A fluorescent method for detecting low-grade 11patUPD
mosaicism in Beckwith–Wiedemann syndrome
Silvia Russo
a,
*
, Monica Mencarelli
a
, Florinda Cavalleri
a
, Angelo Selicorni
b
,
Francesca Cogliati
a
, Lidia Larizza
a,c
a
Laboratory of Molecular Biology, Istituto Auxologico Italiano, Milan 20135, Italy
b
II Pediatric Department, University of Milan, Milan, Italy
c
Department of Biology and Genetics, University of Milan, Milan, Italy
Received 29 April 2002; accepted for publication 31 July 2003
Abstract
The quantitative evaluation of mosaicism for uniparental disomy (UPD) involving a restricted chromosomal region requires the
availability of a sensitive and reproducible method that is capable of detecting even a small percentage of disomic cells and avoiding false
positive and false negative results. The occurrence of UPD is usually monitored by means of the parent–proband segregation analysis of
microsatellites mapping to the target region. We here describe the quantitative blood cell evaluation of segmental mosaic UPD11, a marker of
Beckwith–Wiedemann syndrome, by means of the segregation analysis of 11p15 microsatellites using both radioactive and fluorescence-
based techniques. As the greater amplification efficiency of the shorter allele in heterozygous subjects may bias the correct evaluation of
disomy, the mean short/long allele ratio was established at three loci of each of 30 normal heterozygous subjects, as well as the peak A
s
=A
l
area in the presence of 50% of each allele. The interval was defined using a 5% level of significance.
The results show that the fluorescence-based technique is superior to radioactivity in detecting the subtle allelic imbalances present in low-
grade mosaicism conditions.
q 2003 Elsevier Ltd. All rights reserved.
Keywords: Fluorescence-based and radioactive methodology; Mosaicism; Uniparental disomy; Beckwith –Wiedemann syndrome
1. Introduction
Beckwith–Wiedemann syndrome (BWS, MIM#130650),
the most common of the overgrowth syndromes, is
characterised by anterior abdominal wall defects, macro-
glossia and gigantism [1,2], and increased susceptibility
(7.5%) to childhood tumours including Wilms’ tumours,
adrenocortical carcinomas and hepatoblastomas [3,4]. Its
presentation is sporadic in most patients but familial in 15%
of cases [5]. It is a genetically heterogeneous and complex
imprinted disorder: linkage studies offamilial cases [6,7] and
cytogenetic 11p anomalies in sporadic cases [8 –11] indicate
the etiological involvement of the 11p15.5 region, which
contains a cluster of at least eight imprinted genes [11].The
known pathogenetic mechanisms of BWS are: (a) rare
(,1%) chromosomal abnormalities affecting 11p15, such as
maternally derived translocations/inversions [12]; (b) partial
or complete trisomy 11 with paternal duplication (, 1%)
[13–15]; (c) uniparental paternal disomy (UPD) of 11p15
(20%) [16]; and (d) maternally inherited germline mutations
in the coding region of p57
KIP2
(5–20%) [19,20]. There may
also be still undiscovered etiologies having consequences
such as the bi-allelic expression of paternally expressed
11p15.5 genes, including insulin growth factor 2 (IGF2),
which is normally transcribed only from the paternal allele at
a frequency ranging from 20% [17] to 80% [12,18], and that
of a long QT intronic transcript 1 (LT1) transcribed in the
antisense orientation within KCNQ1, a gene encoding a
voltage-gated potassium channel that encompasses all of the
translocation 11p15 breakpoints in 50% of the patients [18].
The aim of this study was to establish the most suitable
technical approach to testing BWS patients with paternal
UPD, a mechanism which, after karyotyping, is the first step
in the diagnostic flow chart of our laboratory. All of
the reported BWS UPD cases are mosaic for paternal 11p15
0890-8508/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.mcp.2003.07.002
Molecular and Cellular Probes 17 (2003) 295–299
www.elsevier.com/locate/ymcpr
*
Corresponding author. Tel.: þ39-255192860; fax: þ 39-25456913.
E-mail address: s.russo@auxologico.it (S. Russo).