Not only Alagille syndrome. Syndromic paucity of interlobular bile ducts secondary to HNF1β deficiency: a case report and literature review

Abstract

Background
paucity of interlobular bile ducts is an important observation at liver biopsy in the diagnostic work-up of neonatal cholestasis. To date, other than in the Alagille syndrome, syndromic paucity of interlobular bile ducts has been documented in four cholestatic neonates with HFN1β mutations. A syndromic phenotype, known as renal cysts and diabetes syndrome (RCAD), has been identified. This is usually characterized by a wide clinical spectrum, including renal cysts, maturity-onset diabetes of the young, exocrine pancreatic insufficiency, urogenital abnormalities and a not well established liver involvement. Herein we report a novel case of paucity of interlobular bile ducts due to an HFN1β defect.

Case presentation
A 5-week-old boy was admitted to our department for cholestatic jaundice with increased gamma-glutamyl transpeptidase and an unremarkable clinical examination. He had been delivered by Caesarian section at 38 weeks’ gestation from unrelated parents, with a birth weight of 2600 g (3rd percentile). Screening for cholestatic diseases, including Alagille syndrome, was negative except for a minor pulmonary artery stenosis at echocardiography and a doubt of a thoracic butterfly hemivertebra. The finding of hyperechogenic kidneys with multiple bilateral cortical cysts at ultrasound examination, associated with moderately impaired renal function with proteinuria, polyuria and metabolic acidosis, was suggestive of ciliopathy. A liver biopsy was performed revealing paucity of interlobular bile ducts, thus the diagnosis of Alagille syndrome was reconsidered. Although genetic tests for liver cholestatic diseases were performed with negative results for Alagille syndrome (JAG1 and NOTCH2), a de-novo missense mutation of HNF1β gene was detected. At 18 months of age our patient has persistent cholestasis and his itching is not under satisfactory control.

Conclusions
Alagille syndrome may not be the only syndrome determining paucity of interlobular bile ducts in neonates presenting with cholestasis and renal impairment, especially in small for gestational age newborns. We suggest that HNF1β deficiency should also be ruled out, taking into consideration HNF1β mutations, together with Alagille syndrome, in next generation sequencing strategies in neonates with cholestasis, renal impairment and/or paucity of interlobular bile ducts at liver biopsy.

Full text

C A S E R E P O R T Open Access
Not only Alagille syndrome. Syndromic
paucity of interlobular bile ducts secondary
to HNF1βdeficiency: a case report and
literature review
Michele Pinon
1*
, Michele Carboni
1,2
, Davide Colavito
3
, Fabio Cisarò
1
, Licia Peruzzi
4
, Antonio Pizzol
1,2
,
Giulia Calosso
1,2
, Ezio David
5
and Pier Luigi Calvo
1
Abstract
Background: paucity of interlobular bile ducts is an important observation at liver biopsy in the diagnostic work-up
of neonatal cholestasis. To date, other than in the Alagille syndrome, syndromic paucity of interlobular bile ducts
has been documented in four cholestatic neonates with HFN1βmutations. A syndromic phenotype, known as renal
cysts and diabetes syndrome (RCAD), has been identified. This is usually characterized by a wide clinical spectrum,
including renal cysts, maturity-onset diabetes of the young, exocrine pancreatic insufficiency, urogenital abnormalities
and a not well established liver involvement. Herein we report a novel case of paucity of interlobular bile ducts due to
an HFN1βdefect.
Case presentation: A 5-week-old boy was admitted to our department for cholestatic jaundice with increased
gamma-glutamyl transpeptidase and an unremarkable clinical examination. He had been delivered by Caesarian
section at 38 weeks’gestation from unrelated parents, with a birth weight of 2600 g (3rd percentile). Screening
for cholestatic diseases, including Alagille syndrome, was negative except for a minor pulmonary artery stenosis
at echocardiography and a doubt of a thoracic butterfly hemivertebra. The finding of hyperechogenic kidneys
with multiple bilateral cortical cysts at ultrasound examination, associated with moderately impaired renal
function with proteinuria, polyuria and metabolic acidosis, was suggestive of ciliopathy. A liver biopsy was
performed revealing paucity of interlobular bile ducts, thus the diagnosis of Alagille syndrome was reconsidered.
Although genetic tests for liver cholestatic diseases were performed with negative results for Alagille syndrome
(JAG1 and NOTCH2), a de-novo missense mutation of HNF1βgene was detected. At 18 months of age our patient
has persistent cholestasis and his itching is not under satisfactory control.
Conclusions: Alagille syndrome may not be the only syndrome determining paucity of interlobular bile ducts in
neonates presenting with cholestasis and renal impairment, especially in small for gestational age newborns. We
suggest that HNF1βdeficiency should also be ruled out, taking into consideration HNF1βmutations, together
with Alagille syndrome, in next generation sequencing strategies in neonates with cholestasis, renal impairment
and/or paucity of interlobular bile ducts at liver biopsy.
Keywords: Paucity of interlobular bile ducts, HNF1βmutations, Alagille syndrome, Ciliopathy, Renal cysts
* Correspondence: michele.pinon@gmail.com
1
Pediatric Gastroenterology Unit, Regina Margherita Children’s Hospital,
Azienda Ospedaliero-Universitaria Città della Salute e della Scienza, University
of Turin, Piazza Polonia 94, 10126 Turin, Italy
Full list of author information is available at the end of the article
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Pinon et al. Italian Journal of Pediatrics (2019) 45:27
https://doi.org/10.1186/s13052-019-0617-y

Background
Neonatal cholestasis is characterized by conjugated
hyperbilirubinemia and manifests clinically with jaun-
dice, pruritus, failure to thrive, fat-soluble vitamin defi-
ciency and xanthomas. There may also be hypocholic or
acholic stools in the presence of functional or anatomic
biliary obstruction. Diagnostic work-up is of paramount
importance to exclude biliary atresia, as the timing of
surgical intervention directly impacts clinical outcomes.
Liver biopsy adds essential information to the diagnos-
tic evaluation in persistent neonatal cholestasis, prompt-
ing clinicians to consider biliary atresia if ductular
proliferation is present. As recently reported, paucity of
interlobular bile ducts (PILBD) is not such a rare finding
at histology, especially in infants with cholestasis (70/
632 of pediatric liver biopsies, not considering graft ver-
sus host disease, drugs, chronic rejection) [1]. Cholesta-
sis due to PILBD is caused by an alteration in the
anatomic integrity of the biliary tract with absence of, or
a marked decrease in, the number of interlobular bile
ducts. PILBD can be only documented histologically as a
loss of intrahepatic bile ducts in more than 50% of the
portal tracts in a biopsy specimen containing at least 10
portal tracts. Two PILBD categories have been recog-
nized: syndromic (S-PILBD) and nonsyndromic
(NS-PILBD). S-PILBD is associated to Alagille syndrome
(AGS) and is variably characterized by the presence of at
least three of the five following features: PILBD-associated
chronic cholestasis, peripheral pulmonary artery stenosis,
vertebrae segmentation anomalies, characteristic facies
and posterior embryotoxon. Moreover, renal and vascular
alterations are often present in numerous AGS patients,
even if they are not included in the diagnostic criteria.
AGS is commonly associated with mutations in JAG1
gene, which encodes a protein involved in Notch signaling
(AGS type 1), or in NOTCH2 gene (AGS type 2).
NS-PILBD is a non-specific condition of unknown eti-
ology and is not associated with systemic malformations
or other disorders that induce biliary ductopenia. Despite
medical treatment, end stage liver disease due to persist-
ent cholestasis may occur in children with PILBD [2], as
well as intractable pruritus, affecting the quality of life,
failure to thrive and osteodystrophy [3]. Taken together
(S-PILBD and NS-PILBD), liver transplantation is neces-
sary in 30–40% of these patients and PILBD accounts for
5–10% of all the indications for pediatric liver transplant-
ation [1,3,4]. That is why PILBD is an important finding
in the diagnostic work-up of neonatal cholestasis, which
should alert clinicians to consider a diagnosis of AGS, to-
gether with the result of echocardiogram, imaging of the
vertebrae and ophthalmologic examination.
To date, to the best of our knowledge, only AGS
has been associated with syndromic PILBD and there
are few reports on NS-PILBD of unknown origin [5].
However, although no other underlying causes of syn-
dromic PILBD have yet been well established, PILBD
has been documented in a low number of cholestatic
neonates with HNF1βmutations [6–10].
Case presentation
A 5-week-old boy was admitted to our department for
jaundice and failure to thrive. He had been delivered in
another neonatal centre by Cesarean section, from non-
consanguineous healthy parents, at 38weeks of gestation,
with an Apgar score of 9/9. His birth weight was 2600 g
(3rd percentile), length 49 cm (27th percentile) and cranial
circumference 32.5 cm (5th percentile). Urinary Cyto-
megalovirus test was negative, as was his family history for
known diabetes, hepatic or renal disease; he had a healthy
8-year-old brother. The baby had been discharged from
the other centre, in a satisfactory condition, on the 4th
day of life.
Our physical examination was unremarkable, except for skin
and scleral jaundice. The baby also had hypocholic stools.
Routine blood tests confirmed cholestatic jaundice (total bili-
rubin 11.95 mg/dL, conjugated bilirubin 6.69 mg/dL) with in-
creased gamma-glutamyl transpeptidase (GGT 221 U/L),
which persisted after ursodeoxycholic acid treatment (20 mg/
Kg/day). Fat-soluble vitamins supplementation was started
and cow’s milk with highly hydrolyzed proteins enriched with
medium chain triglycerides was recommended.
Routine screening for cholestatic diseases, including
primary investigations for Alagille syndrome, was nega-
tive except for a minor pulmonary artery stenosis at
echocardiography and a doubt of a thoracic butterfly
hemivertebra. Abdominal ultrasound (US) examination
revealed a normal liver for size and echogenicity, normal
biliary intrahepatic and extrahepatic tree, regular liver
vessel flow and hyperechogenic kidneys, with multiple
bilateral cortical cysts (maximum size 2 mm). Renal
function was impaired (serum creatinine 0.59 mg/dL, es-
timated glomerular filtration rate 35 ml/min/1.73m
2
,
Chronic Kidney Disease KDIGO stage 3), with metabolic
acidosis and tubular proteinuria; he also had polyuria (7
mL/Kg/h) during hospitalization with depressed breg-
matic fontanelle. The laboratory tests performed during
hospitalization and follow-up are reported in Table 1.
The presence of hyperechogenic kidneys, with multiple
bilateral cortical cysts at US examination, associated
with a moderate alteration of renal function, were sug-
gestive of ciliopathy. Hepato-biliary scintigraphy showed
no passage of bile. A liver biopsy was performed, reveal-
ing PILBD with biliary stasis (Fig. 1). The association of
cholestasis and PILBD, other than the renal involvement,
led us to reconsider the diagnosis of AGS and to per-
form genetic tests for liver cholestatic diseases.
Thebabycontinuedtohavehypocholic stools and persist-
ent cholestasis whilst hospitalized. Although renal function
Pinon et al. Italian Journal of Pediatrics (2019) 45:27 Page 2 of 9

was stable, a central venous access device was necessary for
the first few weeks to treat the dehydration caused by poly-
uria. Moreover, a mild hyperparathyroidism was documented
and subcutaneous erythropoietin (EPO) administration was
started to treat a progressive anemia.
He was discharged from our department at the age of
2 months. At the first monthly follow-up, renal function
and proteinuria were stable. Because of the persistence
of hyponatremia and metabolic acidosis with hyperkale-
mia, supplemental oral rehydration with sodium chloride
and administration of sodium bicarbonate were contin-
ued. EPO treatment was still necessary to maintain nor-
mal hemoglobin levels. During the following months, his
cholestasis remained stable, although the onset of itching
Table 1 Laboratory tests
Tests Reference range 5 weeks 2 months 6.5 months 9 months 11 months 14 months 16 months 18 months
Liver function tests
Bilirubin total (mg/dL) < 1 11.95 15.5 4.3 1.6 1.3 2.2 2.5 2.8
Conjugated bilirubin (mg/dL) < 0.2 6.68 13.5 4.1 1.5 1.2 2.1 2.2 2.4
AST (IU/L) < 50 210 276 86 75 92 189 135 87
ALT (IU/L) < 40 320 349 47 63 90 332 177 77
GGT (IU/L) < 50 221 1631 986 872 1129 1629 969 657
bile acids (mg/dL) < 10 –150 264 –117 304 253 132
Albumin (g/dL) 3.6–5.2 –3.7 3.3 3.7 4 4 4.2 4.2
Cholesterol (mg/dL) < 200 –361 –270 –349 256 –
HDL cholesterol (mg/dL) > 40 - - - - - 133 101 -
LDL cholesterol (mg/dL) < 90 - - - - - 179 131 -
Triglycerides (mg/dL) < 105 –– 135 138 119 187 120 78
Kidney function tests
Serum creatinine (mg/dL) 0.18–0.33 0.58 –0.5 0.51 0.5 0.47 0.46 0.50
eGFR* (ml/min/1.73m
2
)37–55 56 62 66 67 66
Uric acid (mg/dL) < 6 –– – 3.6 3.8 3.2 2.5 2.8
PrU/CrU** (mg/mg) < 0.2 1.7 –0 0 0 0 0.2 0
Hemoglobin (g/dL) 10.5–13.5 –9 10.7 9.9 13.6 11.1 11.6 10.8
*estimated glomerular filtration rate (Schwartz’s eGFR = 0.413 x length/sCr)
**proteinuria/creatininuria index
Fig. 1 Histology of liver biopsy aPaucity of intrahepatic bile ducts with mild Kupffer cell activation, mild hepatocitic polymorphism, focal
eosinophilic degeneration with a Councilman body; compatible with lobular light hepatitis. H&E 250X. bInterlobular portal tract with a ductular
reaction resembling a ductal plate malformation. Cytokeratin 7250X.
Pinon et al. Italian Journal of Pediatrics (2019) 45:27 Page 3 of 9

required rifampicin administration. His bilirubin level
then decreased significantly, reaching a plateau (total
bilirubin 2.5 mg/dL, conjugated 2.2 mg/dL) and the
color of his stools normalized. There were no alterations
in hepatic synthesis indexes or alpha-fetoprotein levels
and no signs of portal hypertension. Renal function had
a slight improvement, then stabilized to mild chronic
renal failure (Chronic Kidney Disease KDIGO stage 2).
US examination revealed an enlarged liver with a slightly
inhomogeneous structure, but no focal liver lesions. It
also revealed bilateral hyperechogenic kidneys of re-
duced size, whereas cysts were no longer documented. A
reduction of fecal pancreatic elastase, with an increase in
fecal fat excretion, was observed, probably due to an ini-
tial pancreatic exocrine dysfunction. As no pancreatic
hypoplasia was evidenced at US examination, a magnetic
resonance cholangiopancreatography (MRCP) has been
programmed.
Genetic tests for liver cholestatic diseases revealed nega-
tive results for AGS (JAG1 and NOTCH2). However, sub-
sequent whole exome sequencing (WES) analysis and
interpretation, together with variant prioritization analysis,
highlighted the presence of a previously described [11–13]
missense heterozygous mutation in the HNF1βgene,
p.Arg276Gln (c.827G > A). The mutation is located in
exon 4 of the HNF1βgene within the DNA binding do-
main, leading to the substitution of glutamine by arginine
at codon 276 (R276Q). As this novel mutation was absent
in the proband’s parents, we concluded that the patient
was a carrier of a de-novo mutation. To the best of our
knowledge, this is the first patient reported to be a carrier
of the p.Arg276Gln mutation presenting with renal in-
volvement associated with early onset cholestasis.
At time of writing the baby is 18 months of age, with
persistent cholestasis and pruritus, and a normal neuro-
logical development.
Discussion and conclusions
To date, neonatal cholestasis has been identified in 5
subjects with pathogenic HNF1βmutations, in most
cases de-novo deletions [6–10]; to the best of our know-
ledge, our case is the 6th one. Liver biopsy documented
the presence of PILBD, histologically similar to AGS, in
5/6 patients including our case [6–9]. This datum is cur-
rently lacking in the remaining case [10].
The hepatocyte nuclear factor 1β(HNF1β), also known as
transcription factor 2 (TCF2), is a key regulator of organogen-
esis for organs derived from the ventral endoderm [14]andit
is involved in transcriptional and functional regulation of the
liver, kidneys, urogenital tract and pancreas [15]. To date,
more than 50 heterozygous mutations in the HNF1βgene
(17q12) have been described in adults and young children, in-
cluding missense mutations, small insertions-deletions or
whole-gene deletions [15]. HNF1β-related disorders are
inherited with an autosomal dominant pattern, even if most of
themutationsarereportedtobede-novo (ashighas50%of
cases) [15]. HNF1βmutations were first recognized in a small
group of patients with maturity-onset diabetes of the young,
defined as MODY type 5, a monogenic form of early-onset
diabetes mellitus with onset before the age of 25 [16]. Ap-
proximately 50% of the patients with HNF1B mutations go on
to develop diabetes, most likely as a result of impaired insulin
secretion due to pancreatic hypoplasia, together with insulin
resistance. The co-occurrence of non-diabetic renal disease in
these patients led to the discovery of the importance of renal
involvement in the presence of an HNF1βdefect [17], prob-
ably one of the most commonly known monogenic causes of
developmental renal disease [15]. Cystic disease is the predom-
inant renal HNF1β-associated phenotype, characterized by
cortical small cysts, usually noted after birth, even if enlarged
bilateralhyperechogenickidneysmaybeobservedbyprenatal
ultrasonography. Clinical presentation varies a great deal and
can range from normal or mild alteration of renal function to
chronic kidney failure, up to end-stage renal disease, dialysis
or renal transplantation [15]. Electrolyte abnormalities, such as
hypomagnesemia [18] and hyperuricemia [19]with
early-onset gout, may also be present. The syndrome associ-
ated with HNF1βdefects is termed Renal Cysts and Diabetes
Syndrome (RCAD, OMIM #137920), even if it is characterized
by a larger clinical spectrum, which also includes pancreatic
hypoplasia with exocrine insufficiency [20], urogenital abnor-
malities [21] and a neurological involvement with autism
spectrum disorders and cognitive impairment [22]. Liver in-
volvement is frequently reported as an asymptomatic rise in
the levels of transaminases. Less frequently, it has been de-
scribed as a cholestatic liver impairment, such as neonatal or
late-onset cholestasis [7]. The patients presenting with neo-
natal cholestasis had similar histological results, showing
PILBD associated to marked cholestasis and a variable degree
of periportal fibrosis [6–10],asshowninTable2.Theywere
small for the gestational age (SGA) with a history of intrauter-
ine growth restriction (IUGR), in contrast with most chole-
static neonates. Moreover, they had renal cysts or renal
hyperechogenicity, two of them also had more complex renal
malformations, such as unilateral kidney agenesis and renal
dysplasia, with a variable degree of chronic renal insufficiency
not requiring dialysis or renal transplantation. There was a
long follow-up (> 10 years) in 60% of cases. Diabetes requiring
insulin therapy occurred at an average age of 10 years in 3/5
cases and 2/5 had pancreatic hypoplasia with impaired pancre-
atic exocrine function. Urogenital malformations were present
in only 1/5 cases and a mild cognitive impairment was ob-
served in 2/5. Although there is no data on a
genotype-phenotype correlation, noteworthy is the fact that
the patient reported by Raile et al with whole gene deletion
had the most serious phenotype [6]. A progressive resolution
of cholestasis within the first year of life was observed after
conservative therapy in 3/5 cases, with a persistent mild
Pinon et al. Italian Journal of Pediatrics (2019) 45:27 Page 4 of 9

Table 2 Patients’characteristics of subjects with HNF1βmutations presenting with neonatal cholestasis
Sex/origin
GW/BW g
(DS)
Liver involvement Liver biopsy Renal function and
ultrasound
Pancreatic involvement Growth Urogenital
malformations/
cognitive
impairement
HNF1βmutation Follow-
up
duration
Reference
♂/Japan
39/2390
(−2.26)
- neonatal cholestasis,
acholic stools
- no abnormality of
extrahepatic bile ducts
at explorative surgery
- cholestasis resolution
at 9-month follow-up
with a persistent mild
transaminases
alteration
- transient
hypercholesterolemia
PILBD, marked
cholestasis
- multiple bilateral cysts
(right, four 1–2cm
diameter cysts, left,
one 1 cm diameter
cyst)
- mild chronic renal
insufficiency
diabetes requiring insulin
therapy at 13 years of age
(polyuria and polydipsia,
mild metabolic acidosis)
NA absent/ mild c.457C > A, p.H135N (missense
mutation in exon 2, de novo or
paternal: history of liver
dysfunction and renal
insufficiency in his paternal
family)
13 years Kitanaka
S 2004
[9]
♂/Belgium
(Sardinian
origin)
37/1520
(−3.46)
- neonatal cholestasis,
slightly enlarged liver
- cholestasis resolution
at 1- year follow-up
with a persistent mild
transaminases
alteration
- 3 episodes of
cholangitis
- high triglyceridemia
(300 mg/dL)
PILBD, severe
biliary stasis,
slight periportal
fibrosis
- left kidney agenesis,
enlarged and
hyperechogenic right
kidney, multiple
cortical cysts
- progressive chronic
renal insufficiency
from 19 months
- diabetes requiring insulin
therapy at 5 years of age
without ketoacidosis
- pancreatic atrophy with
progressive exocrine
pancreatic deficiency
requiring enzyme
substitution from the age
of 16 years
final height
of 162.1 cm
(−1.86 SD),
BMI 19.0 Kg/
m
2
(−0.62 SD)
absent/NA 499_504
delGCTCTG
insCCCCT, A167FS
(combination of a deletion and
insertion in exon 2, de novo)
18 years Beckers D
2007 [8]
♂/Germany
35/1780 (−
1.69)
- neonatal cholestasis,
acholic stools
- cholestasis resolution
at 1 year follow-up
with a persistent mild
transaminases
alteration
- hypercholesterolemia
(292 mg/dL) and
hypertriglyceridemia
(307 mg/dL)
PILBD - severe malformations
of both kidneys (cystic
kidney dysplasia and
hydronephrosis due
to urethral stenosis)
- chronic renal
insufficiency
- diabetes requiring insulin
therapy at 13 years of age
- pancreatic hypoplasia
with progressive exocrine
pancreatic deficiency
final height
of 133.9 cm
(−6.7 SD),
BMI 17.3 Kg/
m
2
(−2.1 SD)
inguinal hernia,
abdominal
testis/delayed
psychomotor
development
HNF1βdeletion exons 1–9, de
novo
18 years Raile K
2009 [6]
♀/Czech
Republic
38/2360 (−
1.60)
- neonatal cholestasis,
acholic stools
- Kasai
portoenterostomy at
32 days of age as
extrahepatic bile ducts
were not visualized at
explorative surgery
- progressive increase in
liver function tests,
mainly cholestatic
- multiple cysts in the
PILBD,
cholestasis
without signs
of bile duct
proliferation
- multiple bilateral
cortical cysts (maximal
diameter 5 mm),
prenatally
hyperechogenic
kidneys
- normal renal function
by 2-year follow-up
-mild
hypomagnesemia
- pancreatic hypoplasia
(absent body and tail)
without exocrine
pancreatic deficiency
- normoglycaemia by 2-
year follow-up
growth
along the
3rd centile
absent/absent 1698 kb deletion including
HNF1β, de novo
2 years Kotalova
R 2015
[7]
Pinon et al. Italian Journal of Pediatrics (2019) 45:27 Page 5 of 9

Table 2 Patients’characteristics of subjects with HNF1βmutations presenting with neonatal cholestasis (Continued)
Sex/origin
GW/BW g
(DS)
Liver involvement Liver biopsy Renal function and
ultrasound
Pancreatic involvement Growth Urogenital
malformations/
cognitive
impairement
HNF1βmutation Follow-
up
duration
Reference
left hepatic lobe
(diameter from 2 to 7
mm)
♂/France
35/NA
- neonatal cholestasis
without acholic stools
- hepatocellular
carcinoma with
elevated alpha-
fetoprotein levels at
16 months of age re-
quiring liver transplant
- no relapse at 1-year
follow-up
- multinodular
hepatic tumor
and
micronodular
cirrhosis at
the explant
-no
information
available on
PILBD
- renal
hyperechogenicity
- transient renal failure
NA NA NA/NA 1.5 Mb deletion including
HNF1β
2 years de
Leusse C
[10]
♂/Italy
38/2600 (−
1.27)
- neonatal cholestasis,
hypocholic stools
- persistent cholestasis
and pruritus at 18-
month follow-up
- hypercholesterolemia
(256 mg/dL) and
hypertriglyceridemia
(120 mg/dL)
PILBD, biliary
stasis
- hyperechogenic
kidneys, with multiple
bilateral cortical cysts
(maximum size 2 mm)
- chronic renal
insufficiency
initial pancreatic exocrine
dysfunction without
pancreatic hypoplasia at US
growth
along the
10th centile
absent/absent c.827G > A, p.R276Q (missense
mutation in exon 4, de novo)
18
months
Present
report
NA: Information not available, GW: Gestation weeks, BW: Birth weight, PILBD: Paucity of interlobular bile ducts, BMI: Body mass index, US: Ultrasound
Pinon et al. Italian Journal of Pediatrics (2019) 45:27 Page 6 of 9

alteration of transaminases at follow-up. The 4th pa-
tient underwent Kasai portoenterostomy at 32 days of
age as extrahepatic bile ducts were not visualized at
explorative surgery, with a consequent reduction of
liver function tests to mildly elevated values. The 5th
patient had a completely different clinical course, in
as much as there was a diagnosis of hepatocellular
carcinoma with elevated alpha-fetoprotein levels at 16
months of age. He was transplanted and the histo-
logical evaluation of the explanted liver showed
micronodular cirrhosis [10].
Our patient’s clinical course was similar to 4/5 cases
previously reported in literature [6–9], i.e. the presence
of renal cysts with a moderate alteration of renal func-
tion and an incipient pancreatic insufficiency. Uric acid
and magnesium levels were in the normal range, there
were no urogenital malformations or evident neuro-
logical deficits. As would be expected, considering the
short follow-up and young age of the patient, there was
no diabetes at time of writing. Conversely, our patient
differed from the other 3/5 cases as his cholestasis did
not resolve within the first year of life, but it was stable
at 18 months with persistence of poorly controlled itch-
ing, despite medical therapy.
The hepatic phenotype is consistent with the paucity
of bile ducts observed in knock-out mice with a
liver-targeted HNF1βdeletion [23]. It has been reported
that HNF1βcould be necessary for intrahepatic bile duct
morphogenesis during liver formation from the ductal
plate, that is normally detected along the periportal mes-
enchyme during the embryonic period [23]. The inacti-
vation of HNF1βin mice causes severe jaundice and
growth retardation; histological analysis has demon-
strated the persistence of the ductal plate after birth to-
gether with a strong decrease in intrahepatic bile ducts,
most likely responsible for PILBD. Other abnormalities
in mice include gallbladder and extrahepatic bile duct
epithelial dysplasia and poor formation of interlobular
arteries [23].
A biliary extrahepatic involvement has also been re-
ported in humans, as in the patient with neonatal chole-
stasis and PILBD who underwent Kasai portoenterostomy
[7]. Another report described biliary abnormalities, identi-
fied by MRCP, in six patients with HNF1B mutations.
Most of them had varying types of bile duct cysts (BDCs)
in the extrahepatic bile ducts, with an atypical morphology
for any Todani classification [24].
All these alterations seem to have an underlying devel-
opmental origin from anomalies in ductal plate remodel-
ing, resulting in ductal plate malformations (DPMs),
characterized by the persistence of post-natal embryonic
biliary structures, biliary cell clusters or duct-like struc-
tures [25]. HNF1βcould well play a pivotal role as a
regulator of primitive ductal structures (PDS). According
to a new pathogenic classification, DPMs are not the re-
sult of a lack of PDS remodeling, but rather the common
endpoint of different defects of differentiation, matur-
ation, expansion, polarity and/or ciliogenesis of PDS,
affecting distinct stages of bile duct morphogenesis: e.g.
mice with HNF1βdeficient livers showed a normal dif-
ferentiation, but an abnormal PDS maturation [26].
These developmental anomalies represent the liver in-
volvement in a wide variety of diseases that affect vari-
ous organs, generally classified as ciliopathies [27]. The
role of HNF1βin ciliogenesis has been evidenced by
electron microscopy, demonstrating a reduction of nor-
mal primary cilia on the epithelia cells of cholangiocytes
in liver biopsies from three adults with late-onset chole-
stasis and no structural intra- or extrahepatic bile duct
defects [28]. That is why HNF1βis considered a ciliopa-
thy gene included among the genetic defects of syn-
dromic ciliopathies with liver involvement [29].
Abnormal biliary structures or bile duct cysts are a com-
mon finding in most ciliopathies [27]. However, in the
presence of HNF1βdefects, these abnormalities may in-
volve a paucity or a complete lack of intrahepatic bile
ducts. What we deduced from our case and other similar
ones reported, is that a ciliopathy should also be consid-
ered when liver biopsy shows ductopenia, with negative
investigations for AGS.
Next generation sequencing (NGS) strategies, such as
WES or whole genome sequencing (WGS), are promis-
ing to discriminate neonatal monogenic cholestatic dis-
orders and should play a pivotal role in the evaluation of
cholestatic neonates in addition to liver biopsy results.
In silico gene panel technology is another effective tool
to perform targeted analysis of WES or WGS data [30].
We are of the opinion that it is advisable to take into
consideration HNF1βgene mutations in WES or WGS
data analysis, together with AGS gene defects, in neo-
nates with cholestasis, renal impairment and/or PILBD
at liver biopsy. Moreover, HNF1βgene should be in-
cluded in NGS-expanded panels created for cholestatic
disorders.
As for AGS, we also advocate an early genetic test in
the presence of extrahepatic involvement to exclude a
misdiagnosis of biliary atresia, avoiding the need for un-
necessary explorative surgery [31].
The importance of clinical examination and timely
follow-up must not be underestimated, as HNF1βmuta-
tions may lead to serious extrahepatic manifestations.
Further studies in larger patient series are required so as
to better define the prognosis of these patients, also con-
sidering the recent report of a cholestatic infant with he-
patocellular carcinoma [10].
In conclusion, HNF1βdeficiency is probably associ-
ated to a more prevalent and complex biliary phenotype
than previously reported, with important clinical
Pinon et al. Italian Journal of Pediatrics (2019) 45:27 Page 7 of 9

implications. HFN1βdefects should be considered in ne-
onates with cholestasis and renal impairment, especially
in SGA and IUGR newborns with a family history of
renal disease or diabetes. Ductopenia is an important
finding in the diagnostic work-up of neonatal cholestasis
that, however, requires thorough investigation to rule
out causes other than AGS. Therefore, HNF1βdefi-
ciency should be taken into consideration as one of the
underlying causes of S-PILBD, in addition to AGS.
Hopefully, our findings may add further information
to the scarce documentation of this rare disease.
Abbreviations
AGS: Alagille Syndrome; BDCs: bile duct cysts; DPMs: ductal plate
malformations; EPO: erythropoietin; GGT: gamma-glutamyl transpeptidase;
HNF1β: hepatocyte nuclear factor 1β; IUGR: intrauterine growth restriction;
MODY5: maturity-onset diabetes of the young; MRCP: magnetic resonance
cholangiopancreatography; NGS: next generation sequencing; PDS: primitive
ductal structures; PILBD: paucity of interlobular bile ducts; RCAD: renal cysts
and diabetes; SGA: small for the gestational age; TCF2: transcription factor 2;
US: ultrasound; WES: whole exome sequencing
Acknowledgments
the authors wish to thank Ms. Barbara Wade for her linguistic advice.
Funding
no funding.
Availability of data and materials
data sharing not applicable to this article as no datasets were generated or
analyzed during the current study.
Authors’contributions
MP, MC and PLC conceived the study, analysed and interpreted the data and
drafted the manuscript. DC and ED performed respectively the genetic and
histologic analysis and interpreted the data. FC, LP, AP and GC critically
reviewed the manuscript. All authors have read and approved the final
submitted manuscript.
Ethics approval and consent to participate
parental informed consent for publication was obtained.
Consent for publication
written informed consent was obtained from the patient’s legal guardians
for publication of this case report and any accompanying images.
Competing interests
the authors declare that they have no competing interests.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Pediatric Gastroenterology Unit, Regina Margherita Children’s Hospital,
Azienda Ospedaliero-Universitaria Città della Salute e della Scienza, University
of Turin, Piazza Polonia 94, 10126 Turin, Italy.
2
Postgraduation School of
Pediatrics, Regina Margherita Children’s Hospital, Azienda
Ospedaliero-Universitaria Città della Salute e della Scienza, University of Turin,
Turin, Italy.
3
Research & Innovation (R&I Genetics) srl, Padua, Italy.
4
Pediatric
Nephrology Unit, Regina Margherita Children’s Hospital, Azienda
Ospedaliero-Universitaria Città della Salute e della Scienza, University of Turin,
Turin, Italy.
5
Department of Pathology, Azienda Ospedaliero-Universitaria Città
della Salute e della Scienza, University of Turin, Turin, Italy.
Received: 7 November 2018 Accepted: 11 February 2019
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