SHORT REPORT
Dissection of the MYCN locus in Feingold syndrome
and isolated oesophageal atresia
Marie Cognet1, Agne´s Nougayrede1, Vale´rie Malan1,9, Patrick Callier2, Celia Cretole3, Laurence Faivre2,
David Genevieve4, Alice Goldenberg5, Delphine Heron6, Sandra Mercier7, Nicole Philip8, Sabine Sigaudy8,
Alain Verloes9, Sabine Sarnaki3, Arnold Munnich1,10, Michel Vekemans1,10, Stanislas Lyonnet1,10,
Heather Etchevers1, Jeanne Amiel1,10 and Loı¨c de Pontual*,1,11
Feingold syndrome (FS) is a syndromic microcephaly entity for which MYCN is the major disease-causing gene. We studied
the expression pattern of MYCN at different stages of human embryonic development and collected a series of 17 FS and
12 isolated oesophageal atresia (IOA) cases. An MYCN gene deletion/mutation was identified in 47% of FS cases exclusively.
We hypothesized that mutations or deletions of highly conserved non-coding elements (HCNEs) at the MYCN locus could lead
to its misregulation and thereby to FS and/or IOA. We subsequently sequenced five HCNEs at the MYCN locus and designed
a high-density tiling path comparative genomic hybridization array of 3.3Mb at the MYCN locus. We found no mutations or
deletions in this region, supporting the hypothesis of genetic heterogeneity in FS.
European Journal of Human Genetics advance online publication, 12 January 2011; doi:10.1038/ejhg.2010.225
Keywords: Feingold syndrome; MYCN; genetic heterogeneity
INTRODUCTION
Feingold syndrome (FS, MIM164280) combines characteristic digital
anomalies (ie, brachymesophalangy of the second and fifth fingers and
brachysyndactyly of the toes), microcephaly, oesophageal/duodenal
atresia, and variable learning disabilities.1 FS has been mapped to
2p23–242 and is the consequence of MYCN gene (MIM 164840) loss-
of-function either by germline deletions or by coding-sequence
mutations.3,4 Conversely, MYCN amplification is a prognostic factor
for a bad outcome and is found in about 10% of neuroblastomas.5
In this study, we studied the expression pattern of MYCN at
different stages of human embryonic development, and screened a
cohort of 17 patients suspected of FS and 12 patients with isolated
oesophageal atresia (IOA). We identified a heterozygous mutation/
deletion in seven FS cases (47%) and no mutation or deletion in IOA.
Some highly conserved non-coding elements (HCNEs), able to direct
N-myc expression, have been identified in transgenic mice6–8 We
hypothesized that deregulation of tissue- or stage-specific MYCN
expression following mutation or disruption of regulatory HCNEs
at the MYCN locus could lead to FS and/or IOA. We subsequently
sequenced five HCNEs at the MYCN locus and searched for small
deletions in the 3.3-Mb vicinity of MYCN.
PATIENTS AND METHODS
A total of 29 patients were included in the study: 17 patients with possible FS
(Table 1) and 12 patients with IOA. Diagnostic criteria for FS were the presence
of three or more of the core features: (i) microcephaly, (ii) brachymesophalangy
of the second and fifth finger, (iii) 2/3 or 4/5 toe syndactylies, and (iv) oesophageal
or duodenal atresia. Whereas postnatal microcephaly was constant after 3 years of
age, head circumference was normal at birth in three cases. All patients showed
mild-to-moderate mental retardation and eight developed postnatal growth
retardation. Brachymesophalangy of the second and fifth finger was noted in
15 cases, syndactylies in 12 cases, and oesophageal atresia in 14 of the 17 cases
(Figure 1, Table 1). Additional features are listed in Table 1. All IOA cases were
sporadic (10 type III and 2 type I), with no additional malformations.
Blood samples were obtained with informed consent and DNAwas extracted
according to standard protocols. DNA sequencing of the three coding exons
and intronic flanking regions was performed by the fluorometric method on
both strands (ABI BigDye Terminator Sequencing Kit V.2.1, Applied Biosys-
tems). Comparative genomics analysis of the MYCN locus indicated five
HCNEs with 475% identity over 350 bp across humans, rhesus, dog, and
mouse (Figure 1). These HCNEs were studied by direct sequencing in all
patients with no coding-sequence mutation (primers available on request).
A 3.3-Mb region extending 1.94Mb centromeric (5¢) and 1.36Mb telomeric
(3¢) to MYCN (chr2: 12 800 000–16 590 000; NCBI Build 36.1) was studied by
fine-tiling array-based comparative genomic hybridization (CGH; NimbleGen
Systems, http://www.nimblegen.com/products/cgh/human.html#cnv) on 6 FS
and 10 IOA patients with no MYCN coding-sequence mutation, as well as 550
normal-banded chromosomes on blood karyotype. The average spacing of
probes in Nimblegen fine-tiling array is 52 bp. A deletion was considered when
at least 10 probes were abnormal, giving a deletion detection resolution of
about 500 bp at the MYCN locus. Genome-wide array-CGH with a resolution
of 50 kb was performed in the five FS patients with no MYCN mutation and
normal Nimblegen fine-tiling array, using the Agilent Human Genome CGH
Microarray Kit 244 K (Agilent Technologies, Santa Clara, CA, USA).
To study MYCN expression during human development, embryos were
collected from terminated pregnancies in agreement with French bioethics laws
Received 7 April 2010; revised 19 October 2010; accepted 19 October 2010
1Unite´ INSERM U-781, Universite´ Paris Descartes, Paris, France; 2Service de Ge´ne´tique, Hoˆpital d’enfants, Dijon, France; 3Services de Chirurgie pe´diatrique, Hoˆpital Necker-
Enfant Malades, AP-HP, Paris, France; 4De´partement de Ge´ne´tique Me´dicale, Centre de re´fe´rence anomalies du de´veloppement, Hoˆpital Arnaud de Villeneuve, Montpellier,
France; 5Service de Ge´ne´tique, Hoˆpital Charles Nicolle, Rouen, France; 6Service de Ge´ne´tique, Hoˆpital de la Pitie´ Salpeˆtrie´re, Paris, France; 7Service de Ge´ne´tique, Hoˆpital Sud,
Rennes, France; 8Service de Ge´ne´tique, Hoˆpital de la Timone, Marseille, France; 9Service de Ge´ne´tique, Hoˆpital Robert Debre´, Paris, France; 10Services de Ge´ne´tique et
cytoge´ne´tique, Hoˆpital Necker-Enfant Malades, AP-HP, Paris, France; 11Services de Pe´diatrie, Hoˆpital Jean Verdier, AP-HP, Bondy, France
*Correspondence: Professor L de Pontual, De´partement de Ge´ne´tique, Hoˆpital Necker-Enfants Malades, 149, rue de Se´vres, 75743 Paris Cedex 15, France. Tel: +33 14 449 5648;
Fax: +33 14 449 5150; E-mail: loic.de-pontual@inserm.fr
European Journal of Human Genetics (2011), 1–5
& 2011 Macmillan Publishers Limited All rights reserved 1018-4813/11
www.nature.com/ejhg

(94-654 and 04-800) and the Necker Hospital ethics committee. Probe
synthesis and hybridization were carried out as described previously.9
RESULTS
Direct sequencing and searching for deletion in MYCN locus
We identified a heterozygous coding-sequence mutation in seven
patients (five novel, Table 1). All mutations resulted in a premature
stop codon that removed the basic helix-loop-helix (b-HLH) and the
leucine-zipper (LeuZ) domains or modified a conserved amino acid
essential for DNA binding (Figure 2). One patient had a deletion of
425 kb encompassing the MYCN gene alone. Six mutations occurred
de novo and one was inherited from the affected father (AO28,
Table 1), who showed brachymesophalangy of the second and fifth
fingers, syndactyly between the fourth and fifth toes, microcephaly,
and mild mental retardation. Additional features observed in patients
harbouring a MYCN coding-sequence mutation or deletion were
congenital heart malformations (two cases), kidney hypoplasia (two
cases), asplenia (one case), and diaphragmatic hernia (one case). This
last malformation had never been reported previously and CGH
analysis showed no additional rearrangements in this patient. The
MYCN locus was further investigated in patients with no coding-
sequence mutations; we sequenced five HCNEs identified in the
MYCN locus (Figure 2) and identified no nucleotide variations in
either FS or IOA patients. Fine-tiling array-based CGH identified no
micro-rearrangements in the 3.3-Mb region encompassing MYCN.
Genome-wide array-CGH 244 K was normal in the five patients with
no MYCN coding-sequence mutation and normal Nimblegen fine-
tiling array (Table 1).
Table 1 Clinical features in the series of 17 FS patients with and without MYCN mutation
Mutated patients AO2 AO28 AO37 A056 A060 A065 A067 A068 Total
Sex F F M M F M F M 4M/4F
Familial history  +   +  +  3/8
Head circumference at birth 2 3 4 3 2 4 3 2 8/8
Postnatal microcephaly (SD) 3 3 4 3 2 4 3 2 8/8
Weight and size at birth 50th c. 25th c. 25–50th c. 2550th c. 25–50th c. 25–50th c. 25–50th c. 25–50th c.
Postnatal growth retardation (SD) 2 2.5 2 2 1 2 0 0 5/8
Mental retardation Mild Moderate Mild Mild Mild Moderate Mild Mild 8/8
Micrognatia      + +  2/8
Brachymesophalangy II et V + + + + + + + + 8/8
Toe syndactyly 2/3  +  + + + +  5/8
Toe syndactyly 4/5 + + +   + +  5/8
Oesophageal atresia + + + + + + +  7/8
Duodenal atresia         0/8
Renal hypoplasia +  +      2/8
Congenital cardiac defect ASD VSD       2/8
Deafness         0/8
Asplenia  +       1/8
Result of MYCN gene screening c.1180G4A c.1293delC c.1110insG c.928-930insGT c.474-514del c.1177C4T c.134dupC del 2p24.3 8/8
Non-mutated patients AO3 AO4 AO22 AO35 AO36 AO39 AO41 AO42 AO43 Total
Sex M F F M M F M F F 4M/5F
Familial history      +a +b   2/9
Head circumference at birth –2.5 0 2 0 2.5 0 –3 4 1 5/9
Postnatal microcephaly (SD) –2.5 2 2.5 2 2.5 –3 –3 4 2 9/9
Weight and size at birth 25–50th c. 50th c. 50th c. 50th c. 50th c. 50th c. 25–50th c. 50th c. 50th c.
Postnatal growth retardation (SD) 1 1  2 1.5 0 2.5 3 0 3/9
Mental retardation Mild Moderate Mild Mild Mild Mild Moderate Mild Mild 9/9
Micrognatia  +  +      2/9
Brachymesophalangy II et V +  + + + + + +  7/9
Toe syndactyly 2/3  + +   +  +  4/9
Toe syndactyly 4/5    +  +    2/9
Oesophageal atresia + +  + + +  + + 7/9
Duodenal atresia    +      1/9
Renal hypoplasia          0/9
Congenital cardiac defect     VSD, MA, AC    VSD 1/9
Deafness    +      0/9
Asplenia          0/9
Result of MYCN gene screening          0/9
Result of Nimblegen fine-tiling array Normal Normal Normal Normal Normal Normal NP NP NP 0/6
Result of 244K genome wide array Normal Normal Normal Normal Normal NP NP NP NP 0/5
Abbreviations: AC, aortic coarctation; ASD, atrial septal defect; del, deletion; F, female; M, male; MA, mitral atresia; VSD, ventricular septal defect.
aThe father and a sister of AO39 are microcephalic and have digital anomalies (brachymesophalangy of the second and fifth fingers and brachysyndactyly of the toes). The sister has also learning
disabilities.
bThe mother of AO41 is microcephalic and has anomalies in the hand (brachymesophalangy of the second and fifth fingers).
MYCN in Feingold syndrome and isolated oesophageal atresia
M Cognet et al
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European Journal of Human Genetics