Introduction
Intestinal neuronal dysplasia type B (INDB; OMIM
601223) is a malformation of the enteric nervous system (ENS) that
accounts for over 95% of cases of isolated IND (1) and is characterized by malformation of
the submucosal plexus. Children with INDB present with intractable
constipation and grossly slowed intestinal transit time. The
histological features of INDB include hyperplasia of the submucosal
plexus, giant ganglia, ectopic ganglion cells at the muscular and
mucosa layers, and increased acetylcholinesterase activity in the
lamina propria and around submucosal blood vessels, which indicates
immaturity of the ENS (1). The
clinical picture of INDB resembles Hirschsprung disease (HSCR; OMIM
142623), a congenital disorder characterized by the absence of
intramural ganglion cells in the myenteric and submucosal plexuses
along a variable portion of the distal intestine. INDB does not
include a region of aganglionosis, in absolute contrast to HSCR,
but as in HSCR, it is reported to sometimes show increased
extrinsic nerve fibers in the affected gut (2). In addition, some investigators have
reported that 25–35% of patients with HSCR have associated INDB
(2). The lack of unified criteria
for the diagnosis of INDB has led to doubt regarding whether INDB
exists as a distinct histopathology entity. Moreover, the clinical
features of INDB are shared in common with immaturity of the ENS
and arise in healthy individuals at an early age. However, a
concerted effort has been made by scientists and clinicians to
establish some generally accepted criteria for the diagnosis of
INDB (2). This has led to great
advances in the understanding of its pathological basis.
The etiopathogenesis of INDB remains obscure. It is
generally accepted that it is caused by a delay in ENS maturation,
although its association with intestinal chronic obstruction and
HSCR indicates that it may arise as a secondary response to
obstruction or inflammation of the bowel, either in the foetal or
postnatal period (3). The genetic
basis and congenital origin of INDB were determined based on a
study of affected monozygotic twins and on reports of families in
which several members had biopsy-proven INDB across multiple
generations, although with no specific identified genetic
alterations (4,5). Several genes have been described to
play some aetiological role in HSCR pathogenesis, usually related
to the developmental programme of neural crest cells (6). Since HSCR and INDB are ENS disorders
that frequently occur in combination, common molecular pathways
involved in the genesis of the two pathologies may exist. However,
no mutations have been found in the most relevant genes implicated
in HSCR, such as RET and GDNF, in several INDB
patient series (7,8), or in other screened genes (5,9,10).
By contrast, a certain combination of single nucleotide
polymorphisms (SNPs) in the RET protooncogene was found to
be associated with the INDB phenotype in a previous study performed
by our group (8).
The mutation spotting lethal (sl) at Ednrb
which, in homozigosis, leads to aganglionosis in rats, provokes, in
heterozygosis, hyperganglionosis and giant ganglia in the
submucosal plexus (11). In
addition, giant ganglia have also been observed in the aganglionic
region of Edn3-deficient mice (12). The EDNRB and EDN3
genes have previously been evaluated as susceptibility genes for
INDB, with no mutations identified (7). However, the sensibility of the
technique used was not sufficient to completely exclude these genes
as INDB susceptibility factors. In addition, the absence of coding
mutations in these genes does not rule out the existence of other
variants that may confer susceptibility to INDB, as is the case
with the RET high-susceptibility-risk haplotype for HSCR
(13). The aim of the present
study was to determine whether the EDNRB/EDN3 signalling pathway
plays a role in the pathogenesis of INDB. To this end, a mutational
screening of its coding sequence was conducted, and the identified
polymorphisms and haplotypes were evaluated as susceptibility
factors for this disease.
Patients and methods
The study comprised a total of 23 patients
presenting with INDB histologically diagnosed using the criteria
updated by Meier-Ruge et al (2). Of note, one of the INDB patients
included in this series had a monozygotic twin, also affected by
INDB.
Triads composed of the patient and both parents were
complete for 22 of the patients. In addition, a control group
comprising 150 unrelated, race, age and gender-matched individuals,
without any symptoms suggestive of ENS alterations, was analyzed.
Informed consent was obtained from all the participants for
clinical and molecular genetic studies. The study conformed to the
tenets of the declaration of Helsinki and was approved by the
Hospitales Universitarios Virgen del Rocío IRB. Mutational
screening was carried out by denaturing high performance liquid
chromatography (dHPLC) and sequence analysis, as previously
described (14).
Large-scale genotyping of EDNRB and
END3 polymorphisms was performed in the INDB triads and
controls as previously described (14). Allelic, genotypic and haplotypic
frequencies and distributions were compared between patients and
controls by SPSS version 15.0 for Windows. In addition, previously
obtained data from 196 isolated HSCR patients were used (14). In each analysis, statistical
significance was calculated using Pearson’s χ2 test,
with statistical significance set at α=0.05. Haplotypes comprising
the EDNRB and EDN3 polymorphisms analysed were
generated based on the results of the complete triads when
available (patient, father and mother), which allowed us to
reconstruct and compare the transmitted vs. non-transmitted
alleles. With these results, we proceeded to compare the
distribution of haplotypes among the patients and controls.
Statistical estimates were calculated using the method outlined
above.
Results
The mutational screening of the EDNRB and
EDN3 coding regions in 23 INDB patients revealed the
presence of nine sequence variants, three of which had never been
previously reported (Table I).
Since both the silent changes and intronic variants generate no
variation at the protein sequence level, it was more probable that
their pathogenic mechanism, if any, would operate by affecting
transcript stability, RNA splicing or DNA-protein binding. Thus, as
a first approach, those variants were submitted to several splice
site and transcription factor binding site sequence prediction
interfaces (http://www.fruitfly.org/seq_tools/splice.html;
http://www.fruitfly.org/cgi-bin/seq_tools/promoter.pl;
http://www.ebi.ac.uk/asd-srv/wb.cgi).
None of these variants were predicted to have any impact on the
expression and maturation of mRNA in silico.
 | Table I.Sequence variants detected in
EDNRB and EDN3 mutational screening in INDB
patients. |
Table I.
Sequence variants detected in
EDNRB and EDN3 mutational screening in INDB
patients.
| Gene | Nucleotide
change | Amino acid
change | Novel/previously
described | Allelic frequency in
control population (%) |
|---|
| EDNRB | | | | |
| c.732G>A | T244T | Novel | 0.5 |
| c.802-139A>G | | Novel | 0.0 |
| c.802-122T>C | | rs9530703 | 6.7 |
| c.831A>G | L277L | rs5351 | 56.5 |
| c.914G>A | S305N | rs5352 | 2.3 |
| EDN3 | | | | |
| c.53-57C>T | | Novel | 4.0 |
| c.365+23G>A | | rs11570257 | 4.0 |
| c.621-80T>C | | rs11570349 | 0.0 |
|
c.*240delCC | | rs34516274 | 28.5 |
The most notable result was a heterozygous
conservative substitution of serine to asparagine at codon 305 of
the EDNRB gene in two independent patients. This variant was
previously described in an HSCR patient and was proposed to be a
causative mutation due to the hypothetical disruption of a putative
phosphorylation site and the absence of the variant in 50 control
individuals tested (15). However,
in the present study, we detected this variant in 5 out of 150
control individuals in a heterozygous state. These results are in
accordance with the available data on the genotypic frequency of
S305N in a European population (http://www.ncbi.nlm.nih.gov/SNP). Thus, it seems that
the S305N variant is a rare polymorphism rather than a mutation
related to HSCR or INDB.
The genotypic data of three EDNRB SNPs
(IVS1-4125C>T, c.561C>T and c.*1985 G>A) and three
EDN3 SNPs (IVS2+7474T, IVS2-3935 C>G and IVS5+236 G>A)
in the INDB patients and controls were obtained using TaqMan
technology. These data, together with the available data from 196
HSCR patients previously obtained by our group (14), were used to analyse the allelic
(Table II) and genotypic (Table III) distributions among the patient
and control groups. No statistical differences were found in the
distribution of any of these variants or the generated haplotypes
comprising them when INDB patients were compared to HSCR patients
or when INDB patients were compared to control individuals.
Additionally, no statistical differences were obtained in a
transmission disequilibrium test (TDT) analysis (data not
shown).
| Table II.Allelic distribution and frequency of
EDNRB and EDN3 genotyped variants among patient and control
groups. |
Table II.
Allelic distribution and frequency of
EDNRB and EDN3 genotyped variants among patient and control
groups.
| Variant | INDB vs. HSCR
(%) | INDB vs. controls
(%) |
|---|
| EDNRB | | |
| c.484-4125
C>T | | |
| C | 37 (80.4) vs. 186
(84.5) | 37 (80.4) vs. 170
(83.3) |
| T | 9 (19.6) vs. 34
(15.5) | 9 (19.6) vs. 34
(16.7) |
| χ2=0.40,
p=0.5266483 | χ2=0.22,
p=0.63794669 |
| I187I | | |
| C | 46 (100) vs. 219
(99.5) | 46 (100) vs. 202
(100) |
| T | 0 (0) vs. 1
(0.5) | 0 (0) vs. 0 (0) |
| Fisher’s p=1 | NA |
| c.+1985 G>A | | |
| G | 35 (87.5) vs. 188
(88.7) | 35 (87.5) vs. 172
(86.0) |
| A | 5 (12.5) vs. 24
(11.3) | 5 (12.5) vs. 28
(14.0) |
| χ2=0.05,
p=0.8302636 | χ2=0.06,
p=0.8014447 |
| EDN3 | | |
| c.365+7474
T>C | | |
| T | 32 (66.7) vs. 154
(80.5) | 32 (66.7) vs. 127
(63.5) |
| C | 16 (33.3) vs. 64
(19.5) | 16 (33.3) vs. 73
(36.5) |
| χ2=0.30,
p=0.5866268 | χ2=0.63,
p=0.4262079 |
| c.366-3935
C>G | | |
| C | 37 (80.4) vs. 182
(75.9) | 37 (80.4) vs. 176
(83.8) |
| G | 9 (19.6) vs. 38
(24.1) | 9 (19.6) vs. 34
(16.2) |
| χ2=0.14,
p=0.7108324 | χ2=0.31,
p=0.5792162 |
|
c.*231+236 G>A | | |
| G | 32 (69.6) vs. 169
(76.8) | 32 (69.6) vs. 156
(83.9) |
| A | 14 (30.4) vs. 51
(23.2) | 14 (30.4) vs. 30
(16.1) |
| χ2=1.08,
p=0.2978297 | χ2=2.16,
p=0.1415837 |
| Table III.Genotypic distribution and frequency
of EDNRB and EDN3 genotyped variants among patient and control
groups. |
Table III.
Genotypic distribution and frequency
of EDNRB and EDN3 genotyped variants among patient and control
groups.
| Variant | DNI B vs. HSCR
(%) | DNI B vs. controls
(%) |
|---|
| EDNRB | | |
| c.484-4125
C>T | | |
| CC | 16 (69.6) vs. 79
(71.2) | 16 (69.6) vs. 70
(69.6) |
| CT | 5 (21.7) vs. 29
(26.1) | 5 (21.7) vs. 30
(29.4) |
| T | 2 (8.7) vs. 3
(2.7) | 2 (8.7) vs. 2
(2.0) |
| χ2=0.08,
p=0.7721618 | χ2=0.32,
p=0.5695065 |
| I187I | | |
| CC | 23 (100) vs. 109
(99.1) | 23 (100) vs. 101
(100) |
| CT | 0 (0) vs. 1
(0.9) | 0 (0) vs. 0
(0.0) |
| T | 0 (0) vs. 0
(0.0) | 0 (0) vs. 0
(0.0) |
| Fisher’s p=1 | NA |
| c.+1985
G>A | | |
| GG | 15 (75.5) vs. 82
(87.4) | 15 (75.5) vs. 72
(72.0) |
| GA | 5 (25.5) vs. 24
(22.6) | 5 (25.5) vs. 28
(28.0) |
| AA | 0 (0) vs. 0
(0.0) | 0 (0) vs. 0
(0.0) |
| χ2=0.05,
p=0.8302636 | χ2=0.08,
p=0.7838616 |
| EDN3 | | |
| c.365+7474
T>C | | |
| TT | 10 (41.7) vs. 60
(55.0) | 10 (41.7) vs. 42
(40.0) |
| TC | 12 (50) vs. 34
(31.2) | 12 (50) vs. 43
(41.0) |
| CC | 2 (8.3) vs. 15
(13.8) | 2 (8.3) vs. 20
(19.0) |
| χ2=3.13,
p=0.20867686 | χ2=1.70,
p=0.42667582 |
| c.366-3935
C>G | | |
| CC | 14 (60.9) vs. 75
(68.2) | 14 (60.9) vs. 75
(60.0) |
| CG | 9 (39.1) vs. 32
(29.1) | 9 (39.1) vs. 46
(36.8) |
| GG | 0 (0) vs. 3
(2.7) | 0 (0) vs. 4
(3.2) |
| χ2=1.40,
p=0.49628003 | χ2=0.01,
p=0.9197185 |
|
c.*231+236 G>A | | |
| GG | 11 (47.8) vs. 66
(60.0) | 11 (47.8) vs. 61
(62.2) |
| GA | 10 (43.5) vs. 37
(33.6) | 10 (43.5) vs. 34
(34.7) |
| AA | 2 (8.7) vs. 7
(6.4) | 2 (8.7) vs. 3
(3.1) |
| χ2=1.16,
p=0.55955014 | χ2=1.02,
p=0.3119877 |
Discussion
The pathogenesis of INDB is not completely
understood and its aetiology remains unknown, although several
pieces of evidence suggest that it has a genetic basis. The
presence of histological features characteristic of INDB in murine
models with heterozygous mutations at Ednrb and Edn3
(11,12) indicate that this signalling pathway
may be implicated in the pathogenesis of INDB, as is the case in
HSCR. However, the results presented here and in previous reports
(7) fail to reveal any causal
variant in IDNB patients. We also failed to find any association
between SNPs or the haplotypes comprising them and the disease. The
lack of associations between INDB and the EDNRB or
EDN3 genes suggests that the endothelin signalling pathway
is not implicated in the pathogenesis of this disease. However, the
small patient sample presented here does not allow this hypothesis
to be completely ruled out. In fact, a low number of patients is a
recurring problem in the analysis of the genetic basis of IDNB
(5,7,9,10),
due to the low prevalence of the disease and the difficulties
involved in the clinical diagnosis and management of patients.
Several animal models with mutations of various
genes involved in ENS development, such as Hox1L11,
Spry2, Ednrb and Edn3, share histological
features with the INDB phenotype in humans (11,16–18),
suggesting that hyperganglionosis in ENS could be due to defects in
a variety of such genes, alone or in combination. For this reason,
it would be useful to completely characterize the genetic
background of patients with INDB and to analyse whether different
genes interact in order to increase the number of nerve cells and
ganglia. Given the negative results obtained to date in studies of
the molecular causes of INDB (5,7,9,10),
we suggest the adoption of a completely new experimental approach
to elucidate the genetic basis of INDB. In this vein, animal models
with deleted Bmp4, a protein implicated in ENS formation, present
hypoganglionosis (19). It is
tempting to speculate that the overexpression of BMP4 protein may
cause an increase in the number and size of enteric ganglia. In
fact, overexpression of Ntf3 promotes hyperplasia of the myenteric
plexus in rats (20). In the same
manner, we propose that INDB arises as a consequence of gain of
function mutations in genes related to ENS development. Therefore,
a different molecular approach to studying the genetic basis of
INDB, which includes screening for genetic duplications or enhancer
mutations, may be required.
Although the genetic basis of INBD remains to be
elucidated, it must be acknowledged that our results and those
presented elsewhere (5,7,9,10)
also support the possibility that INDB is a secondary consequence
of environmental factors, such as intestinal stenosis or intestinal
obstruction (21). It is well
known that individuals are not only a result of their genetic
information, but also of interaction with their surrounding
environment. Despite the importance that intestinal obstructive
lesions may play in the INDB phenotype, we believe that, before
ruling out a genetic basis, it is necessary to completely analyse
the genetic factors that may be associated with this disease by
evaluating an adequate number of patients and by adopting
alternative experimental approaches.
Acknowledgements
We would like to thank all the
patients who participated in the study, as well as their families
for their collaboration. This study was funded by the Fondo de
Investigación Sanitaria, Instituto de Salud Carlos III (ISCIII),
Spain (PI070080, and PI071315 for the E-Rare project), Consejería
de Salud (PI0249-2008) de la Junta de Andalucía, Spain; and
Consejería de Innovación Ciencia y Empresa (CTS-2590) de la Junta
de Andalucía, Spain. The CIBER de Enfermedades Raras is an
initiative of ISCIII. A.S.M. is a pre-doctoral fellow funded by
ISCIII, Spain.
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