various genetic tests are available and required to char-acterise PWS and AS. In devising a testing protocol, it isimportant to recall what each test can do. Methylationanalysis can diagnose PWS and AS unequivocally when theabnormal pattern is present. Methylation does not define themechanism, as it is abnormal in the common large deletion,UPD and IC defects[10]. Methylation is normal in ASpatients with UBE3A mutation. FISH with a single probewill detect a deletion but this one result can not beextrapolated further into the critical region—and a FISHdeletion per se does not distinguish PWS from AS. DNAtesting is required to show UPD. When an imprinting defectis suspected, further specialised testing is necessary if the ICdefect is to be characterised. At present>95% of IC defectsare microdeletions withinSNRPN, but very rare patients

* Corresponding author.E-mail address: ellies@chw.edu.au (A. Smith).

Annales de Génétique 45 (2002) 189–191

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© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.PII: S 0 0 0 3 - 3 9 9 5 ( 0 2 ) 0 1 1 3 6 - X

have been reported who have PWS/AS by some other ICdefect [11,12].

The dilemma for most diagnostic genetic laboratories isto separate out the few patients with an IC defect from thelarge number of patients referred with PWS or AS, themajority of whom will have the large common deletion.This important step is undertaken variably in differentcentres and has included the cytogenetic and/or FISHtesting of parents [13] as well as detailed DNA work [4,7].To address this point, our protocol was based on therationale that in a patient with proven PWS or AS (byabnormal methylation) the use of two FISH probes, onewithin the SNRPN region and one outside, would establishthe size of the deletion.

2. Methods

Our method was that after routine cytogenetic analysis,PWS and AS patients with an abnormal methylation testresult were evaluated further with FISH, performed on thesuspension remaining from the cytogenetic harvest, retainedin fixative at –20 °C [14]. Deletion for SNRPN, present inall patients, was the criteria for use of the second probe.Slides were dropped again using the same suspension.

3. Results

We evaluated 55 patients in this way—38 with PWS(aged 2 weeks to 44 years), and 17 patients with AS(aged 2–30 years) (Table 1). The second probe wasGABR�3 (Oncor) (seven patients) and D15S10 (Vysis)(39 patients)—these being located 40–100 kb telomeric toSNRPN. All patients deleted for the first probe demonstrateda deletion with the second probe, confirming the largecommon deletion and excluding an imprinting defect. Therewere five patients (9% of this cohort) for whom there wasinsufficient suspension left for testing with the secondprobe. The results of the cytogenetic test were availablewithin 2 weeks and the two FISH tests within a furtherweek.

4. Discussion

To determine the mechanism in DNA proven PWS/AS,we recommend a protocol of deletion detection with FISHstarting with SNRPN, as shown in the flow chart (Fig. 1).The benefit of following this protocol was in cost saving,speed of a result and reduction in the number of parentsrequired to be tested. These two FISH procedures do notrequire a recollection from the patient or another cytoge-netic set-up and harvest. When a deletion is detected onFISH with SNRPN, the alternative approach of testing thefather in PWS and the mother in AS, still results in twoFISH tests, and includes an additional cytogenetic set-upand harvest. It also adds considerably to the time taken. Ininterpreting the results, if the initial FISH test with SNRPNis normal, studies for UPD should be initiated. This willrequire parental blood samples. If biparental chromosomesare demonstrated, one is altered to an IC defect. If SNRPNis deleted and the second probe is non-deleted, one is alsoaltered to an IC defect. Special arrangements will berequired to elucidate the type of IC defect present (ifpossible). For suspensions which are inadequate for asecond FISH procedure a decision would need to bemade—either not to proceed further, recollect blood fromthese patients for a second FISH test or test the parent.

When a patient with suspected PWS or AS comes forgenetic testing, the results of this protocol will provide adefinitive diagnosis in PWS for 99.5% of patients and in ASfor ∼ 80%, but the importance of these results also lies ingiving recurrence risk estimates.

References

[1] V.A. Holm, S.B. Cassidy, M.G. Butler, J.M. Hanchett, L.R. Green-swag, B.Y. Whitman, F. Greenberg, Prader–Willi syndrome: consen-sus diagnostic criteria, Pediatrics 91 (1993) 398–402.

[2] C.A. Williams, H. Angelman, J. Clayton-Smith, D.J. Driscoll,J.E. Hendrickson, J.H.M. Knoll, R.E. Magennis, A. Schinzel,J. Wagstaff, E.M. Whidden, R.T. Zori, Angelman syndrome: con-sensus for diagnostic criteria, Am. J. Med. Genet. 56 (1995)237–238.

Table 1Further testing in PWS and AS patients with abnormal methylation. Nopatient in this cohort (n = 55) was suspected of an IC defect

Males Females Total

PWS deleted with two probes 12 18 30PWS non-deleted SNRPN → UPD 2 1 3PWS insufficient for two probes 3 2 5AS deleted with two probes 8 8 16AS non-deleted SNRPN → UPD 0 1 1AS insufficient for two probes 0 0 0

Fig. 1. Flow chart of testing. Pt, patient; RR, recurrence risk.

190 A. Smith et al. / Annales de Génétique 45 (2002) 189–191

[3] R.D. Nicholls, S. Saitoh, B. Horsthemke, Imprinting in Prader–Williand Angelman syndromes, T.I.G. 14 (1998) 194–200.

[4] K. Buiting, S. Saitoh, S. Gross, B. Dittrich, S. Schwartz,R.D. Nicholls, B. Horsthemke, Inherited microdeletions in theAngelman and Prader–Willi syndromes define an imprinting centreon human chromosome 15, Nat. Genet. 9 (1995) 395–400.

[5] S.L. Christian, W.P. Robinson, B. Huang, A. Mutirangura,M.R. Line, M. Nakao, U. Surti, A. Chakravarti, D.H. Ledbetter,Molecular characterisation of two proximal deletion breakpointregions in both Prader–Willi and Angelman syndrome patients, Am.J. Hum. Genet. 57 (1995) 40–48.

[6] J.M. Amos-Landgraf, Y. Ji, W. Gottlieb, T. Depinet, A.E. Wandstrat,S.B. Cassidy, D.J. Driscoll, P.K. Rogan, S. Schwartz, R.D. Nicholls,Chromosome breakage in Prader–Willi and Angelman syndromesinvolves recombination between large, transcribed repeats at proxi-mal and distal breakpoints, Am. J. Hum. Genet. 65 (1999)370–386.

[7] B. Dittrich, K. Buiting, B. Korn, S. Rickard, J. Buxton, S. Saitoh,R.D. Nicholls, A. Poustka, A. Winterpacht, B. Zabel,B. Horsthemke, Imprint switching on human chromosome 15 mayinvolve alternate transcripts of the SNRPN gene, Nat. Genet. 14(1996) 163–170.

[8] K. Buiting, B. Dittrich, S. Gross, C. Lich, C. Farber, T. Buchholz,E. Smith, A. Reis, J. Burger, M.M. Nothen, U. Barth-Witte,B. Jannsen, D. Abeliovich, I. Lerer, A.M.W. van den Ouweland,D.J.J. Halley, C. Schrander-Stumpel, H. Smeets, P. Meinecke,S. Malcolm, A. Gardner, M. Lalande, R.D. Nicholls, K. Friend,A. Schulze, G. Matthijs, H. Kokkonen, P. Hilbert, L. van Malder-gem, G. Glover, P. Carbonell, P. Willems, G. Gillessen-Kaesbach,B. Horsthemke, Sporadic imprinting defects in Prader–Willi syn-

drome and Angelman syndrome: implications for imprint switching,genetic counselling and prenatal diagnosis, Am. J. Hum. Genet. 63(1998) 170–180.

[9] J.E. Ming, N. Blagowidow, J.H.M. Knoll, L. Rollings, P. Fortina,D.M. McDonald-McGinn, N.B. Spinner, E.H. Zackai, Submicro-scopic deletion in cousins with Prader–Willi syndrome causes agrandmatrilineal inheritance pattern: effects of imprinting, Am.J. Med. Genet. 92 (2000) 19–24.

[10] American Society of Human Genetics/American College of MedicalGenetics Test and Technology Transfer Committee, Diagnostictesting for Prader–Willi and Angelman syndromes: report of theASHG/ACMG test and technology transfer committee, Am. J. Hum.Genet. 58 (1996) 1085–1088.

[11] M. Runte, C. Farber, C. Lich, M. Zeschnigk, T. Buchholz, A. Smith,L. van Maldergem, J. Burger, F. Muscatelli, G. Gillessen-Kaesbach,B. Horsthemke, K. Buiting, Comprehensive methylation analysis intypical and atypical PWS and AS patients with normal biparentalchromosomes 15, Eur. J. Hum. Genet. 9 (2001) 519–526.

[12] K. Buiting, A. Barnicoat, C. Lich, M. Pembrey, S. Malcolm,B. Horsthemke, Disruption of the bipartite imprinting center in afamily with Angelman syndrome, Am. J. Hum. Genet. 68 (2001)1290–1294.

[13] K.G. Monaghan, D.L. Van Dyke, G. Feldman, A. Wiktor, L. Weiss,Diagnostic testing: a cost analysis for Prader–Willi and Angelmansyndromes, Am. J. Hum. Genet. 60 (1997) 244–247.

[14] A. Smith, Z.M. Deng, M. Prasad, L. Robson, T. Woodage, R. Trent,Comparison of high resolution cytogenetics, fluorescence in situhybridisation (FISH) and DNA studies to validate the diagnosis ofPrader–Willi and Angelman syndromes, Arch. Dis. Child. 72 (1995)397–402.

A. Smith et al. / Annales de Génétique 45 (2002) 189–191 191