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Dissection of the MYCN locus in Feingold syndrome and isolated oesophageal atresia

Abstract

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 3.3 Mb at the MYCN locus. We found no mutations or deletions in this region, supporting the hypothesis of genetic heterogeneity in FS.

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, 7, 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.

Table 1 Clinical features in the series of 17 FS patients with and without MYCN mutation
Figure 1
figure1

Patient AO68 with FS and MYCN deletion. Note the round face, brachymesophalangy of the second and fifth fingers, and short feet with brachydactyly.

Blood samples were obtained with informed consent and DNA was 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 Biosystems). Comparative genomics analysis of the MYCN locus indicated five HCNEs with >75% 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 1.94 Mb centromeric (5′) and 1.36 Mb 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 (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).

Figure 2
figure2

Schematic representation of the MYCN locus (6647 bp). The deletion and mutations identified in five FS patients and HCNEs with >75% identity over 350 bp across humans, rhesus, dog, and mouse (ECR browser software) are shown.

MYCN expression in early human development

Additional features observed in patients with an MYCN mutation motivated the study of MYCN expression at different stages of human embryonic development (Figure 3). At Carnegie stage (CS) 13, MYCN appears ubiquitously expressed, with higher expression in the limb-bud mesenchyme (Figures 2a and b). At CS 15, MYCN is differentially and highly expressed in the CNS/PNS, the oesophageal and bronchic epithelia, Rathke's pouch, sympathetic ganglia, and both ectodermal and mesenchymal components of the forelimb. At CS 17 and 18, MYCN is highly expressed throughout the CNS/PNS and in both Rathke's pouch and the corresponding precursor of the neurohypophysis, the infundibulum (Figures 3t and u), the smooth muscle of the umbilical arteries, the adrenal gland, and the hindgut as well as other sites (Figures 3q–u). However, despite low levels of cardiac expression seen in situ at CS 13, we no longer observed any cardiac expression at CS 18 (Figures 3v and w).

Figure 3
figure3

MYCN expression in early human development. Antisense and sense (negative control, b, d, f) riboprobes are presented side by side in panels a to p. (a) At CS 13 (37 days’ gestation) MYCN is ubiquitously expressed. (c) CS 15 (36 days’ gestation), transverse section in head, showing MYCN expression is still widespread but is particularly strong in the brain and craniofacial mesenchyme, as well as in the precursors to the pituitary gland. (e) CS 15, transverse section through the cervical neural tube. (g) CS 15, section at the level of the forelimb. As compared with the control (h), no expression is observed in the liver, but is specific to both epithelia and mesenchyme of bronchi, oesophagus (i, j), and forelimb bud (m, n), as well as at levels of the central and peripheral nervous system (CNS/PNS; il, o, p). (q) Hematoxylin–eosin stain of sagittal section at CS 17 and 18 (42–45 days’ gestation). MYCN expression in an adjacent section (r) shows continued but diminished expression in the CNS/PNS and craniofacial mesenchyme, and presence of transcripts in the adrenal gland, hindgut, and genital tubercule. (t) MYCN continues to be expressed in the developing pituitary, unlike in the ventricular myocardium (v vs w). Abbreviations: ad, adrenal gland; aer, apical ectodermal ridge; ao, aorta; br, bronchi; da, dorsal aorta; di, diencephalon; drg, dorsal root ganglia; eg, enteric ganglia; g, gut; g/IX/X, cranial ganglia IX/X; gt, genital tubercule; h, heart; hg, hindgut; inf, infundibulum; lb, limb bud; lv, liver; mes, mesencephalon; mx, maxilla; nt, neural tube; oe, oesophagus; rp, rathke's pouch; rh, rhombencephalon; sc, spinal cord; sg, sympathetic ganglion; st, stomach; tel, telencephalon; tg, tongue; umb, umbilical cord. Scale bars: 0.5 mm, except for qs, 1 mm.

Discussion

We identified an MYCN mutation in 50% of our cases (8/17). No major phenotypic differences could be found among the core features of FS retrospectively, between patients with and without a MYCN mutation (Table 1). Only syndactyly of toes 4 and 5 was more frequent in the group with MYCN mutations. The high frequency of oesophageal atresia in our series is due to a recruitment bias through paediatric surgeons. Importantly, no additional malformations were present in the group of patients without mutations. Although head circumference can be normal at birth, postnatal microcephaly is constant in our series. Most patients were sporadic cases, contrasting with a previous report.4 This discrepancy could be ascribed to both a recruitment bias for familial cases before the gene was identified, and the fact that, clinically, the entity is better recognized since then. Several additional congenital malformations have been reported in FS; ie, vertebral malformations, congenital cardiac defects, and renal hypoplasia.4 Renal hypoplasia needs to be detected early on in order to prevent renal failure.2 One of our patients presented asplenia. This has not hitherto been reported in FS but is present in the N-myc hypomorphic mouse model.10 A diaphragmatic hernia was detected in the same patient at birth. Facial features reported in FS are tenuous and combine short palpebral fissures, broad nasal bridge, and micrognathia.

We studied the pattern of expression of MYCN at different stages of normal human embryonic development. MYCN is widely expressed in forelimb mesenchyme at the stages we studied, consistent with the constant distal bone malformations observed in FS. Expression in Rathke's pouch raises the question of involvement of the pituitary gland in the growth deficit. We observed MYCN expression in both bronchial tubes and the oesophagus at CS 15, but not in the diaphragm at CS 17 and 18. N-myc knockout mice had been generated concomitantly by three independent groups.11, 12, 13 Embryonic lethality was consistently observed between embryonic days E10.5 and E12.5 of gestation, with developmental delay and small size of mesonephros, lung, heart, and gut. Interestingly, mutant mice with 25% of wild-type levels of N-myc protein die at birth and are unable to breathe because of a severe deficiency in lung-branching morphogenesis.10

The molecular mechanisms underlying the regulation of MYCN expression have not been totally elucidated. It has been shown, by replacing endogenous N-myc coding sequences by the c-myc ones, that c-myc can complement N-myc functions.14 Therefore, the specificity of both genes resides in their controlled expression patterns. No mutation/deletion of MYCN regulatory elements could be identified in humans. Altogether, these results are suggestive of genetic heterogeneity in FS.

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Correspondence to Loïc de Pontual.

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Cognet, M., Nougayrede, A., Malan, V. et al. Dissection of the MYCN locus in Feingold syndrome and isolated oesophageal atresia. Eur J Hum Genet 19, 602–606 (2011). https://doi.org/10.1038/ejhg.2010.225

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Keywords

  • Feingold syndrome
  • MYCN
  • genetic heterogeneity

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