Download A novel CDKN1C variant uncovered in a patient with Beckwith

A novel CDKN1C variant uncovered in a patient with Beckwith-Wiedemann syndrome.
BASTAKI FATMA1, SAIF FATIMA1, AL-ALI MAHMOUD TALEB2 AND HAMZEH ABDUL
REZZAK†2
1
Pediatric Department, Latifa Hospital, Dubai Health Authority, Dubai, UAE
2
Centre for Arab Genomic Studies, Dubai, UAE
†Corresponding
Author
Address for corresponding author is: Centre for Arab Genomic Studies, P.O. Box 22252, Dubai,
United Arab Emirates
Telephone: +971-4-398 6 777, Fax: +971-4-3980 999
Email: [email protected]
Author
Phone no.
Email address
Fatma Bastaki
+971 4 2193023
[email protected]
Fatima Saif
+971 4 2193023
[email protected]
Mahmoud Taleb Al Ali
+9714398 6 777
[email protected]
Abdul Rezzak Hamzeh
+9714398 6 777
[email protected]
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A novel CDKN1C variant uncovered in a patient with Beckwith-Wiedemann syndrome.
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Abstract
The cyclin-dependent kinase inhibitor; CDKN1C negatively regulates cell proliferation by
binding to and inhibiting several G1 cyclin/CDK complexes. Loss-of-function mutations in
CDKN1C cause an over-growth syndrome named Beckwith-Wiedemann (BWS). The study was
conducted in Latifa hospital, Dubai where diagnosis took place. Molecular elucidation of the
novel mutation was carried out by amplifying and direct sequencing of the CDKN1C gene.
In this study, we report a novel mutation (c.703C>T) in CDKN1C harbored by an Emirati child
with BWS. This is a nonsense mutation affecting codon 235 of CDKN1C (p.Gln235X) that
appeared de novo in the patient (heterozygous), while both parents had wild-type copies of the
gene. The clinical and molecular consequences of this mutation are provided and discussed in
the light of previously reported CDKN1C mutations.
Keywords
p57KIP2, Overgrowth syndrome, novel nonsense mutation
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Introduction
Beckwith-Wiedemann syndrome (BWS) is a disorder characterized by overgrowth which
manifests itself throughout prenatal and postnatal periods as macrosomia, macroglossia and
abdominal wall defects leading to omphalocele and diastasis recti. The clinical presentation of
BWS (OMIM 130650) is markedly variable, and its multigenic and complex molecular causation
is based on aberrant genomic imprinting. Dysregulation of imprinted growth regulatory genes on
chromosome 11p15 is behind this generalized overgrowth, which can lead to additional features
including; hemihypertrophy, neoplasia (e.g. Wilms tumor, hepatoblastoma and gonadoblastoma),
adrenocortical cytomegaly, hepatomegaly, pancreatic hyperplasia and renal medullary dysplasia.
Other clinical findings include cryptorchidism, nevus flammeus, neonatal hypoglycemia and ear
anomalies; such as ear lobe creases. In most of the cases (85%), BWS is sporadic, but familial
transmission accounts for 10-15% of the cases. Several genetic and/or epigenetic aberrations are
associated with BWS; a minority of these aberrations involves chromosomal rearrangements.
Most of BWS pathogenesis is accounted for by epigenetic errors affecting the BWS-associated
region on 11p15.5 (1). The latter region encompasses a cluster of genes; one of which is
CDKN1C (p57KIP2, a cyclin-dependent kinase inhibitor). Point mutations in the CDKN1C seem
to cause BWS in 5-10% of sporadic BWS patients and in 40% of the cases with positive family
history (2). CDKN1C is a negative regulator of cell proliferation, because it is a strong, tightbinding inhibitor of several G1 cyclin/CDK complexes. The protein encoded by CDKN1C is
316 amino acids long and it has three structurally distinct regions: a CDK-inhibitory domain, a
domain that is rich in proline-alanine repeats, named the PAPA region, and a C-terminal
conserved motif, named QT box (3). CDKN1C is preferentially transcribed from the maternal
allele, and consequently it is generally considered to be "paternally imprinted" (1). Intriguingly,
patients with CDKN1C mutations have a higher risk of abdominal wall defects, but a lower risk
for developing tumors when compared to BWS cases that are caused by other mutations within
11p15.5. In cases resulting from molecular anomalies in CDKN1C, it appears that mutations
were either inherited from the mother or had emerged de novo on the maternal allele, although
this is not always the case (4). In this study we report a novel mutation in the CDKN1C gene
harbored by an Emirati BWS patient in a heterozygous state. The mutation, which originated in
the patient de novo, is a C to T transversion at position c.703, resulting in a premature
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termination of the protein. Here we present clinical as well as molecular data from this BWS
patient and his novel CDKN1C mutation.
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Case Report
The patient is a 9-year-old Emirati boy who was born at 37 weeks gestation by elective caesarean
section for omphalocele which was detected antenatally. The parents are healthy and nonconsanguineous. His Apgar score was 8 and 9 at 1 and 5 minutes, respectively, his weight was 4
kg, and his head circumference was 36 cm (both are above the 50th percentile).
Physical examination at birth revealed a dusky, distressed baby with dysmorphic features (Figure
1: panels A, B and D) including mid-face hypoplasia, infraorbital creases, facial nevus
flammeus, macroglossia, posterior helical ear pit, large Omphalocele (covered by Wharton jelly)
and bilateral undescended testis. His systemic examination was unremarkable. He was
ventilated for severe respiratory distress, received Surfactant and started on Dextrose 10 % for
hypoglycemia. The Omphalocele was surgically repaired on the second day following birth; it
contained small bowel as well as small extra liver lobe.
Echocardiogram of the patient revealed a small muscular ventricular septal defect, which closed
spontaneously. His six-monthly abdominal ultrasound (Figure 1; panel C) revealed a nonprogressive cyst in the left upper quadrant of the abdomen as well as mild hepatomegaly.
Levels of alpha-fetoprotein were repeatedly normal.
Genetic Findings
Upon informed consent, peripheral blood samples were collected from the affected child and his
parents. Thereafter, genomic DNA was extracted from blood samples according to standard
protocols. Exon 2 and exon 3 of the CDKN1C gene were amplified by PCR and sequenced
directly; these steps were carried out and validated at the Bioscientia Centre for Human Genetics,
Ingelheim, Germany. The consequence of the mutation unraveled in this study was assessed
using ExPASy translation tool (http://web.expasy.org/translate/) and PolyPhen-2 which predicts
the functional effects of human nonsynonymous SNPs (http://genetics.bwh.harvard.edu/pph2/).
The results confirmed the presence of the novel CDKN1C mutation c.703C>T (p.Gln235X) in
the patient, while no such mutation could be found in the parents (Figure 2; panels B and C).
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Figure 1: Clinical features of the affected child; A, B and D show a number of dysmorphic
features such as mid-face hypoplasia, infraorbital creases, facial nevus flammeus, macroglossia,
posterior helical ear pit. C: Abdominal ultrasound scan showing the cyst in the left upper
quadrant cyst in the patient.
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Discussion
The child had met his key developmental milestones, but altogether the abovementioned clinical
findings were suggestive of BWS, therefore samples from the patient and both his parents were
subjected to molecular analysis of the gene CDKN1C. The patients harbors a novel CDKN1C
mutation c.703C>T (p.Gln235X), for which he is heterozygous. It affects exon 2 and it alters
codon 235 (CAG to TAG) resulting in translation termination at this position. As the mutation
was not present in the parents, it is assumed that it emerged de novo in the patient. Notably, this
particular variant in CDNK1C was not reported on in any of the relevant databases such as
dbSNP, 1,000-genomes and EVS.
Figure 2: A; Schematic representation of human CDKN1C. Previously reported, BWSassociated truncation mutations that abolish the QT box are depicted below the protein with
filled arrowheads. The position of the novel variant reported in this study is indicated with an
arrow above the protein. B and C; Sequence chromatograms of the mutated region in CDKN1C
in the patient (B) and his mother (C). The patient harbors a heterozygous mutation (c.703C>T)
in this gene that results in (p.Gln235X), while the mother has only the WT version of this
paternally imprinted gene.
The protein encoded by CDKN1C strongly inhibits the cyclin/CDK complexes of the G1 phase,
therefore it decelerate the progression into the cell cycle. As a matter of fact, the C-terminal QTbox of CDKN1C can bind PCNA (proliferating cell nuclear antigen), which plays a crucial role
in DNA replication (5). A number of mutations across the length of CDKN1C were previously
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uncovered, and these mutations were quite informative as to the function of CDKN1C in the cell.
In general, loss-of-function mutations promote cell proliferation giving rise to an over-growth
phenotype as in BWS. Almost all BWS-related CDKN1C mutations are either missense
alterations affecting the N-terminal region or nonsense ones that produce truncated proteins of
various lengths. In contrast, gain-of-function mutations of CDKN1C are linked to stunted
growth as in patients with IMAGe and Russell Silver syndromes (6). These gain-of-function
mutations tend to be missense ones, affecting the PCNA-binding domain of the protein and
disrupting its ubiquitination.
This study unraveled a novel variant affecting codon 235, which is located just before the start of
the PCNA-binding domain in CDKN1C, see Figure 2; panel A. The molecular alteration leads
to creating a stop codon, and most probably, to the production of a truncated protein that
completely lacks the QT box. The clinical presentation of the patient indicates that this mutation
resulted in a loss-of-function of CDKN1C, and this is in line with three other comparable
mutations that have been reported previously (2, 7, 8). The three mutations are all nonsense, and
are located around the boundary between the PAPA and QT domains (Figure 2; panel A), as is
the case with the mutation reported in this study. The previously reported mutations introduce
translational termination signals at codons 232, 241 and 247, thus they probably abolish the QT
domain. Moreover, they produce a similar range of phenotypic entities to our mutation within
the usual phenotypic gamut of BWS. Based on these published reports and the clinical
presentation of the patient, it can be postulated that the novel CDKN1C mutation; c.703C>T
(p.Gln235X) is the cause of BWS in our Emirati patient.
The patient's main BWS symptom was omphalocele, and interestingly; CDKN1C mutations
appear to cause high frequency of omphalocele in patients (9). Additionally, our patient suffered
cryptorchidism, which corroborates studies suggesting that genital anomalies were underreported
previously (10). Although the patient showed no abnormal signs in relation to AFP tumor
marker levels, but it is imperative that he is followed up medically for any signs of neoplasia, as
BWS patients at a higher risk for developing tumors. In this context it is particularly important
to fully investigate the epigastric cyst that is uncovered by abdominal USS. Such medical
follow-up is recommended to continue until the age of 10 years, despite the fact that studies
which focus on patients with sporadic mutations of CDKN1C do not support increased cancer
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susceptibility in these patients (7). This study with its novel finding can help direct molecular
investigations for the new cases that emerge locally and regionally, in order to elucidate the
underlying causality properly and efficiently.
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Acknowledgements
Authors would like to thank the patient and his family for full cooperation. Thanks also go to
and Sheikh Hamdan Bin Rashid Al Maktoum Award for Medical Sciences for continuous and
comprehensive support. Authors have no conflict of interest to report.
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