Atypical Molecular Findings Identify Limits ofTechnical Screening Tests for Prader-Willi andAngelman Syndrome Diagnoses

Perrine Malzac, Anne Moncla, Karinne Pedeillier, Catherine Vo Van, Lydie Girardot, andMarie-Antoinette Voelckel*Departement de Genetique Medicale, Hopital d’Enfants de la Timone, Marseille, France

INTRODUCTION

Prader-Willi syndrome (PWS) and Angelman syn-drome (AS) are caused, respectively, by absence of pa-ternal or maternal contributions of the same chromo-some region 15q11–q13. This region was described tobe associated with genomic imprinting [Lalande, 1996;Glenn et al., 1996]. Criteria for the clinical diagnosishave been previously described [ASHG/ACMG, 1996;Williams et al., 1995].

It is well accepted that DNA methylation analysis isvery useful as an initial screening test for PWS and AS.This strategy is recommended by ASHG/ACMG (1996).Differential methylation is assayed by cleaving DNAwith methylation-sensitive restriction enzymes fol-lowed by Southern blotting using 15q11–q13-specificprobes, such as ZNF127 (D15S9), PW71 (D15S63), exon1 of SNRPN gene, and intron 5 of SNRPN [Driscoll etal., 1992; Dittrich et al., 1992; Sutcliffe et al., 1994;Glenn et al., 1993]. The PW71 and exon 1 of SNRPNprobes are currently used, because these give a clearlydetectable difference in methylation pattern betweenpaternal and maternal alleles using genomic DNA fromleucocytes.

For AS, the differential methylation tests identifythree of the four major classes of genetic defects–deletion, uniparental disomy, and imprinting muta-tions–but are not effective for approximately 20–30% ofAS patients. The latter cases can have a substantialrecurrence risk (up to 50%), and, in a few of these, amutation in the newly described UBE3A gene (E6AP)was found [Kishino et al., 1997; Matsuura et al., 1997].Nearly all cases of PWS can be detected by this mo-lecular screening [Gillessen-Kaesbach et al., 1995;Kubota et al., 1996; Buchholz et al., 1997].

In our department, we have established a strategy(consistent with the literature) for detecting molecularabnormalities in PWS and AS so that we may performgenetic counseling and estimate recurrence risk

[ASHG/ACMG, 1996]. This strategy consists of meth-ylation screening followed by fluorescence in situ hy-bridization (FISH) analysis and associated microsatel-lite studies.

CASE REPORTS AND DISCUSSIONWe would like to draw attention to two examples

observed in our series of 118 PWS and 89 AS patients.The first concerns a boy with AS, who was diagnosed in1993. Clinically, the index case presented the typicalmanifestations of this syndrome. In a first molecularapproach, the pattern of allele-specific methylationwith probe PW71 was found to be normal. We theninvestigated the segregation haplotypes with seven mi-crosatellites (D15S11, D15S128, D15S210, D15S113,GABRB3, 155.1 CA, and 85 CA) and found no evidenceof deletion or uniparental disomy (Fig. 1). These resultssuggested that this affected child could be classifiedamong cases with no identifiable defect.

Then, following our protocol, the diagnosis of AS inthis child was confirmed by careful clinical evaluation.Subsequently, in the course of testing with the SNRPNprobe all of those cases that were normal with PW71,abnormal methylation was observed at the latter sitein this patient.

This AS patient, therefore, appears to display abnor-mal methylation at the 58-end of SNRPN but not atPW71. This result was repeated with different South-ern blots and was confirmed in genomic DNA from fi-broblasts (Fig. 2). The patient’s mother showed a nor-mal pattern of methylation at both sites. Another simi-lar case was recently described with an atypicaldeletion detected by FISH analysis (cosmid D15S113)and polymorphism analysis (D15S210 and D15S113)[Gilbert et al., 1997].

On one hand, these discordances seem to be rare butlead us to point to the interest of preferentially testingAS patients with SNRPN probe. On the other hand,patients initially classified in the group without iden-tifiable molecular defect after PW71, FISH, and micro-satellite tests must be analyzed again with SNRPN,avoiding a useless mutation screening of UBE3A.

In the second example, a PWS male presented withneonatal hypotonia and feeding problems, hypogonad-ism, and typical appearance. The diagnosis of PWS was

*Correspondence to: M-A Voelckel, Ph.D., Laboratoire de Ge-netique Moleculaire, Departement de Genetique Medicale, Hopi-tal d’Enfants de la Timone, 264 rue Saint-Pierre, 13 385 MarseilleCedex 5, France. E-mail: mvoelcke@ap-hm.fr

Received 28 October 1997; Accepted 26 March 1998

American Journal of Medical Genetics 78:242–244 (1998)

© 1998 Wiley-Liss, Inc.

confirmed by the manifestation of obesity with hyper-phagia at the age of 3 years. The karyotype was nor-mal, and DNA analyses were performed to assay for amolecular defect.

The first analyses were performed in 1993 with het-eropolymorphisms and restriction fragment lengthpolymorphisms with the probes D15S11 and D15S10

and showed the presence of two copies by quantitativeSouthern blotting. Methylation studies with the PW71and exon 1 of SNRPN probes were normal. We thenperformed FISH analysis using the standard chromo-some 15q11–q13 probes and observed a deletion of the15q11–q13 region in 20% of the cells. Somatic mosa-icism was described previously by Cassidy et al. [1984]in two patients using high-resolution prometaphasebanding techniques. Recently, Mowery-Rushton et al.[1996] reported four cases (one typical and three atypi-cal PWS) in whom mosaicism for the deletion was iden-tified by FISH analysis. The results obtained with thisPWS patient indicate that analysis of allele-specificmethylation at SRNPN and PW71 is not sensitiveenough to identify cases of mosaic deletion, because thenormal cell population masks the uniparental methyl-ation of the deleted chromosome 15 in these cases.

The PW71 methylation test is a powerful laboratorytool and can be very helpful to the physician. However,a negative PW71 methylation test cannot rule out asure clinical diagnosis. In our experience, in AS andPWS, a precise clinical examination remains essentialwhen the molecular methylation screening is negative.For a very suggestive clinical diagnosis, FISH analysisin at least 100 metaphases and SNRPN methylationtest (in various tissue types) could be performed as ad-ditional data, and, in AS, before beginning the muta-tion screening in UBE3A.

ACKNOWLEDGMENTS

We wish to thank Dr. Marc Lalande for helpful dis-cussion.

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Fig. 2. Southern hybridization pattern using (1) DNA probe PW71B(D15S63) with double-digest HindIII + HpaII in lymphocytes, (2) DNAprobe SNRPN exon 1 with double-digest XbaI + NotI in lymphocytes, and(3) DNA probe SNRPN exon 1 with double-digest XbaI + NotI in fibro-blasts. The large fragments 6.6 kb in 1 and 4.2 kb in 2 and 3 represent themethylated site of maternal origin; the small fragments 4.7 kb in 1 and 0.9kb in 2 and 3 represent the unmethylated site of paternal origin. The blacksquares represent the patient with AS; N, normal individual with normalpattern; AS, methylation pattern of a control patient with AS; PWS, meth-ylation pattern of a control patient with PWS.

Fig. 1. Segregation of chromosome 15q11–q13 microsatellite genotypesin the patient with AS and his parents. The results are given as the physi-cal cartography in the following order: centromere–D15S11–D15S63–SNRPN–D15S128–D15S210–D15S113–GABRB3–155.1CA–85CA–telomere.

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