Pediatr Surg Int (2013) 29:855–872
DOI 10.1007/s00383-013-3351-3
REVIEW ARTICLE
Classification and diagnostic criteria of variants
of Hirschsprung’s disease
Florian Friedmacher • Prem Puri
Published online: 14 August 2013
Ó Springer-Verlag Berlin Heidelberg 2013
Abstract ‘‘Variants of Hirschsprung’s disease’’ are con-
ditions that clinically resemble Hirschsprung’s disease
(HD), despite the presence of ganglion cells in rectal suc-
tion biopsies. The diagnosis and management of these
patients can be challenging. Specific histological, immu-
nohistochemical and electron microscopic investigations
are required to characterize this heterogeneous group of
functional bowel disorders. Variants of HD include intes-
tinal neuronal dysplasia, intestinal ganglioneuromatosis,
isolated hypoganglionosis, immature ganglia, absence of
the argyrophil plexus, internal anal sphincter achalasia and
congenital smooth muscle cell disorders such as mega-
cystis microcolon intestinal hypoperistalsis syndrome. This
review article systematically classifies variants of HD
based on current diagnostic criteria with an additional
focus on pathogenesis, epidemiology, clinical presentation,
management and outcome.
Keywords Hirschsprung’s disease
Intestinal
neuronal dysplasia
Ganglioneuromatosis

Hypoganglionosis
Immature ganglia
Internal anal
sphincter achalasia
Megacystis microcolon
intestinal hypoperistalsis syndrome
F. Friedmacher
P. Puri (&)
National Children’s Research Centre, Our Lady’s Children’s
Hospital, Crumlin, Dublin 12, Ireland
e-mail: prem.puri@ucd.ie
P. Puri
Conway Institute of Biomolecular and Biomedical Research,
School of Medicine and Medical Science, University College
Dublin, Dublin, Ireland
Introduction
The typical patient with Hirschsprung’s disease (HD) is
either a newborn presenting with delayed passage of
meconium and abdominal distension or a young child with
severe chronic constipation. 80–90 % of all HD cases
produce clinical symptoms and are diagnosed in the neo-
natal period. The diagnosis of HD is confirmed by histo-
logical and histochemical evaluation of rectal suction
biopsies, which demonstrate the absence of ganglion cells
in the submucosa and increased acetylcholinesterase
(AChE) activity in the lamina propria. However, there are a
number of patients who clinically resemble HD despite the
presence of ganglion cells in rectal biopsies. Various terms
such as ‘‘chronic idiopathic intestinal pseudo-obstruction’’,
‘‘intestinal hypoperistalsis syndrome’’ or ‘‘pseudo-Hirsch-
sprung’s disease’’ have been used to describe these
conditions. The diagnosis and management of these
patients can be challenging for both pediatric surgeons and
gastroenterologists.
During the past three decades, our group has focused its
research interest in delineating these conditions based on
specific histological, immunohistochemical and electron
microscopic studies. In 1997, we suggested that ‘‘Variant
Hirschsprung’s disease’’ [1, 2] may be a more appropriate
description for this heterogeneous group of functional
bowel disorders (Table 1) in patients who continue to have
persistent bowel symptoms despite a ganglionic rectal
biopsy. At present, there are only a few articles in the
literature which have attempted to standardize the termi-
nology of HD and allied intestinal disorders [3, 4]. This
review article systematically classifies variants of HD
based on current diagnostic criteria (Fig. 1) with an addi-
tional focus on pathogenesis, epidemiology, clinical pre-
sentation, management and outcome.
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Intestinal neuronal dysplasia
Intestinal neuronal dysplasia (IND) was first described in
1971 by Meyer–Ruge as a hyperplastic malformation of the
enteric plexus [5]. Shortly afterwards, Puri et al. [6]
reported a case of IND in association with HD, who had
rectosigmoid aganglionosis combined with IND of the
descending and transverse colon. Fadda et al. [7] subse-
quently classified IND into two clinical and histological
distinct subtypes: IND type A (IND A), which occurs in
less than 5 % of all IND cases, is characterized by con-
genital aplasia or hypoplasia of the sympathetic innerva-
tion. Patients with IND A typically present in the neonatal
period with abdominal distension, bowel obstruction and
episodes of diarrhea with hemorrhagic stools. IND type B
(IND B) is characterized by hyperplasia of the parasym-
pathetic submucosal plexus and accounts for more than
Table 1 Variants of Hirschsprung’s disease
Intestinal neuronal dysplasia
Intestinal ganglioneuromatosis
Isolated hypoganglionosis
Immature ganglia
Absence of the argyrophil plexus
Internal anal sphincter achalasia
Megacystis microcolon intestinal hypoperistalsis syndrome
Fig. 1 Diagnostic algorithm for investigating chronic constipation and functional bowel obstruction in newborn infants and young children
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95 % of all IND cases. Typical histological features of IND
B are hyperganglionosis, giant ganglia, ectopic ganglion
cells and increased AChE activity in the lamina propria and
around submucosal blood vessels [2]. IND occurring in
association with HD is always IND B.
Pathogenesis of IND
There still remains a controversy surrounding the existence
of IND as a distinct histopathological entity [3, 8–10]. It
has been suggested by several authors that the observed
changes seen in IND may be either a variant of normal
bowel development or a secondary acquired phenomenon
caused by congenital obstruction or inflammation [11, 12].
An underlying autoimmune mechanism has also been
proposed [13]. However, multiple familial cases of IND
have been described in the literature, suggesting that there
might also be a genetic component [14, 15]. The strongest
evidence that IND is a real entity arose from experimental
animal models. Hox11L1 is a homeobox gene involved in
the proliferation and differentiation of peripheral neural
crest cells. Two different Hox11L1 knockout mouse mod-
els have been generated [16, 17]. In both cases, homozy-
gous mutant mice developed megacolon at the age of
3–5 weeks. Histological and immunohistochemical evalu-
ation revealed hyperplasia of ganglia similar to the phe-
notype observed in human IND. Another animal model
resulting in a phenotype similar to IND was reported in rats
Pediatr Surg Int (2013) 29:855–872
with a heterozygous mutation of the endothelin B receptor
(EDNRB) showing features of hyperganglionosis, giant
ganglia and hypertrophied nerve fiber strands in the sub-
mucosal plexus [18]. However, mutational screening of
Hox11L1 and EDNRB genes in human patients with IND
demonstrated no mutations in these genes [19–21].
Epidemiology of IND
Intestinal neuronal dysplasia occurs with an incidence of
approximately 1 in every 7,500 newborns [22]. However,
the frequency of isolated IND varies highly between the
different centers worldwide with reported rates between 0.3
and 40 % of all rectal suction biopsies [12, 23, 24]. IND
proximal to a segment of aganglionosis is not uncommon
and has been suggested as a possible cause of persistent
bowel problems after definitive surgery for HD [25]. Some
authors have found IND associated with HD in up to 44 %
of patients, while others have rarely observed this combi-
nation [26–30]. The high variability of patient age, speci-
men type and staining methods has resulted in considerable
confusion in the literature regarding the accurate diagnostic
criteria [31].
Clinical presentation of IND
The majority of patients with IND present with chronic
constipation with or without abdominal distension, thus
resembling HD [32], but with a normal barium contrast
enema. It has been shown that intestinal obstruction is the
most characteristic clinical symptom of IND in infants and
young children [33]. Furthermore, there is a high incidence
of additional congenital anomalies, currently ranging from
25 to 30 % [34]. The most common ones are anorectal
malformations, intestinal malrotation, megacystis, con-
genital short bowel, hypertrophic pyloric stenosis, necro-
tizing enterocolitis and Down syndrome [35, 36].
Diagnosis of IND
Rectal suction biopsy is the method of choice for the diag-
nosis of IND. It is essential to include a sufficient amount of
submucosa in the biopsy specimens. The diagnosis of IND
was previously based on qualitative criteria, thus resulting in
a high intraobserver variation [37, 38]. Therefore, the debate
about the existence of IND as a distinct histopathological
entity remains controversial, which is mainly due to the lack
of consensus in diagnostic criteria [11, 39]. IND was initially
diagnosed on the basis of AChE histochemistry of nerve
fibers in rectal suction biopsies [40, 41] (Fig. 2a, b). How-
ever, since the AChE activity in the lamina propria mucosae
has been shown to be an age-dependent phenomenon that
disappears on maturation of the submucosal plexus [42–44],
857
more specific staining techniques were required to assess
accurately the enteric nervous system [45]. Meier-Ruge et al.
[46–48] have proposed enzyme histochemistry for lactate
dehydrogenase, succinate dehydrogenase and nitric oxide
synthase to evaluate and diagnose IND B quantitatively.
Various other neuronal and glial markers such as nicotin-
amide adenine dinucleotide phosphate diaphorase (NADPH-
d) (Fig. 2c, d), neural cell adhesion molecule (NCAM),
neuron-specific enolase (NSE), cathepsin D, protein gene
protein 9.5 (PGP9.5), S-100 protein, peripherin, synapto-
physin and cuprolinic blue have also been used [49–52].
Cuprolinic blue staining has been suggested as it stains the
whole population of ganglion cells [52, 53], but only the cell
bodies and not the axons, which makes it relatively easy to
distinguish between the individual cells. Furthermore, a
defective innervation of the neuromuscular junction within
the affected bowel segment of patients with IND has been
shown [54]. Abnormal submucosal vasculature is a further
histological finding in isolated IND and IND associated with
HD and may be a useful additional diagnostic feature [55]. In
addition, a reduced number of c-Kit positive interstitial cells
of Cajal (ICCs) has been demonstrated in the myenteric
plexus and muscle layers [56, 57]. More recently, a marked
reduction in PTEN expression in the submucosal and
myenteric plexuses of patients with IND has been discov-
ered, which may explain the observed motility dysfunction
[58]. Due to the controversy in staining techniques and age-
dependent AChE expression, the most commonly used
diagnostic criteria at present are: (1) more than 20 % of 25
submucosal ganglia must be giant ganglia containing 9 or
more ganglion cells and (2) the patient must be older than
1 year, as before that age, giant ganglia may be misinter-
preted due to the fact that immature ganglia often have an
incomplete differentiation in nerve cells [59–61]. Most
patients with IND do not display any specific radiological
features on barium contrast enemas other than rectosigmoid
distension. The rectosphincteric reflex has often been shown
to be present, absent or atypical in these patients [34].
Management of IND
There is broad compliance with the recommendation that
the management of IND B should be conservative in the
first instance, consisting of laxatives and enemas [62]. The
majority of patients have been shown to respond well to
this treatment strategy. However, if bowel symptoms per-
sist longer than 6 months of conservative bowel manage-
ment, surgical treatment options should be considered [63].
Myectomy of the internal anal sphincter has been per-
formed by several authors with satisfactory results [2].
Some investigators also recommend injection of botulinum
toxin into the internal anal sphincter. Resection of the
affected bowel segment and pull-through procedure is
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Fig. 2 AChE staining of a normal rectal suction biopsy (a). Rectal
suction biopsy from a patient with IND showing hyperganglionosis,
giant ganglia and increased AChE activity in the lamina propria (b).
rarely indicated in infants and children with IND [34]. In
adolescent and adult patients with IND, resection of the
dilated and redundant colon segment is often the only
successful therapeutic option [64]. The indication for sur-
gery should not be determined on the basis of histopa-
thological findings alone, instead the decision must be
based on the individual patient’s clinical symptoms and
distress [65].
Outcome of IND
An intensive long-term follow-up involving a team of
experienced pediatric surgeons and gastroenterologists is
necessary in patients with IND and chronic constipation
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NADPH-d staining of a normal submucosal plexus (c). Submucosal
plexus of a patient with IND showing giant ganglia (d)
[66]. Gillick et al. [67] reported functional outcome in 33
patients with IND. After a mean follow-up of 2.4 years,
64 % of their patients had a good response to conservative
management with normal bowel habits and did not require
any surgical intervention. However, 36 % underwent
myectomy of the internal anal sphincter after failed con-
servative treatment. Seven out of these 12 patients had
normal bowel habits after surgery and two were able to stay
clean with regular enemas. Three patients continued having
persistent constipation after myectomy and subsequently
underwent resection of redundant and dilated sigmoid
colon, which resulted in normal bowel habits. Schärli et al.
[29] achieved satisfactory results in 90 % of their patients
within 6 months after internal sphincter myectomy.
Pediatr Surg Int (2013) 29:855–872
Intestinal ganglioneuromatosis
Intestinal ganglioneuromatosis (GNM) is characterized by
diffuse proliferation of nerve fibers with significant
hyperplasia of submucosal and myenteric ganglion cells
causing thickening of the bowel wall [68]. This extremely
rare but severe neoplastic condition leads to chronic bowel
obstruction and is frequently associated with multiple
endocrine neoplasia type 2B (MEN 2B), neurofibromatosis
1 or Cowden syndrome [69].
Pathogenesis
The pathogenesis of intestinal GNM is related to complex
hyperplasia of peptidergic, cholinergic and probably
adrenergic nerve fibers and neurons [70]. Transmural GNM
mainly originates from the myenteric plexus, while
mucosal GNM predominantly affects the submucosal
plexus and is often associated with von Recklinghausen’s
disease [70]. Mutation analysis in patients with MEN 2B
further confirmed a de novo germline Met918Thr mutation
in exon 16 of the RET proto-oncogene [71], suggesting a
clear genetic component to this condition. In addition, a
recent experimental study in mice revealed that deletion of
the PTEN gene on chromosome 10 disrupts the develop-
ment of the enteric nerve system resulting in a phenotype
similar to human intestinal GNM [72].
Epidemiology of intestinal GNM
The exact incidence of intestinal GNM is unknown, but it
has been reported that the frequently associated MEN 2B
syndrome occurs in approximately 1:4,000,000 live births
[71]. Furthermore, it can be estimated that intestinal GNM
is present in 90 % of patients with MEN 2B [73].
Clinical presentation of intestinal GNM
The vast majority of patients with intestinal GNM present
with severe chronic constipation and abdominal distension
due to intestinal obstruction [73, 74], similar to those with
HD. Constipation can alternate with episodes of diarrhea
[75, 76]. The similarity of the gastrointestinal symptoms
between patients with IND and MEN 2B-associated
intestinal GNM has suggested that these two conditions
could be the result of mutations affecting the same domain
of the RET proto-oncogene [23]. Gastrointestinal dysmo-
tility is a common initial presentation of patients with MEN
2B, but the rarity of this syndrome often delays the diag-
nosis. Further findings are mucosal neuromas of the lips
and tongue, as well as medullated corneal nerve fibers,
distinctive facies with enlarged lips and an asthenic
‘‘marfanoid’’ body habitus [71].
859
Diagnosis of intestinal GNM
The diagnosis of intestinal GNM is mainly based on
clinical presentation and histological examination of
rectal suction or full-thickness biopsies showing massive
proliferation of submucosal and myenteric plexuses
comprising thick nerve trunks with scattered mature
neurons, giant ganglia with often 15–40 nerve cells and a
high AChE activity [77–80]. Intestinal GNM appears to
be largely confined to the colon and rectum, unlike
neurofibromatosis, which occurs more commonly in the
small intestine [81]. Although AChE histochemistry has
been suggested to show the typical submucosal and
myenteric changes in this condition, highlighting the
thickness of the nerve fibers, it can also be quite easily
appreciated in standard H&E-stained paraffin sections
[82]. In addition, NSE, synaptophysin and S-100 protein
immunostaining has been introduced to evaluate and
diagnose intestinal GNM [70]. It has also been demon-
strated that the submucosal hyperplasia can be extensive,
but not as prominent as the one described in the
myenteric plexus [82, 83]. As intestinal GNM is fre-
quently associated with MEN 2B, the diagnosis should
prompt additional molecular, endocrinological and
oncological evaluation [84, 85]. In general, a mutational
analysis of the RET proto-oncogene is strongly recom-
mended in all patients with intestinal GNM and MEN
2B as well as their family members [76].
Management of intestinal GNM
Surgical resection of the affected bowel segment is not
always mandatory in patients with MEN 2B-associated
intestinal GNM [86]. It has been shown that in the majority
of cases, the gastrointestinal symptoms can be managed
with daily laxatives and enemas [87]. However, some
patients ultimately require surgery for severe intestinal
bowel obstruction or stricture formation [88]. Furthermore,
all patients with intestinal GNM that carry MEN 2B
mutations should undergo a prophylactic total thyroidec-
tomy to prevent development of medullary thyroid carci-
noma [73, 80].
Outcome of intestinal GNM
Early diagnosis and treatment of patients with MEN 2B-
associated intestinal GNM are essential for long-term sur-
vival. A yearly follow-up with monitoring of basal plasma
calcitonin and CEA levels for possible tumor recurrence is
recommended [80]. In addition, continued surveillance of
the adrenal glands with abdominal ultrasound scanning
and urine analysis of catecholamine metabolites includ-
ing metanephrine, normetanephrine, dopamine and
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vanillylmandelic acid is required as patients with MEN 2B
have at least a 50 % risk of developing pheochromocytoma
[79].
Isolated hypoganglionosis
Isolated hypoganglionosis (HG) is a rare entity and has been
classified as a ‘‘hypogenetic type’’ of intestinal innervation
disorders. The clinical presentation of patients with isolated
HG is similar to those with classical HD with non-specific
symptoms of severe constipation or bowel obstruction [1,
89]. It has been demonstrated that congenital and acquired
HG are two distinct entities with different clinical charac-
teristics and histological findings [90]. There is only a
limited number of cases in the published literature as iso-
lated HG is one of the rarest subtypes of intestinal inner-
vation disorders and there remains a controversy regarding
it as a distinct isolated histopathological entity [11].
Pathogenesis of isolated HG
The pathogenesis and genetic basis of isolated HG both
remain unclear. Several authors have performed mutational
analysis of the RET gene, which has been reported to be
associated with HD, but neither causative missense muta-
tion nor neutral substitutions were found [91, 92]. Rolle
et al. [93] have previously shown that some cases of iso-
lated HG exhibit deficient expression of c-Kit positive
ICCs within the myenteric plexus and the smooth muscle
layer, which may contribute to the motility dysfunction in
the hypoganglionic bowel segment [57]. A lack or reduced
expression of NCAM-positive nerve fibers within the
lamina propria, muscularis mucosae as well as circular and
longitudinal muscle layers of patients with isolated HG has
also been described [89, 94, 95].
Epidemiology of isolated HG
Reports of finding isolated HG in rectal biopsies are rare and
vary between 0.3 and 6.4 % [24, 26, 30]. Since 1978, there
have been a total number of 92 cases published in the English
literature [96]. Thirty-two percent of them were diagnosed in
the newborn period. However, the median age at diagnosis was
4.8 years, which was due to the fact that in some cases the
diagnosis was not made until the patients were adolescents.
Clinical presentation of isolated HG
The most common presenting symptoms of isolated HG are
severe chronic constipation with intestinal obstruction and
enterocolitis, thus resembling classical symptoms of HD.
The median age at diagnosis is considerably higher in
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patients with isolated HG compared to patients with HD,
which is predominantly diagnosed during the newborn
period. Enterocolitis of the newborn has been reported to
be the most serious complication of isolated HG [96].
Diagnosis of isolated HG
It is unclear whether isolated HG represents a severe form
of intestinal dysganglionosis or solely an abnormality of
the enteric nervous system that leads to severe constipation
[11]. Hence, the diagnosis can be difficult and a consensus
in diagnostic criteria still remains to be found. A full-
thickness bowel specimen is required for the definitive
diagnosis of isolated HG [97–100]. The vast majority of
reported cases have been evaluated by immunohisto-
chemical staining showing sparse and small myenteric
ganglia, absent or low AChE activity in the lamina propria
as well as hypertrophy of muscularis mucosae and circular
muscle [101]. Meier-Ruge et al. [102] found significant
histopathological differences between resected bowel
specimens from patients with isolated HG and normal
bowel tissue using AChE staining. They showed a 40 %
reduction in the number of nerve cells, accompanied by a
doubled distance between ganglia and a three times smaller
plexus area in the hypoganglionic bowel segment. These
observations currently form the basis for the histopathol-
ogical diagnosis of isolated HG. It has also been suggested
that the size of the myenteric plexus may be an indicator of
clinical severity [103]. Thus, several neuronal markers
have been introduced to facilitate the diagnosis of isolated
HG. To determine the muscular nitrergic innervation and
differentiation of mature from immature ganglia in patients
with isolated HG, NADPH-d staining has been employed
to demonstrate a reduced number of positive nerve fibers in
the muscularis mucosae with absent or sparse submucosal
and myenteric ganglion cells [104] (Fig. 3a–d). Further-
more, c-Kit staining has been used to investigate the
expression of ICCs and thus intestinal pacemaker activity,
which is markedly decreased or even absent in patients
with isolated HG [56, 93, 105].
Management of isolated HG
The management of isolated HG is similar to that of HD.
According to the literature, a majority of patients undergo
resection of the affected bowel segment with subsequent
pull-through procedure [96, 106].
Outcome of isolated HG
The postoperative outcome after resection of the hypo-
ganglionic segment usually is good [107]. Typical com-
plications of isolated HG are enterocolitis, chronic
Pediatr Surg Int (2013) 29:855–872
Fig. 3 NADPH-d staining in whole mount preparation of a normal myenteric plexus (a, c). Myenteric plexus of a patient with isolated HG
showing markedly reduced number of ganglion cells (b, d)
constipation, overflow encopresis and the need for redo
pull-through operation due to residual hypoganglionosis
[34]. An overall mortality rate of 8 % has recently been
reported with the majority of patients who died being
newborns suffering from severe enterocolitis [34].
Immature ganglia
Immature ganglia (IG) are usually found in biopsies from
premature infants presenting with functional bowel
obstruction. It has been reported that delayed maturation of
ganglion cells in the submucosal and myenteric plexuses is
the most common cause of chronic constipation during the
first year of life [85].
861
Pathogenesis of IG
A combination of large (fully mature) and small (imma-
ture) ganglion cells can be found at birth [108]. In the early
postnatal period, ganglion cells in the submucosal plexus
are generally less developed than the ones in the myenteric
plexus [109]. It has further been demonstrated that this
immaturity is a physiological, age-dependent phenomenon
and maturation of IG strongly correlates with the age of the
patient [3, 109]. Strong evidence supporting this theory has
arisen from several animal studies showing postnatal
maturation of the submucosal and myenteric plexuses
[110–112]. Therefore, the finding of immature ganglion
cells may be an indicator of transient functional immaturity
of the bowel [108].
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Epidemiology of IG
There is not much epidemiological data on the incidence of
IG. Ure et al. [30] found four (2.8 %) cases of immature
ganglion cells in their cohort of 141 patients with intestinal
neuronal malformations. More recently, Puri et al. [34]
reported ten (5.6 %) cases of IG in bowel specimens of 178
patients with variants of HD.
Clinical presentation of IG
Patients with IG usually present with a history of chronic
constipation or functional bowel obstruction resembling
HD. Further clinical features may include slow-transit
peristalsis and insufficient defecation [113].
Diagnosis of IG
In general, the diagnosis of IG can be made from rectal
suction biopsies. The ganglion cells appear very small
and have a less significant nucleus with an inconspicuous
nucleolus [109, 114] (Fig. 4a, b). However, it is often
not possible with AChE histochemistry to distinguish
between these small ganglion cells and the supporting
enteric glial cells. Thus, NADPH-d and NCAM staining
have been suggested as neuronal markers to show the
small ganglion cells more clearly [1, 101]. Enzyme
histochemistry for succinate and lactate dehydrogenase is
also commonly used to determine IG showing an absent
or weak positive reaction [3, 46]. In addition, cathepsin
D has been recommended to evaluate the maturation of
immature ganglion cells in more detail [50]. Another
helpful biomarker to detect immature enteric ganglion
cells is Bcl2 [115], which clearly discriminates these
immature small neurons.
Management of IG
The management of patients with IG is conservative with
use of laxatives and enemas [1, 2].
Outcome of IG
The majority of patients with IG can successfully be
managed with conservative treatment until their ganglion
cells are fully mature. It is recommended to repeat the
biopsy 12–18 months after initial investigation [3].
Absence of the argyrophil plexus
The lack of argyrophil cells in the myenteric plexus, which
is also known as absence of the argyrophil plexus (AP), is a
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Pediatr Surg Int (2013) 29:855–872
rare cause of constipation and functional bowel obstruction
in infants and children.
Pathogenesis of absence of the AP
In the normal myenteric plexus there are two distinct sub-
types of nerve cells, which can be distinguished by their
affinity for silver stains: (1) argyrophil cells and (2) argen-
taffin cells. Argyrophil cells usually comprise 5–20 % of the
total number of myenteric neurons [116]. The processes of
these cells along with extrinsic and parasympathetic fibers
form a complex neuronal network within the myenteric
plexus, which controls gastrointestinal peristalsis and transit
time. Argyrophil cells coordinate the activation of argen-
taffin cells, which secrete specific neurotransmitters and
ultimately cause contraction or relaxation of muscle fibers
within the bowel wall [116]. A distinct time-lag has been
demonstrated between the appearance of both cell types with
argyrophil cells appearing earlier than argentaffin cells [117].
It has been shown that there is a caudocranial gradient in the
differentiation of these neuron cells in the human bowel [117,
118]. Therefore, it is suspected that the disruption of this
differentiation process may lead to an absence of the AP.
Epidemiology of absence of the AP
There is a paucity of published data on the incidence of this
very rare condition. In 1997, Puri et al. [1] reported three
cases with absence of the AP in their series of patients with
functional bowel disorders. Familial cases in the offspring
of consanguineous parents and recurrence in siblings sug-
gests that the absence of the AP may be inherited in an
autosomal-recessive manner [116, 119].
Clinical presentation of absence of the AP
The clinical symptoms of patients with absence of the AP are
highly similar to HD presenting with severe constipation,
moderate abdominal distension and lack of peristalsis [120].
Diagnosis of absence of the AP
The absence of argyrophil cells and their neuronal pro-
cesses can only be demonstrated using silver impregnation
[121] (Fig. 5a, b) of full-thickness biopsies, while con-
ventional H&E staining, AChE histochemistry and immu-
nohistochemistry with several neuronal markers fail to
show this abnormality [1, 101].
Management of absence of the AP
The majority of patients with absence of the AP can be
managed conservatively with laxatives and enemas, but in
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Fig. 4 H&E (a) and AChE (b) staining in a patient with IG showing immature ganglion cells

Fig. 5 Silver staining showing normal AP (a) and absence of argyrophil cells (b) in a patient with absence of the AP

some cases internal sphincter myectomy or formation of a
colostomy may be necessary due to persistent constipation
[1, 120].
Outcome of absence of the AP
Conservative and surgical treatment of patients with
absence of the AP usually results in a satisfactory outcome
[1, 120].
Internal anal sphincter achalasia
Internal anal sphincter achalasia (IASA) is a condition with
clinical presentation similar to HD [122–124], but in con-
trast to HD, ganglion cells are present in rectal suction
biopsies. Previously, IASA was referred to as ultrashort-
segment HD, which is characterized by an aganglionic
segment of 1–3 cm above the pectinate line, normal AChE
activity in the lamina propria and increased AChE activity

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Fig. 6 NADPH-d staining of normal IAS (a). Reduced NADPH-d positive innervations (b) in a patient with IASA
in the muscularis mucosae [125]. Several investigators
have suggested that IASA is more accurate for this path-
ological entity as many patients with absence of the rec-
tosphincteric reflex on anorectal manometry showed
presence of ganglion cells combined with normal AChE
activity in rectal biopsies [126–128].
Pathogenesis of IASA
The exact pathogenesis of IASA remains unclear despite
the attempts of several investigators to determine the
pathophysiological mechanisms in more detail. It has
been suggested that age-related changes in the develop-
ing intramuscular innervation of the internal anal
sphincter (IAS) may form the basis for the observed
motility dysfunction [129]. By analyzing the NADPH-d
activity (Fig. 6a, b), Hirakawa et al. [130] reported
absent to marked reduction of nitrergic innervation
within the IAS of patients with IASA as the underlying
pathomechanism leading to spasm or increased tone. A
defective innervation of the neuromuscular junction of
the IAS with decreased expression of PGP9.5 and syn-
apsin-1 has also been detected [131]. Kobayashi et al.
[94] further demonstrated absent to markedly reduced
NADPH-d and NCAM activity in the IAS of patients
with IASA. In addition, a reduced number of c-Kit
positive ICCs have been found in the IAS of patients
with IASA [132]. The deficiency in nitrergic innervation
and ICCs may explain the impaired anal relaxation in
patients with IASA [133].
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Pediatr Surg Int (2013) 29:855–872
Epidemiology of IASA
Since 1973, a total number of 395 cases with IASA have
been reported in the literature [134]. However, the exact
incidence of IASA is unknown.
Clinical presentation of IASA
The clinical presentation of IASA is in most cases similar
to that of HD. Patients with IASA usually present with
severe constipation with or without soiling. About one-
third of these patients have a history of abdominal disten-
sion and failure of laxative therapy [125, 126].
Diagnosis of IASA
The diagnosis of IASA is based on clinical symptoms
combined with finding of absence of the rectosphincteric
reflex on rectal balloon inflation with marked increased
rhythmic activity on anorectal manometry (Fig. 7a, b),
presence of ganglion cells and normal AChE activity in
rectal suction biopsies as well as reduction of nitrergic
innervation within the IAS.
Management of IASA
Posterior IAS myectomy has been recommended for the
treatment of IASA [135–137]. More recently, intrasphinc-
teric injection of botulinum toxin has been introduced as a
therapeutic alternative [138–143].
Pediatr Surg Int (2013) 29:855–872 865

Fig. 7 Anorectal manometry

showing evidence of the
rectosphincteric reflex in a
normal IAS (a). Absence of the
rectosphincteric reflex on rectal
balloon inflation with marked
increased rhythmic activity of
the IAS (b) in a patient with
IASA

Fig. 8 Voiding
cystourethrogram (a) and
barium contrast enema
(b) showing massively enlarged
bladder and microcolon in a
patient with MMIHS

Outcome of IASA indicated that posterior IAS myectomy appears to be a

more effective treatment option resulting in a better func-
The vast majority of patients with IASA have regular tional outcome compared to intrasphincteric botulinum
bowel movements after treatment irrespective of the ther- toxin injection [134]. The rate of transient fecal inconti-
apeutic approach [34, 135]. However, it has recently been nence, non-response and subsequent surgical procedures

123
866
Fig. 9 Electron microscopy of smooth muscle cells in normal bowel (a). Central core degenerations resulting in empty vacuoles (asterisks) and
increased connective tissue (triangles) in smooth muscle cells (b) of a patient with MMIHS
were significantly higher after injection of botulinum toxin,
whereas long-term improvements were significantly more
frequent following IAS myectomy. Interestingly, no dif-
ferences were found in the postoperative use of laxatives or
enemas, postoperative soiling as well as constipation
between both procedures.
Megacystis microcolon intestinal hypoperistalsis
syndrome
Megacystis microcolon intestinal hypoperistalsis syndrome
(MMIHS) is the most severe form of functional bowel
obstruction in the newborn [144]. This rare condition is
characterized by massive abdominal distension caused by a
large-dilated non-obstructed bladder, microcolon with
malrotation and decreased or absent intestinal peristalsis
[34] (Fig. 8a, b).
Pathogenesis of MMIHS
Megacystis microcolon intestinal hypoperistalsis syndrome
was first described in 1976 by Berdon et al. [145], who
observed above characteristic features in five newborn
girls. Since then, various hypotheses have been proposed to
explain the pathogenesis of MMIHS. Genetic [146], myo-
genic [147–149], neurogenic [150, 151] and hormonal
[151, 152] etiologies have been discussed. However, most
of these theories are derived from case reports due to the
rarity of this condition. Thus, the exact etiology still
remains unclear. At present, several candidate genes for
MMIHS have been identified. Gene knockout of the
123
Pediatr Surg Int (2013) 29:855–872
nicotinic acetylcholine receptor (nAChR) in transgenic
mice resulted in a phenotype similar to that of human
MMIHS [153]. Furthermore, it has been shown that a lack
of expression of the a3, b2 and b4 subunits of nAChR in
small bowel tissue from patients with MMIHS contributes
to the pathogenesis of this rare condition [154, 155].
CHRNA3 and CHRNB4 genes, which are both coding for
the b4 subunit of nAChR, are additional strong candidates
[156]. Much published data also points to a dysfunction
within the smooth muscle layer. Puri et al. [157] reported
vacuolar changes in the smooth muscle cells within the
bowel and bladder wall of patients with MMIHS (Fig. 9a,
b). Therefore, it has been suggested that the observed
smooth muscle myopathy may be the underlying cause of
MMIHS [148, 158]. The expression of several contractile
and cytoskeleton proteins such as aSMA, calponin, cal-
desmin and desmin has also been found to be absent or
decreased in the colonic smooth muscle tissue of patients
with MMIHS [147, 159, 160]. Further support of this
theory is derived from the findings of abnormal synapto-
physin distribution in the circular muscle layer of bowel
and bladder specimens, as well as increased connective
tissue and atrophic smooth muscle fibers [161]. Moreover,
a decreased expression of ICCs in the bladder has been
observed [56, 159, 162]. In addition to the smooth muscle
dysfunction, other studies have focused on neuronal dif-
ferences in MMIHS [163]. Previously, immaturity and
malfunction of autonomic nerve endings in the whole
gastrointestinal tract have been demonstrated [150]. Axo-
nal dystrophy and additional defects of the autonomic
innervation in the intestine of patients with MMIHS have
also been reported [151, 164]. Furthermore, an intramural
Pediatr Surg Int (2013) 29:855–872
inflammatory process that affects the gastrointestinal and
urinary tract has been proposed in the pathogenesis of
MMIHS [152].
Epidemiology of MMIHS
Since 1976, there have been a total number of 227 patients
with the diagnosis of MMIHS reported in the published
literature [165]. Among them, a male-to-female ratio of
1:2.4 has been observed, suggesting a distinct female pre-
dominance. In addition, it has been reported that male
patients with MMIHS seem to have a shorter life expec-
tancy than affected females. This is most likely due to a
more severe form of this condition in males compared to
females [166, 167]. Furthermore, occurrence of familial
cases in the offspring of consanguineous parents and
recurrence in siblings indicates that MMIHS may be
inherited in an autosomal-recessive manner [168–170].
Clinical presentation of MMIHS
The enlarged bladder with hydronephrosis, microcolon and
intestinal malrotation can often be demonstrated on pre-
natal ultrasound or MRI scans [146]. The clinical presen-
tation of MMIHS is similar to that of other severe neonatal
intestinal obstruction, typically presenting with massive
abdominal distension, which is a consequence of the large-
dilated non-obstructed bladder with or without upper uri-
nary tract dilatation. The majority of patients with MMIHS
are unable to void spontaneously and require either vesi-
costomy or catheterization. It has been shown that the
detrusor muscle in these patients is strikingly abnormal,
which is most likely the cause for the voiding dysfunction
[171]. Additional common findings are bile-stained vom-
iting, absent or decreased bowel sounds and failure to pass
meconium [165].
Diagnosis of MMIHS
There should be an emphasis on prenatal diagnosis of
MMIHS to allow for adequate prenatal counseling to
enable future parents to make an informed decision
regarding the continuation of the pregnancy, given the poor
prognosis of this severe condition. The diagnosis of
MMIHS is usually made on the basis of prenatal ultrasound
findings and characteristic clinical presentation in the
immediate neonatal period. An enlarged bladder and
hydronephrosis can already be found on fetal ultrasound
studies [172]. Analysis of enzymatic changes in amniotic
fluid in combination with MRI scans can further contribute
to the prenatal diagnosis of MMIHS [173]. Histological
evaluation of myenteric and submucosal plexuses has
revealed normal ganglion cells in 77 % of the investigated
867
bowel specimens from patients with MMIHS. The
remaining 23 % were shown to have various neuronal
abnormalities including hyper-/hypoganglionosis and
immature ganglia [34]. In addition, some authors found
significant anomalies in smooth muscle cells from bowel
and bladder specimens, such as vacuolar degeneration as
well as thinning of the longitudinal muscle [149, 157].
Management of MMIHS
The management of patients with MMIHS is frustrating. A
number of prokinetic drugs and gastrointestinal hormones
have been tried without any success [174]. Surgical treat-
ment usually has to be performed for malrotation, bowel
obstruction and megacystis to achieve decompression of
bowel and bladder. However, in most cases, these inter-
ventions do not result in any improvement in enteral food
intake, functional bowel obstruction or bladder function.
Consequently, the majority of patients with MMIHS are
maintained on total parenteral nutrition (TPN), which leads
to further comorbidities, such as catheter sepsis, dyslipi-
demia, TPN-associated liver disease and eventually chronic
liver failure [165]. In patients with irreversible intestinal
pathology and failure of TPN, intestinal and multivisceral
transplantation has recently been introduced as a valuable
therapeutic alternative. At present, 12 multivisceral trans-
plantations have been reported in MMIHS patients [34].
Outcome of MMIHS
In recent years, the survival rate of MMIHS has consid-
erably improved from 12.6 % initially to 55.6 %. Between
1977 and 2011, an overall survival rate of 19.7 % has been
reported with the oldest survivor being 24 years old. The
most frequent cause of death in MMIHS patients has been
shown to be overwhelming sepsis, followed by multiple
organ failure and malnutrition [165]. A 3-year survival rate
of 50 % with all survivors tolerating enteral feeding and
showing adequate gastric emptying has recently been
reported [175]. These improvements in outcome have
mainly been attributed to more specialized care, innova-
tions in parenteral nutrition and introduction of multivis-
ceral transplantation.
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